imm\ PROBLEMS IN PHOTOSYNTHESIS Publication Number 367 AMERICAN LECTURE SERIES® A Monograph in The BANNERSTONE DIVISION of AMERICAN LECTURES IN BIOCHEMISTRY AND BIOPHYSICS Edited by W. BLADERGROEN, Ir., M.A., Ph.D. Sandoz Ltd. Basle, Switzerland PROBLEMS IN PHOTOSYNTHESIS By W. BLADERGROEN, Ph.D. CHARLES C THOMAS • PUBLISHER Springfield • Illinois • U.S.A. CHARLES C THOMAS ■ PUBLISHER Bannerstone House 301-327 East Lawrence Avenue, Springfield, Illinois, U.S.A. Published simultaneously in the British Commonwealth of Nations by BLACKWELL SCIENTIFIC PUBLICATIONS, LTD., OXFORD, ENGLAND Published simultaneously in Canada by THE RVERSON PRESS, TORONTO This book is protected by copyright. No part of it may be reproduced in any manner without written permission from the publisher. © 1960, by CHARLES C THOMAS • PUBLISHER Library of Congress Catalog Card Number: 59-8491 With THOMAS BOOKS careful attention is given to all details of manufacturing and design. It is the Publisher's desire to present books that are satisfactory as to their physical qualities and artistic possibilities and appropriate for their particular use. THOMAS BOOKS will be true to those laws of quality that assure a good name and good will. Printed in the United States of America Dedicated to the Founder of Enzyme Chemistry and Cell Physiology OTTO HEINRICH WARBURG Director of ttie Max Planck Institute for Cell Physiology, Berlin-Dahlem ^ //^r^ (If U-Lt Preface Civilized man, long having abandoned his worship of fire and sun, is scarcely further advanced than his ancestors, since the fundamental relation between the sun, the green plant and himself is still shrouded in mystery. Not until the time comes when he can reproduce this ap- parently simple process of photosynthesis in his laboratory, independent of the green plant, can Man claim to be free from the vestiges of his ancestral worship of sun and fire. When that day comes the energy awaiting his disposal is enormous. Tang Pei-Sung {Green Thraldom. Allen & Unwin Ltd., London 1949) In studying photosynthesis we have to distinguish between two main problems: energetics and chemistry. Broadly speaking, the chemical part of the process begins when the energetical part is completed. After light has exerted its photochemical action, chemical reactions set in which enable the plant to synthesize organic substance. A critical and objective review of the work done on the photochemical energetic part of photosynthesis would sug- gest that the work has been completed by Warburg and Burk. By contrast, the study of the chemistry of photosynthesis is still in its infancy, in spite of the many important investigations. that have been carried out. It is self-evident that in a book of this size it is not possible to refer to all the work done in the field. In making a selection I have given preference to those investigations which may be considered to-day to be classical and those which seem to make new contributions to the understanding of Nature's most important achievement. The descriptions of basic experiments are, in most instances, illustrated by numerical examples. I gratefully acknowledge the kindness of the editors and publishers of Angewandte Chemie, Federation Proceedings, Journal of General Physi- ology, Naturwissenschaften, Science and Zeitschrift fiir Naturforschung in granting permission for the reproduction of various diagrams which appeared in their publications. My sincere thanks are due to Mr. N. K. Tregilgas for his valuable help in preparing the English manuscript. W. Bladergroen Basle- Binningen Vll 7 V X V A cal Chi Chi* (ChlC02)*orC02* G h i H N Units and Symbols photosynthetic quotient efficiency wave-length frequency quantum yield quantum requirement energy-rich phosphate bond Angstrom unit = 10~^ cm = 4.186 X 10^ erg chlorophyll excited chlorophyll photolyte of photosynthesis free energy (AG = free energy change) Planck's constant = 6.626 X 10"" erg-sec absorbed light intensity in jA quanta/min incident light intensity in jul quanta/min Avogadro's number = 6.02 X 10"^^ ADP ATP Ph A— S— H DPN+, DPNH TPN+, TPNH FAD FMN \i TPP + TA TK adenosine diphosphate adenosine triphosphate inorganic phosphate coenzyme A diphosphopyridine nucleotide and its reduced form triphosphopyridine nucleotide and its reduced form flavine adenine dinucleotide flavine mononucleotide lipoic acid thiamine diphosphate transaldolase transketolase 1 jumole = 22.4 /il 1 m1 = 0.045 Mmole IX Contents Page Preface vii Units and Symbols ix Chapter 1 . Introductory Remarks § 1 . The Over-all Reaction 3 § 2. Chlorophylls and Carotenoicls 5 § 3. The Structure of the Chloroplast 8 § 4. The Cytochrome Systems in Plants 10 Chapter 2. Some Photochemical Considerations § 5. The Einstein Law 13 § 6. Examples of Photochemical Reactions 14 § 7. The Energy Turnover of the Photochemical Reaction 16 § 8. The Nature of the Free Radicals 17 § 9. Migration of Energy 19 § 10. Free Radicals as Intermediates in Photosynthesis 21 §11. Excited States of Molecules 23 §12. The Excited State of the Chlorophyll Molecule 26 § 13. Interactions Between Chlorophyll and Other Pigments 30 § 14. Some Final Remarks 31 Chapter 3. The Energetics of Photosynthesis A. Energy Turnover § 15. Chlorella and Its Cultivation 35 §16. Principles of Manometry 38 § 17. Light Absorption and Its Measurement 40 § 18. Theory of Manometry 46 § 19. The Two-vessel Method 48 § 20. The Use of Carbonate Buffer Solutions 51 § 21. Energy Turnover 53 § 22. Application of the Quantum Theory 55 § 23. Photooxidation of Chlorophyll 57 § 24. A Chemical Actinometer 59 § 25. Photodissociation of Iron Carbonyl Compounds 61 B. The Quantum Requirement § 26. Quantum Requirement and Efiiciency 64 § 27. The Significance of Compensation 66 § 28. Some Instructive Experiments 69 § 29. The Significance of Carbon Dioxide Pressure 72 § 30. The Action of Blue-green Light 75 XI 7«44l xu PROBLEMS IN PHOTOSYNTHESIS §31. The Nature of the Photosynthesis Enzyme 79 § 32. The Significance of Vanadium 81 § 33. The Hyposulfite Method ■ • ■ ^- § 34. The Carbon Dioxide Outburst 83 § 35. The One-Quantum Reaction of Photosynthesis 84 § 36. Quantum Requirement and Photosynthetic Quotient 87 § 37. Splitting of Photosynthesis into Light and Back Reactions 88 § 38. Some Mathematical Considerations 90 § 39. Stoichiometric Oxygen Production 92 § 40. Oxygen Capacity and Quantum Requirement 95 § 41. Oxygen Capacity and Induced Respiration 97 § 42. Photosynthesis and Respiration 100 § 43. Some Final Remarks 104 Chapter 4. The Chemistry of Photosynthesis A. Introductory Notes § 44. Essentials of Glycolysis 109 § 45. Oxidative Decarboxylation of Pyruvic Acid 112 § 46. The Tricarboxylic Acid Cycle 113 § 47. The Pentose Phosphate Pathway 116 B. The Problem of Water Photolysis § 48. Introductory Remarks 121 § 49. Hill Reactions 124 § 50. Phosphorylations 127 § 51. Light Phosphorylation 129 § 52. Some Final Remarks 134 C. Intermediate Products of Photosynthesis § 53. The Carbon Cycle of Photosynthesis 139 § 54. Is Photosynthesis the Reversal of Respiration? 142 § 55. The Significance of Lipoic Acid 143 § 56. The Significance of Vitamin K 149 § 57. Some Other Investigations 152 § 58. Some Final Remarks 155 D. Carbon Dioxide § 59. The Fluoride Reaction 160 § 60. The Reaction Equations of Photosynthesis 162 § 61. Amino-acids in Chlorella 163 § 62. Lactic Acid Formation in Chlorella 165 § 63. Breakdown and Resynthesis of Glutamic Acid 166 § 64. The Necessity for Glutamic Acid in Photosynthesis 169 § 65. Dissociating Carbon Dioxide 171 § 66. The Carotenoid Oxygenase of Chlorella 173 § 67. Experiments with Quinone 176 § 68. Quinone as a Catalyzer 178 CONTENTS xiii § 69. Quinone Catalysis and Phosphorylation 181 § 70. Some Final Remarks 183 Addendum 187 Author Index 190 Subject Index 194 PROBLEMS IN PHOTOSYNTHESIS CHAPTER 1 Introductory Remarks § 1 The Over-all Reaction The Dutch physician and chemist Ingen-Housz (22) was the first to deter- mine in 1772 that the production of oxygen ("vital air") by the green plant under the influence of light is dependent upon the green parts of the plant. Priestley (30) and van Barneveld (2) had made similar observations in- dependently at about the same time. Other earlier investigators, whose writings were, however, less precise, were Bonnet and Hales. The name chlorophyll was given to the green substance of the leaves by Pelletier and Caventou (29) in 1818. De Saussure (33) in 1804 depicted the over-all reaction of Oo production as follows: light CO2 + H2O — ^ organic substance + O2 He found that the ratio of the COo consumed and the O2 produced was equal to 1. To-day this type of COo-assimilation is called photosynthesis, as there are many other types of C02-assimilation without the absorption of radiation energy. Sachs (31) showed in 1868 that chlorophyll is indispensable for photosynthesis. In 1881 Engelmann (11) succeeded in isolating the chloro- phyll-containing particles — the chloroplasts — from various plants. He was able to show that these isolated chloroplasts develop O^ when illuminated (see § 48). The chemical nature of chlorophyll remained obscure until the funda- mental work of Willstatter and Stoll (47) was published. Conant (6, 7) and especially H. Fischer (12) largely contributed to our present knowledge of the chlorophyll molecule. To-day the reaction equation of photosynthesis is written as follows: light CO2 + H2O -^ — > (CH2O) + O2 - 112000 cal The reaction is endothermic. The product (CH2O) indicates a non-defined primary substance of the carbohydrate group. Production of O2 and fixation of COo are characteristic of the photosynthetic reaction. For every molecule CO2 reduced one molecule Oo is produced. The ratio 7 = COo/Oo is called the assimilatory or photosynthetic quotient. Theoretically it equals 1. In reality this need not be the case, as CO2 can be partly reduced without 3 4 PROBLEMS IN PHOTOSYNTHESIS the formation of carbohydrates, resuking in an assimilatory quotient greater than 1. The quotient may also be smaller than 1, e.g., growing Chlorella reduces the nitrate of the culture medium, resulting in a greater O.- production : NO,- + 2H2O -> NH3 + OH- + 20,. If the quantity of CO. reduced is indicated by x^o^ and the quantity of Oo produced by .Vq,, the general formula for the assimilatory quotient is: 7 = -— (1) Uptake of gas is regarded as negative and removal of gas as positive, so that Xco. and consequendy 7 are always negative. The quantities a'co, and Xq, are expressed in /^mole or, more usually, in ix\ whereby 1 /xmole = 22.4 lA. The reversal of the photosynthetic over-all reaction is the over-all com- bustion reaction of glucose (respiration) : CeHi.Oe + 6O2 > 6C0.2 + 6H,0 + 674000 cal In respiration the ratio of the CO2 produced to the O. consumed is called the respiratory quotient. According to the combustion reaction given above, it equals 1 . In § 54 we shall see whether or not it is correct to consider photo- synthesis as the reversal of respiration, not only with respect to the over-all reactions, but also with regard to the different intermediate steps in the two processes : respiration (CH2O) + 0.2 \ ^ CO.. + H,0 + 112000 cal photosynthesis Photosynthesis is composed of two distinct types of process: a photo- chemical process and a series of dark reacdons which were originally called the Blackman reaction. The photochemical reaction or light reaction is either the photolysis of water or the photolysis of CO2. The dark reactions are far more complex than Blackman (5) presumed. When green cells of Chlorella at constant temperature and in the presence of sufficient CO2 are illuminated with various light intensities /, we obtain various rates v of photosynthesis. Figure 1 shows an assimilation curve obtained by plotting v against i (44, 45, 48). At low intensities the curve is nearly rectilinear and dv/di is constant. As / increases, dv/di decreases and finally becomes zero. Blackman found that the rate of photosynthesis at low intensities is independent of temperature; at higher intensities, however, it increases when the temperature is increased. It thus seems that various processes influence the rate of photosynthesis, depending on the light intensity. One of these processes is of a photochemical nature, dv/di being constant. As this process is independent of temperature, dv/dT = 0. The other process— the Blackman reaction— is not influenced by illumination. It is an INTRODUCTORY REMARKS ordinary, chemical, temperature-dependent reaction, whereby dvjdi = and dvldT > 0. From what has been said it follows that photosynthesis comprises two great problems: energetics and chemistry. The chemical part of photosynthesis begins where the energetical part finishes. When light has exerted its photochemical action — photolysis of water or of CO. — chemical reactions Fig. 1. An assimilation curve. Rates of photosynthesis v plotted against light in- tensities /. set in which enable the plant to build up organic substances. As far as the energetical part of photosynthesis is concerned, a critical and impartial survey of all the work done — in which the problem of the quantum require- ment occupies the most important place — shows that Warburg and Burk found the solution. The chemical part of photosynthesis, however, still remains largely obscure despite important experimental findings. § 2. Chlorophylls and Carotenoids Today seven types of chlorophyll may be distinguished : the chlorophylls a, b, c, d and e, bacteriochlorophyll and bacterioviridin.* Chlorophyll a, which is blue-green, is the most abundant and occurs in all autotrophic organisms except pigment-containing bacteria. Chlorophyll b is yellow- green and is found in higher plants and certain algae. The other chloro- phylls only occur, together with chlorophyll a, in algae. Bacteriochloro- phyll and bacterioviridin are pigments of certain bacteria. As can be seen from Figure 2, chlorophyll is a magnesium complex of a porphyrin. Porphyrins are composed of four pyrrole nuclei which together form a ring system. Chlorophylls a and b have, at the C7 atom, a carboxyl group which is esterified with the high-molecular alcohol phytol. They differ with respect to the C3 atom. This is linked with a methyl group in chlorophyll a and with an aldehyde group in chlorophyll b. In earlier work * Literature on plant pigments: 6, 7, 12, 35, 37, 47. PROBLEMS IN PHOTOSYNTHESIS H-C=CH2 R HaC-C'^ 1 C-^ ^C-^ii C-C2H5 \ I I ^4 C— N N-C // \ / \ H-C Mg C-H \ / \^ // Fig. 2. Constitution of chlorophyll. R-e-CHsin H C=n' U—C chlorophyll a; R -=- CHO in chlorophyll b. \/ I II \5 H3C-C IV C;:^ ^C III ^C-CH3 8\ / ^C^ \/ 7C-H Ce CH2 H-C^-5 C=0 ' I 9 CH2 IICOOCH3 COOC20H39 AhIh hhIh hh'hh h'hh h HjC c-cic-c=c-c=c-c=c-cic-c=c-c=c-c=c-c I II I I I I I I I I H2C C I CH3 ' CH3 ' CH3 ' CHa HsQ^ ^CHa H /C C-CH CH2 I I C CH3 H3C c H. H Fig. 3. (3-Carotene consisting of eight isoprene groups, four of which form two ionone rings. HaC-C C C C ^^^^. ^ HC OH HC; :C-CHa \ e CH CH ^. C C C-CH3 Hq, C CH ^V V" c ^c c H c H CHa Fig. 4. Structural relationship between chlorophyll and carotene. Left: chlorophyll without Mg atom, N atoms and side chains. Right: |3-carotene without ionone rings (Szent- Gyorgyi). Conant (6, 7) believed the isocyclic ring with the Cg and Cio atoms to be of importance in photosynthesis. The yellow carotenoids occur in all plants together with the chlorophylls. These substances are unsaturated hydrocarbons composed of isoprene groups. Some are aliphatic molecules with open carbon chains, some are molecules containing one or two ionone rings. Of the more than 60 carot- enoids known jS-carotene is the most abundant. It is composed of eight isoprene groups, four of which form two ionone rings (Fig. 3). It is surprising to see how the chlorophylls and the carotenoids differ in molecular structure. There seems to be no structural relationship whatever between the four pyrrole nuclei coordinated by a metal in the chlorophyll molecule and the isoprene chains of the carotenoids. However, Szent- Gyorgyi (38) pointed out that the carbon chain of j8-carotene — without the ionone rings — when written as a ring bears a striking resemblance to the INTRODUCTORY REMARKS 7 porphyrin structure of the chlorophylls when the Mg and N atoms as well as the side chains are omitted (Fig. 4). There is evidence for assuming that the carotenoids have special tasks in photosynthesis. It has even been stated that they may take an active part in the primary act of the photosynthetic process. Lynch and French (26) observed that the Hill reaction is inhibited when the carotenoids are removed from the chloroplasts by extraction. After the addition of /3-carotene to such chloroplasts the Hill reaction can again be demonstrated. Fujimori and Livingston (15) found that the carotenoids are as efficient as oxygen in quenching the triplet state of chlorophyll a. However, for the time being there is not sufficient evidence to attribute a primary role in photosynthesis to the carotenoids. The findings of Sager and Zalokar (32) support the hypothesis that the carotenoids are not essential for photosynthesis except possibly in catalytic amounts. This is in agreement with Warburg's work on the action of blue-green light and the role of the photosynthesis enzyme to be discussed in Section B of Chapter 3. Nishimura and Takamatsu (28) found that /3-carotene in various leaves — like chlorophyll — is bound to protein, an important discovery which also confirmed Warburg's views on the photosynthesis enzyme (see § 31). The group of carotenoids also includes xanthophyll and related compounds. The light absorption of chlorophyll in the visible spectrum is relatively great. The absorption curve of a solution of chlorophyll a in ethyl ether shows one maximum in the red and another in the violet. In organic solvents the pigment gives a characteristic fluorescence spectrum where — as in the absorption spectrum — the position of the bands depends upon the kind of solvent used. The addition of quinone, oxygen and some other substances inhibits fluorescence to varying degrees, whereas removal of the Mg atom has practically no influence. In vivo, chlorophyll shows very little fluorescence (see § 1 1) and colloidal solutions of chlorophyll do not show fluorescence at all (23). Warburg (46) pointed out that extracts of chlorophyll in organic solvents should not be identified with the functioning chlorophyll in the chloroplasts. In the study of photosynthesis distinction has to be made between "dead" and "living" chlorophyll (see § 59). The carotenoids absorb light in the violet, showing two or three bands, the positions of which vary according to the solvent used. TABLE 1 Conversion factors for chlorophyll concentrations Mmole mg Ml 1/umoIe = 1 mg = 1 Ml = 1.13 0.045 0.89 0.04 22.4 25.2 8 PROBLEMS IN PHOTOSYNTHESIS It is customary to express chlorophyll concentrations in yumole, jA or mg. Table 1 shows the conversion factors for these units. The molecular weight of chlorophyll a (C55H7o05N4Mg) is 892. § 3 The Structure of the Chloroplast The chloroplasts containing the chlorophylls and carotenoids vary in size and number from plant to plant.* In algae each cell may contain just one chloroplast, whereas in higher plants there may be several hundred per cell. The size of the longest axis varies from 2 to 10 /x. The chloroplasts are the site of the photochemical part of photosynthetic activity. In any case, there is no evidence to the contrary or no reason to assume that cytoplasm may also play an active part. Nevertheless, Hill and Whittingham (20), citing older work by Frenkel (13), assume that COo-fixation in photosynthesis may be associated with cytoplasm. It is difficult to accept such a hypothesis since an experimentally well-established stoichiometric relationship exists between CO2 and chlorophyll (see § 59). The chloroplasts possess a membrane about 50 to 100 A thick (39). The chlorophyll is localized in fairly small units — the grana — which are embedded in the stroma. Grana and stroma are composed of proteins and lipoids or lipoproteids. It has been observ^ed that the grana have a lamellae structure probably surrounded by a membrane. Studies by Steinmann (36) with the electron microscope show that the structure of intact chloroplasts of various algae is lamellated throughout (see §51). In such chloroplasts (e.g., Spirogyra) the lamellae are present in bundles between which thin layers of stroma occur (40, 41). From this it follows that distinction has to be made between two groups of chloroplasts. In the granulated chloroplasts, prevalent in higher plants, the lamellated structure is limited to the grana. Thomas et al. (42) propose the name chloroplast be given to this group only and that the lamel- lated chloroplasts be simply called grana. The monolayer state of chlorophyll in living chloroplasts has also been proposed by Trurnit and Colmano (43). According to Frey-Wyssling (14), the chlorophyll-containing lamellae are embedded in lipoid layers which are situated between protein-water layers. Hubert (21) and Wolken and Schwertz (49) have observed similar layer structures. Figure 5 shows the geometry of the chloroplast as described by Wolken and Schwertz. Baas Becking and Hanson (1) arrived at the con- clusion that four chlorophyll molecules form a tetrade in such a way that the isocyclic rings are turned towards each other. It follows from Figure 5 that there is space for one carotenoid molecule per three chlorophyll molecules. This represents a weight ratio chlorophyll/carotenoids of 5 to 1 . However, the spaces between the chlorophyll molecules (15 A X 15 A) allow enough room for four carotenoid molecules, the diameter of which is only 6 to 7 A. The observations, analyses and calculations of Wolken and Schwertz concern * For more detailed information on chloroplast structure see Granick (16), Miihlethaler (27), Wolken and Schwertz (49). INTRODUCTORY REMARKS model of chloroplast protein layer ^n porphyrin head iso-cyclic ring phytol tail lipid layer chlorophyll molecule schematic molecular network carotenoid chlorophyll and carotenoids molecule Fig. 5. The structure of the chloroplast (Wolken and Schwertz, J. Gen. Physiol.). the algae Euglena gracilis and Poteriochromonas stipitata. The figures obtained are not vahd for other plants. According to Hill and Whittingham (20), the molecular ratio chlorophyll/carotenoids in higher plants may be as high as 5 to 1 (weight ratio : 8 to 1 ) . Stoll et al. (37) found that chlorophyll is bound to protein. This may be the reason for the pigment's highly specific activity. The molecular weight of the chlorophyllproteid, which has been called chloroplastin,* is about 5 X 10". As the molecular weight of chlorophyll is about 900, the stoichio- metric ratio between the protein component and the chlorophyll is 5000, if one molecule of chloroplastin contains only one molecule of chlorophyll. However, chloroplasts contain about 56% protein and 7 to 8% chlorophyll; it may therefore be assumed that one molecule of chloroplastin contains nearly 700 chlorophyll molecules. Taking the chloroplastin molecule to be a globular protein, Frey-Wyssling (14) calculated its diameter. This was found to be 100 A, so that such a molecule has a surface area of about 32000 A2. As the surface area of a porphyrin head is 225 A- (see Fig. 5), only 140 chlorophyll molecules can be present on the surface of one chloroplastin particle. However, the numerous side-chains of the polypeptides may also be linked with chlorophyll molecules, so that it may well be that one chloro- plastin particle contains as many as 700 chlorophyll molecules. If only part of the protein in the chloroplast is used for the formation of chloroplastin, this number would, of course, be much higher. Though these considerations remain more or less speculative and exact figures are still lacking, there seems * Lubimenko (24) was the first to prepare aqueous solutions of chlorophyll. He assumed that in these solutions as well as in the leaves chlorophyll is iDound to protein. Chloroplastin contains chlorophylls a and b and the carotenoids in the same proportion as in the leaves. Wolken and Schwertz (50) prepared chloroplastin by extracting chloroplasts with digitonin. 10 PROBLEMS IN PHOTOSYNTHESIS to be some evidence in favour of a morphological picture of an organized chlorophyll aggregate. We shall see in Chapter 4 that some investigators consider such aggregates of great importance from a physiological point of view. In § 2 mention was made of the findings of Nishimura and Takamatsu (28) regarding the /^-carotene-proteid. This compound has a molecular weight of 5.7 X 10' and contains about 3000 /3-carotene molecules per particle. § 4. The Cytochrome Systems in Plants In chlorophyll-free plant material Bhagvat (3, 4) detected the presence of the cytochromes a, b and c, doubtless performing the same functions as in yeast and animal tissues. According to Hill and Scarisbrick (19, 34), the green cells contain another hematin compound, which they called cytochrome f, instead of cytochrome c. Davenport (8) found a cytochrome be in chloro- plasts. As yet, a cytochrome oxidase specific to cytochrome f has not been found. It is assumed that the very small quantities of cytochrome a in chloroplasts are merely due to the surrounding tissues. The etiolated yellow plastides of barley contains the cytochromes a, be and f. In Table 2 the results of these investigations are compiled. It seems that in the chloro- plasts chlorophyll replaces cytochrome a in 500 to 1000 dmes higher quantities. There is perhaps a lunctional relationship between the cytochromes and photosynthesis. It is further assumed (9, 10, 25) that cytochrome f is oxidized by light, so that the cytochrome system of the chloroplasts during illumination acts like the cytochrome system of the mitochondria during respiration. No oxidation of cytochrome f can take place in the dark because there is no specific cytochrome oxidase.* TABLE 2 Cytochrome systems Cytochromes Mitochondria (yeast) Etiolated yellow plastides (barley) Chloroplasts a a b be be c f f Hill (18) pointed out that mitochondria and chloroplasts show some resemblance with respect to the structure-bound cytochromes. The chloro- phylls could be considered to be parts of these structures. It seems as if the chloroplasts belong to the same category as the mitochondria, the a-component of the cytochrome system being replaced by the chlorophylls. For literature on cytochromes in higher plants, see Hartree (17). INTRODUCTORY REMARKS 1 1 REFERENCES 1. 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Soc, 62:147, 1772. 31. Sachs, J.: Handbuch der Experimentalphysiologie der Pflanzen. Engelmann, Leipzig, 1865. 32. Sager, R. and Zalokar, M.: Nature, 182:98, 1958. 33. Saussure, N. Th. de: Recherches chimiques sur la vegetation. Nyon, Paris, 1804. 34. Scarisbrick, R. : Thesis, Cambridge, 1940. 35. Smith, J. H. C. and Young, V. M. K.: Chlorophyll Formation and Accumula- tion in Plants in Radiation Biology III. McGraw-Hill, New York, 1956. 36. Steinmann, E.: Exper. Cell. Res., ,3:367, 1952. 37. Stole, A. and Wiedemann, E.: Fortschritte der chemischen Forschung. Vol. 2., p. 538. Springer, Berlin, 1952. 38. Szent-Gyorgyi, A.: Bioenergetics. Academic Press, New York. 1957. 12 PROBLEMS IN PHOTOSYNTHESIS 39. Thomas, J. B.: Structure and Function of the Chloroplast in Progress in Bio- physics, .5:109, 1955. 40. Thomas, J. B., Haans, A. J. M., van der Leun, A. A. J. and Koning, .1.: Bio- chim. Biophys. Acta, 25:453, 1957. 41. Thomas, .1. B., Minnaert, K. and Elbers, P. F.: Acta Bot. Xeerl., 5:315, 1956. 42. Thomas, J. B., Post, L. C. and Vertregt, N.: Biochem. Biophys. Acta, 73:20, 1954. 43. Trurnit, H. J. and Colmano, G.: Biochim. Biophys. Acta, 31 AM, 1959. 44. Warburg, O.: Naturw., 10:M1, X'^ll. 45. Warburg, O.: Biochem. Zsc/ir., 766:3S6, 1925. 46. Warburg, O. and Krippahl, G.: Zschr. Natiirf., 7 7/^:718, 1956. 47. Willstatter, R. and Stole, A.: Untersuchungen 'iiber das Chlorop/iyll- Springer, Berlin, 1913. 48. Willst.Ktter, R. and Stole, A.: Untersuchungen uber die Assimilation der Kohlen- saure. Springer, Berlin, 1918. 49. Wolken, J. J. and Schwertz, F. A.: J. Gen. Physiol., 37 AW, 1954. 50. Wolken, J. J. and Schwertz, F. A.: Nature, 177:\?)6, 1956. CHAPTER 2 Some Photochemical Considerations § 5 The Einstein Law In a photochemical reaction a molecule or an atom is brought from its normal ground state to an excited state by the absorption of radiation energy. In 1911 Einstein, encouraged by E. Warburg, extended the quantum theory to photochemistry. According to his law of photochemical equivalence, each molecule is excited (activated) by the absorption of one quantum. Thus, one mole (A^ molecules) is excited by the absorption of A^ quanta, A^ being the Avogadro number (A^ = 6.02 X 10-'). We may consider A^ quanta to to be one mole quanta, just as we may say that A' electrons are one mole elec- trons. As one mole electrons is called the electrochemical equivalent (1 faraday), it is customary to say that one mole quanta (1 einstein) is a photo- chemical equivalent. It must be borne in mind that the law of photochemi- cal equivalence is not an arbitrary hypothesis but, as its name indicates, a thermodynamically well-founded law. The energy amount of a photochemical equivalent is : E = Xhv Planck's constant // has the value 6.626 X 10"-' erg-sec and v is the frequency of the radiation expressed in sec^^ It follows from the theory of radiation : \v = c where c is the velocity of light (2.998 X lO^" cm-sec-^) and X is the wave-length in cm. Hence, we find or or ^ 6.02 X W-' X 6.626 X IQ--' X 2.998 X IQi" , , E = — erg/ mole ^ 1.197X10^ / , E = r erg/ mole A or, 1 erg being 0.239 X 10-' cal Z7 2.86 ,, , E = ^T— cal/mole 14 PROBLEMS IN PHOTOSYNTHESIS Expressing wave-lengths in A instead of in cm, we find 2.86 X 10« E = cal/mole (2) The energy values of the photochemical equivalents of various radiations in the visible spectrum are given in Table 3. TABLE 3 Photochemical equivalents of some kinds of radiation used in photosynthesis experiments Photochemical Kind of Radiation Wave-length in A Equivalent in cal/mole red 6800 42100 red 6600 43300 red 6440 44400 yellow 5780 49400 green 5460 52300 blue-green 5090 56100 blue-green 4800 59500 blue-green 4680 61000 blue 4360 65500 The translational kinetic energy of a molecule can be calculated from the equation ?>kT u = 2 The Boltzmann constant k has the value 1.381 X lO-i*^ erg/degree. At 20 °C the energy u amounts to 6 X lO^^"* erg per molecule. When illuminated with red light, one mole absorbs about 40000 cal, i.e., 280 X 10"^^ erg per molecule. This simple calculation shows that the energy of the irradiated molecule is 50 times higher than before. The new energy level corresponds to a temperature of about 1 4000 ° C. This example gives an idea of the rather important difference in energy between the ground state and the excited state of a molecule. § 6 Examples of Photochemical Reactions When n molecules are excited by the absorption of q quanta, the quotient n/q is called the quantum yield It follows from this over-all reaction that the quantum requirement l/

1. The primary photochemical reaction NH, + hv -^ NHo + H is followed by several dark reactions, e.g., 0.8 NH. + 0.8 H -^ 0.8 NH, 0.2 NH. + 0.2 H -^ 0.2 NH + 0.2 Ho 0.2 NH -* 0.1 N,. + 0.1 Ho The sum of the photochemical and the three dark reactions is 0.2 NH., + liv -^ 0.1 No + 0.3 H. so that the quantum requirement is l/> — 5. These examples show that the quantum requirement of photochemical reactions may deviate considerably from the theoretical value owing to secondary dark reactions. Therefore, when it is found that a process occurring with absorption of radiation energy has a quantum requirement which is not equal to 1, it must be assumed that dark reactions are involved. The complex of reactions must then be disentangled until the correct primary photochemical reaction with l/V = 1 is detected. In our example of the photochemical decomposition of NH3 the quantum requirement of the over-all reaction would have other values if the experimental condidons were 16 PROBLEMS IN PHOTOSYNTHESIS changed. We shall see later that in photosynthesis also the value of the quantum requirement of the over-all reaction depends upon the experimental conditions. § 7 The Energy Turnover of the Photochemical Reaction Let us consider the endothermic reaction E cal _ A - > B + C - U cal which proceeds to the right when one mole of the substance A absorbs the radiation energy E. The reaction thus needs E cal/mole but, according to thermochemical measurements, only U cal/mole should be necessary because the reaction would proceed to the left evolving this amount of energy. The ratio -q oi U and E is called the efficiency of the photochemical reaction As an example we may take the photochemical dissociation of hydro- bromic acid 2 HBr ''" , H. + Br. - 24000 cal As in the photochemical dissociation of hydriodic acid, the quantum yield has the value 2. E. Warburg (58), who was the first to measure photochemical yields, irradiated HBr with ultraviolet light of wave-length 2090 A. Accord- ing to equation 2, the photochemical equivalent is 137000 cal so that 2 mole HBr use this amount of radiation energy, although only 24000 cal per 2 mole HBr are necessary for the dissociation of HBr. Thus, we find for the effi- ciency '^4000 In the same way, E. Warburg found an efficiency of only 2.1% for the photo- chemical dissociation of hydriodic acid. The ozonisation reaction of oxygen has the highest efficiency. The pri- mary photochemical reaction is the formation of an excited molecule Oo*. This is followed by two secondary dark reactions O., + hv -^ O2* O2* + Oo -> O.; -t- O O + O2 -^ 0.3 The over-all reaction is represented by 30o + hv -^ 2O3 The quantum yield has the value 3. E. Warburg used light of wave-length 2070 A, corresponding to the quantum energy of 137000 cal/mole. As the SOME PHOTOCHEMICAL CON8IDER.ATIONS 17 endothermic formation of ozone needs 68000 cal per 3 mole O2, the efficiency is 68000 V = 137000 = 50% § 8 The Nature of the Free Radicals Many investigators believe that free radicals have important functions in photosynthesis. Free radicals are atoms or atom groups possessing unpaired electrons in their electronic structures. Using G. N. Lewis' method of writ- ing chemical formulae, each valency electron of an element is indicated by a dot so that the usual bars between the symbols are replaced by two dots representing an electron pair. The formula for ammonia shows that the five valency electrons of the N atom are combined with the three valency electrons of the three H atoms resulting in four electron pairs :N: + 3 H- -* H:N:H H In this formula one of the electron pairs does not serve as a link between two atoms. Both electrons of this so-called lone electron pair belong to the N atom. The formula for the ammonium ion shows the same number of elec- tron pairs H H:N + :H H As only four and not hve electrons belong to the N atom, there is an excess of one positive elementary charge characterized by a plus sign. Coming back to the definition of the free radical, we see that the H atom and the halogen atoms with seven valency electrons must be considered to be free radicals H« :C1. li- the unpaired electron being represented by a bold dot. A free radical with one unpaired electron is called monoradical, whereas a free radical with two unpaired electrons is called a biradical. Thus, the H atom and the halogen atoms are free monoradicals. They are very active substances formed when halogen acids are photochemically dissociated. The primary photochemical reaction described in § 6 can be written as follows lip/it H:I: > H. + •!: Other examples of free monoradicals are HO • and the still somewhat prob- lematic H02* 18 PROBLEMS IN PHOTOSYNTHESIS H:6« and H:6:0« The excited state of a molecule after irradiation corresponds to the state of a free radical. The irradiation of ethylene involves the formation of a free biradical H H H H C::C J^ .C:C. H H H H A free biradical is always produced when a double bond between two C atoms is changed into a single bond C::C > •C:C« In § 9 we shall see that in the oxidation mechanism of nicotinic amide — the active group of DPN+ and TPN+ — a monoradical is formed when the double bond C=N+ is changed into the single bond C — N C::N+ > .C:N: The tendency to form electron pairs must be ascribed to the magnetic properties of the electrons. The electron possesses a spin denoted by +V2 or — i/o- A spinning charged body must have a magnetic momentum and the magnetic momentum of the electron is called the magneton which equals eh/Awmc = 9.21 X 10-'" gauss-cm^ (e = elementary charge, m = electron mass). According to Pauli's principle, two electrons in the same orbit of an atom must have anti-parallel spin so that their magnetic momenta neutralize each other. In the case of an electron pair there can thus be no resulting spin as + V'2 — V2 = and there cannot be an excess of magnetons as + 1 - 1 = 0. Conditions are different in the case of free radicals. The unpaired electron of the free monoradical has a magnetic momentum which is not compensated by the anti-parallel spin of a second electron. The resulting spin is then V2 and the magneton excess is 1 . In the free biradical we find two unpaired electrons having parallel spin so that the resulting spin is 1/2 + \ 2 = 1 and the magneton excess has the value 2. Owing to their magneton excess, the free radicals behave as paramagnetic substances. When brought into a strong magnetic field, the axis of the magnetic momenta in excess will be orientated in the line joining the poles. As in the electron pairs the magnetic momenta neutralize each other, chemical compounds are generally diamagnetic. When brought into a magnetic field, diamagnetic substances obtain magnetic momenta the axis of which will be orientated at right angles to the line joining the poles. In contrast to other gases, O2 behaves like a paramagnetic substance. It is therefore assumed that the molecule contains unpaired electrons SOME PHOTOCHEMICAL CONSIDERATIONS 19 • :0:0: or :0::0 • • • ■ • It may be that the high reactivity of Oo in biological systems is due to its free radical nature. § 9 Migration of Energy According to Michaelis (36,37), the reversible oxidation-reduction ol organic compounds proceeds in two successive, univalent steps. Instead ol the simultaneous removal or uptake of two electrons, as previously assumed, only one electron at a time is removed or taken up so that free radicals are formed as intermediates (65). This is the principle of compulsory, univalent oxidation-reduction. From many examples we may select the hydrogenation of DPN+ or TPN+ which is nothing but the hydrogenation of nicotinic amide H H H H -CONH.. H Jh . ' H — CONH.. + H Ho R R The relevant part of the nicotinic amide molecule is C— N+=CH R The change in the electronic formula shows that an intermediate free mono- radical is formed C:N + ::C:H ^' , C:N:C:H ^' , C:N:C:H R R * R H The positive charge of the N atom is neutralized by the uptake of the first electron. The double bond N=C disappears and the C atom gets an un- paired electron. After the uptake of the second electron the free monoradical is changed into the reduced nicotinic amide (DPNH or TPNH). Transport of single electrons occurs also in semiconductors. In biological systems reacting molecules are mostly bound to proteins and it is supposed that electrons must migrate through the proteins. Such a transfer of electrons shows some analogy to semiconductivity. Semiconductors have electrical properties intermediate between those of metals which conduct electricity very well and those of insulators which conduct electricity very poorly or not at all. C^rystals of germanium, silicon, copper oxide, silver sulfide, lead sulfide etc. are important semiconductors used as transistors in modern radiotechnique.* In the n type semiconductor * For further details on semiconductivity the reader is referred to the excellent paper by Shockley (47). 20 PROBLEMS IN PHOTOSYNTHESIS free electrons (excess electrons) are liberated and migrate through the crystal. Conductivity is then produced by these negative carriers of current. In thep type semiconductor there is an electron deficit. The deficit electrons can be considered to be positively charged "holes" which act as current carriers. In the ?i type semiconductor a current of electrons proceeds from a negative to a positive pole, whereas in the p type semiconductor a current of "holes" proceeds from a positive to a negative pole. Figure 6 shows the principle of a type of transistor. Three zones A, B, and C can be distinguished. A and C are n-conductive and B is/?-conductive. The conducting electrons produced in B migrate through A and the resulting "holes" in B are occupied by electrons coming from C. Fig. 6. The principle of a transistor. Szent-Gyorgyi (50, 51) pointed out the possibility that proteins may have an electronic structure analogous to that of semiconductors. The theoretical considerations of Evans and Gergely (19) supported this hypothesis. Eley et al. (18) found semiconductivity in plasma albumin, fibrinogen and edestin. In their experiments, however, the proteins used underwent such drastic treatment that the data obtained cannot be applied to native proteins. According to Vladimirov and Konev (57), there is insufficient evidence of semiconductivity in proteins. The statement that energy can migrate through a protein molecule is not new. It is well known that light energy can be absorbed in one part of a molecule and provoke chemical change in another part of the same molecule. This has been proved by Warburg and Negelein (59, 61 ) for the CO-compound of the iron oxygenase (cytochrome oxidase). With the quantum require- ment equal to 1, CO is photochemically dissociated from the iron of the heme part, though the light energy is absorbed by tryptophan and tyrosine in the distant protein part of the enzyme. Biicher and Kaspers (10) found a similar behavior in the photodissociation of CO-myoglobin (see also § 25). Besides the semiconductor hypothesis, energy transfer by resonance seems very probable. According to this hypothesis, the energy of an excited oscil- lator is electrodynamically transferred to neighboring oscillators. Such oscillators showing resonance coupling can be atom groups in the same protein molecule or two molecules of different substances oscillating at the same frequency. The energy donors must be fluorescent at frequencies which the energy acceptor is able to absorb, or in other words, the fluorescence spectrum of the energy donor must overlap the absorption spectrum of the energy acceptor. Furthermore, donor and acceptor should be sufficiently SOME PHOTOCHEMICAL CONSIDERATIONS 21 close together (not more than 1000 A apart). These conditions are fulfilled in protein molecules. From a theoretical point of view, Karreman and Steele (29) investigated whether such resonance energy transfer could be possible in the respiration chain. They found that the fluorescence spectra wholly or partly overlap the absorption spectra of the next steps in the chain DPNH — ^ oxidized FAD — > oxidized cytochrome c so that energy transfer by resonance is highly probable. At the beginning of this paragraph it was pointed out that, as a result of theoretical considerations, free radicals are formed as intermediates in biological processes concomitant with the transfer of single electrons. How- ever, until the participation of unpaired electrons was experimentally estab- lished, the compulsory, univalent oxidation-reduction was still unproved. It has been stated in § 8 that an unpaired electron must be influenced by an external magnetic field. Zavoisky (69) discovered that at a definite value H of an external magnetic field a resonance phenomenon — the so-called electron spin resonance — can be observed. This type of resonance is due to the interaction between the external magnetic field and the magnetic momentum of the spinning, unpaired electron. With the aid of special spectrometers, the absorption of short wave radiation energy under the influence of an appropriate external magnetic field is recorded. At a given frequency v the value of H is changed and at a certain value of H the curve shows a deflection from the base line indicating the electron spin resonance and thus the presence of an unpaired electron.* This technique was first applied by Commoner el al. (14, 15, 16) to biological systems such as enzymatic processes, chloroplasts and living cells. The oxidation-reduction reactions of the type substrate-dehydrogenase-DPN are among the simplest biochemical processes in which unpaired electrons occur. Commoner et al. studied the alcohol dehydrogenase system . 1 , T^T^TVTj alcohol dehydropenase ,, , , , ^^^^^^ , ^^ , Alcohol + DPN+ i t > acetaldehyde + DPNH + H + They found that none of the participants in the reaction show electron spin resonance at any value of H. However, when the mixture of alcohol, alcohol dehydrogenase and DPN+ is examined, the curve shows deflection at a definite value of H, indicating electron spin resonance and thus the occurrence of unpaired electrons. The same results were obtained with mixtures of acetaldehyde, alcohol dehydrogenase and DPNH. These and further experiments on other enzyme systems have established that in oxida- tion-reduction reactions unpaired electrons, i.e., free radicals, occur and thus prove the correctness of Michaelis' hypothesis. § 10 Free Radicals as Intermediates in Photosynthesis The work done by Uri (55) seems to confirm that free radicals occur as intermediates in photosynthesis. His experiments are based on the fact * For literature on electron spin resonance, see Wertz (66) . 22 PROBLEMS IN PHOTOSYNTHESIS that free radicals give rise to polymerization of vinyl compounds. The reaction of ferrous ions with H2O2 (6, 7, 20) gives us an interesting example Fe2+ + H,,02 -^ Fe'+ + OH^ + HO- The HO radicals formed react further cither with ferrous ions Fe-'+ + HO. -^ Fe^+ + OH- (a) or with HoOo H2O2 + HO. -^ HoO + HO2. (b) In the presence of sufficient vinyl compounds of the general formula CHo^ CHX the reactions a and h will be suppressed as the HO radical will be used for the polymerization reaction HO. + CH2=CHX -^ HO— CH2— CHX— and HO— CH,— CHX— + CH2=CHX -^ HO— CH2— CHX— CH.— CHX— etc. The property of free radicals of inducing polymerization thus provides an indirect method of determining these substances. Uri found that the addi- tion of methylmethacrylate* reduces the amount of O2 developed in photo- synthesis {Chlorella) to about one-third. The polymer produced from methyl- methacrylate had a molecular weight of about 10^. As the niolecular weight of methylmethacrylate is 100, the polymerization degree was thus 1000. No polymerization was observed in the dark under the same experimental conditions or upon illumination of killed algae. In the absence of CO2 no polymerization could be obtained, probably because photosynthesis proceeds too slowly. The addition of methylmethacrylate had no influence upon the O2 production in the Hill reaction (see § 49) so that apparently no polymers are formed. This negative result was independent of the hydrogen acceptors used and it may thus be concluded that no free radicals are necessary in the Hill reaction. Uri considers this highly improbable and assumes that the free radicals produced during the Hill reaction may not be able to form long chains of polymers but may act as terminal groups of such chains. This behavior has been found for the free radical SON* and for the Br atom (20, 54). There is, however, insufficient evidence to assume this. In § 9 we discussed the work of Commoner et al. (14, 15) dealing with electron spin resonance in enzyme systems. These authors succeeded in observing electron spin resonance in chloroplasts. Calvin and Sogo (12) confirmed these findings so that the theoretical assumption that chlorophyll is brought into its triplet state upon illumination seems to be experimentally proven in vivo.** However, it must be pointed out that electron spin reso- * CH2=C— COOCH3. CH3 ** Fujimori and Livingston (25) succeeded in determining in vitro the half-life of the triplet state of chlorophyll in organic solvents. SOME PHOTOCHEMICAL CONSIDERATIONS 23 nance in chloroplasts is also observed in the dark, though to a lesser degree. Commoner's studies on isolated chloroplasts and also on living Chlorella support the view that in photosynthesis there must be a univalent electron transport and that the chloroplast acts as a semiconductor (5) (see § 55). The question arises whether the electron spin resonance observed in chloroplasts is due to enzymatic processes. However, Calvin and Sogo found that cooling" to —140° C did not influence the phenomenon. § 11 Excited States of Molecules* When a system absorbs light, the equivalent of the energy absorbed is not lost but has to reappear either in another form or as radiation energy. It is changed into thermal energy when the temperature of the system is increased. It is changed into chemical energy when an endothermic chemical reaction is involved, as is the case in photosynthesis. The radiation energy absorbed, however, may also be emitted again as radiation energy; this is called photo- luminescence. Each photoluminescence exhibits some kind of inertia, as some time passes between the removal of the incident light stimulus and the end of the luminescent phenomenon. If this time span is shorter than 10"^ sec, we have fluorescence. If it is longer than 10~^ sec, we speak of phos- phorescence.** Absorption of radiation energy means absorption of a quantum by an electron bringing the latter on to a higher energy level, i.e., bringing it into an excited state. When this process deals with electrons of the outer shells of the atoms, vibration of the nuclei occurs; this means nothing but a change of radiation energy into thermal energy. In molecules with conjugated double bonds*** we have so-called tt electrons which, not being bonded to any definite nuclei, belong to the whole molecule. When radiation energy is absorbed by tt electrons, emission of radiation energy of lower frequency takes place. If the molecule contains weak bonds with a dissociation energy lower than the radiation energy absorbed (excitation energy), the weak bonds may be changed, the absorbed energy being used up. This is a chemical change and no radiation will be emitted. We may illustrate this by the benzene molecule, the bonds of which require a dissociation energy of at least 126000 cal/mole. According to equation 2, the excitation energy of light of the wave-length 2700 A amounts to 106000 cal/mole. The dissocia- tion energy is thus higher than the excitation energy, and fluorescence, but * For literature on the excited states of molecules, see Laidler (34), Pringsheim (39), Reid (40). ** This differentiation according to Forster (21 ), though arbitrary, is less complicated than the definitions given by Perrin (38) and by Lewis and Kasha (30, 35). See also page 25. *** Conjugated double bonds are written as — CH=CH— CH=CH— CH=etc Organic molecules showing photoluminescence may belong to different categories but they have such conjugated double bonds in common. 24 PROBLEMS IN PHOTOSYNTHESIS no chemical reaction, will occur. In the pyridine molecule, however, the aromatic C — N bonds arc much weaker than the C — C bonds of the benzene molecule so that photochemical dissociation, but no fluorescence, has been observed (21). Generally speaking", we have fluorescence when a molecule absorbs radia- tion energy without using it. As Szent-Gyorgyi (52) pointed out, fluorescence may not have any biological significance. The biological role of molecules is not to emit but to transinit radiation energy. As a matter of fact, biologi- cally active molecules should exhibit very little or no fluorescence as some of the energy absorbed may be lost. Chlorophyll, which we must consider to be the most important energy transmitter, shows very little fluorescence in vivo while its fluorescence in organic solvents is particularly impressive (see § 2). •T, ■T, Fig. 7. Schematic representation of photo- chemical excitation. G: ground state. Si and Si: singlet states. 7\ and 7%.' triplet states. A: absorbed radiation. E: energy loss. F: y/f fluorescence. The short arrows indicate conver- sions with change in spin. Upon irradiation with ultraviolet, the red dye Rhodamin B shows orange fluorescence. Figure 7 depicts schematically what happens. The normal ground state of the molecule is indicated by the line G. The arrow GS^i represents the excitation the molecule undergoes in absorbing the radiation energy. By a change of energy — without radiation — the molecule is brought from the state ■5*2 into the state ^'i. Finally the arrow S^G shows the emission of radiation, which is called fluorescence. This type of excitation whereby an electron arrives at a higher energy level and afterwards returns to the ground state is termed singlet excitation. As has been pointed out in § 8, only two electrons with antiparallel spin can be present on the same energy level. In the case of singlet excitation one of the two electrons jumps on to a higher level and afterwards returns to its companion. This process is quite correct from a quantum mechanical point of view (Fig. 8). The possibility exists, however, that the excited electron, on its way to the higher level, obtains opposite spin so that its magnetic momentum gets the same sign as that of the electron left behind. In this case, the excited electron can no longer return to the ground state, as two electrons with parallel spins cannot coexist on the same level. The excited electron must therefore remain on the higher level indicated in Figure 7 by the lines Ti or T^. The electron is then trapped in the high energy level. This is called the triplet state which, due to some loss of energy, lies somewhat lower than the cor- responding singlet state. The trapped electron emitting radiation can SOME PHOTOCHEMICAL CONSIDERATIONS 25 G © (Z) (Z) (Z) (Z) (D (D ® Fig. 8. Fluorescence, a: electron pair in the ground state, b and c: one electron in the singlet state .S".) or Si. d: both electrons in the ground state again. A: absorbed radia- tion. E: energy loss. F: fluorescence. T T, Q) Ph 0(7)0 a bed Fig. 9. Phosphorescence, a: electron pair in the ground state, b and c: one electron with opposite spin in the triplet state To or Ti. d: both electrons in the ground state again (renewed change of spin). A: absorbed radiation. E: energy loss. Ph: phosphores- cence. s © D T Fig. 10. Multiplicity and free radicals. S: singlet state, resulting spin = 0, mag- neton excess = 0. D: doublet state, re- sulting spin = '/o. magneton excess = 1. T: triplet state, resulting spin = 1, mag- neton excess = 2. The two unpaired elec- trons are characteristic of a free biradical. The one unpaired electron is characteristic of a free monoradical. The horizontal lines on the left represent energy levels. return via the singlet state to the ground state only by changing its spin again. Such a triplet-singlet change, corresponding to a photoluminescence of longer duration, is called phosphorescence (Fig. 9) in accordance with the differentiation proposed by Lewis and Kasha (see footnote page 23). The various states including the doublet state are represented in Figure 10. The scheme shows the presence of a monoradical in the doublet state and of a biradical in the triplet state.* Such molecules are paramagnetic owing to the excess of magnetic momenta (see § 8). * Reid (40) points out that there is some tendency to identify the triplet state with the biradical. This may often be justified but a biradical is not necessarily a triplet and triplet states may not be biradicals. 26 PROBLEMS IN PHOTOSYNTHESIS Excited molecules have a longer life in the triplet state than in the singlet state, as the electron is quasi-trapped. The probability of the singlet- triplet change can be considerably increased by the addition of oxygen or iodides. Kasha (31) found that the singlet spectrum of dichloronaphthalene can be changed into the triplet spectrum by the addition of ethyl iodide. Decrease of temperature to far below ° C destroys the fluorescence of dyes and of some biological substances. Phosphorescence occurs instead of fluorescence because the molecules are brought into the triplet state. Chi ^ — >Chl* fluorescence transition into a long lived ^sensitization triplet state Fig. 11. The photochemical behavior of excited chlorophyll. § 12 The Excited State of the Chlorophyll Molecule There are two different ways in which chlorophyll can use light energy: fluorescence and the photochemical process of photosynthesis. The excited chlorophyll molecule may, depending on the conditions prevailing, either show fluorescence or change into long-lived active forms (triplet state), in which absorbed light energy can be stored for photochemical action. The scheme in Figure 1 1 shows both possibilities; the excited chlorophyll molecule is represented by Chi*. The greater the fluorescence, the smaller the photo- synthetic activity (32). Wassink (62) tried to establish a correlation between fluorescence and photosynthesis. Franck (22, 23, 24, 46) considered fluo- rescence can be ignored so that — as has also been pointed out by Uri (54) — there is no evidence to support Wassink's views. Quite a number of hypotheses on the change of Chi* into long-lived forms have been established. The older assumption that Chi* may give off H atoms Chi* -^ H + Chi,, Chl^^ representing an oxidized or a dehydrogenated chlorophyll molecule, is not accepted today (8). It is indeed difficult to imagine how a C — H bond having a bond energy of 87000 cal/mole can be split in the excited molecule by the uptake of only 40000 cal/mole radiation energy (red part of the spectrum). This might be possible in the blue or the violet (photo- chemical equivalent: 70000 cal/mole) together with thermal energy, but SOME PHOTOCHEMICAL CONSIDERATIONS 27 evidence is lacking. Weigi and Livingston (63) established that all the hydrogen-carbon bonds of the chlorophyll molecule are sufficiently stable to prevent hydrogen exchange in neutral organic solvents in the dark. How- ever, when cells of Chlorella or Scenedesmus are illuminated in water containing 3H2O, it seems that tritium is incorporated at the Cio atom of chlorophyll a. These experiments were carried out by Vishniac and Rose (56), who suggested that this light-dependent labeling of chlorophyll by tritium originadng from water may be the first step in the hydrogen transfer reaction chain in photo- synthesis; the adherents of the water photolysis hypothesis assume this takes place. There is insufficient evidence to believe that this labeling of chloro- phyll is related to photosynthesis and it must be very difficult to ascertain that tritium is really incorporated into the chlorophyll molecule and not into impurities. It is interesting to note that a distinct transfer of tritium from chlorophyll a to TPN+ should occur in the light. However, why should tritium not appear in TPNH, according to the reaction equations discussed in § 50 and § 52 ? Another hypothesis is based upon tautomerism Chi* -^ Chi, Chi, being a tautomeric form. According to a further hypothesis, Chi* reacts with the solvent, i.e. Chi* -\- A ^ Chl„, + A,,, or Chi* + A„, -^ Chi,,, + A Instead of the solvent A, a normal chlorophyll molecule may react with Chi* and give rise to a kind of photodismutation Chi* + Chi -^ Chl„, + Chi,,, It may also be that tautomerism and photodismutation are linked together Chi* -^ Chi, Chi, + Chi ^ Chi,, + Chi,,, Chl„, + Chl„, -^ 2 Chi Many experiments have been made to prove that the excited chlorophyll molecules are changed into long-lived forms to provide the starting point of photosynthesis. Though some of the results obtained are of interest, there is insufficient evidence to support this point of view. On the one hand, a direct, photochemical action of chlorophyll with regeneration of the pigment is assumed. On the other hand, the function of chlorophyll is thought to consist in a transfer of excitation energy to appropriate acceptors, making these ready to react chemically. Strehler and Arnold (49) found that after withdrawal of illumination green plants emit for a few minutes — and even for as long as 200 minutes (48) — a 28 PROBLEMS IN PHOTOSYNTHESIS delayed light of very low intensity. According to Arnold and Davidson (2), the emission spectrum of this chemiluminescence — observed with Chlorella — is identical with the fluorescence spectrum of chlorophyll a. The discoverers of this phenomenon attribute it to an enzymatically catalyzed recombination of substances produced early in photosynthesis. The production of this delayed light would thus be partly a reversal of photosynthesis, in which at least one — still unknown — reaction would be reversible. If, however, the spectrum of the delayed light is identical with the fluorescence spectrum, it must be concluded that the chemiluminescence is due to the excited chloro- phyll molecule. Though Brugger and Franck (9) built up a series of hy- potheses, based partly upon the delayed light phenomenon, to try to explain the photochemical part of photosynthesis, the question still arises whether the phenomenon really has anything to do with true photosynthesis. Chlorophyll is ir} vivo a stable substance with respect to air and to light. However, after strong illumination or when photosynthesis is inhibited by poisons, the leaves become yellow or colourless due to photochemical decom- position of the pigment. This bleaching process is accelerated by excess of O2. According to Gaffron (26), the excited chlorophyll gives its excitation energy to an acceptor A Chi* + A -> Chi + .4* and the excited acceptor produced reacts with Oo A* + Go -^ AOi Fig. 12. Kautsky's photooxidation cycle. Kautsky (33) proposes a cyclic process. Substance A undergoes a cyclic transformation represented in Figure 12. The excited chlorophyll activates A to A"^. A metabolite X reacts with A* to produce a reduced form A' and an oxidized form X'. The change of A via A* to A' means nothing but the photooxidation of X. The substance A is regenerated in the dark by oxidation of A' with Oo. This back oxidation has some similarity with the induced respiration discovered by Burk and Warburg (see § 35). Another hypothesis based upon the formation of the HOo radical, has been put forward by Weiss (64). SOME PHOTOCHEMICAL CONSIDERATIONS 29 Chi* + Oo ^ Chl,„ + HO... Chl„, + .1 -^ Chi + A„, H02« -^ ^' 2 HoO + '/iOo A + 1/4O2 -^ A„, + V/'iHoO According to Schenck (41), a photosensitizing reaction produces a biradical Chlj^ of chlorophyll Chi + /if -^ Chlu The biradical Chlj^ has a pronounced affinity to Oo Chlu + 0,> -> ChlH---02 This compound reacts with an acceptor A ChlR---02 + A ^ Chi + .4O2 It would lead too far to discuss the arguments put forward by Schenk to support his hypothesis. They are based upon interesting investigations on the photosensitized autoxidation of ce-terpinene, found in chenopodium oil, and which, in the presence of chlorophyll, results in the production by photo- synthesis of ascaridol. This is an example of a particular photochemical action of chlorophyll characteristic of the chenopodium plants (43, 44, 45). The question arises whether Schenck's findings can be linked with normal photosynthesis. He assumes that Oo is involved in the photolysis of water, a hydroperoxide base being formed Chli;---02 + H2O -^ HO— ChlH---OOH The hydroperoxide base is dehydrogenated by a hydrogen-transferring enzyme or a cytochrome to produce the free radical HO — Chip O — 0». This compound splits off Oo, forming a new free radical HO — Chlj^*, which is then split into normal chlorophyll and an HO radical HO— Chla* -^ Chi + HO« Finally, four HO radicals yield water and Oo, the intermediately formed H2O2 being decomposed by catalase 4 HO. -^ 2 H,02 ^ 2 H2O + O2 For the sake of clarity the reactions involved are depicted again below 4 Chi + 4 //f/ ^ 4 Chin 4 Chlu + 4 O2 ^ 4 ChlR O2 4 CWr O2 + 4 H2O -^ 4 HO— Chhi OOH 4 HO— Chlu OOH -^ 4 HO— Chin O— O. + 4 H 4 HO— ChlR O— O. -^ 4 HO— Chhc + 4 O2 4 HO— Chlu* -^ 4 Chi + 4 HO- 4 HO« -> 2 H2O2 2 H2O2 -^ 2 H2O + O2 30 PROBLEMS IN PHOTOSYNTHESIS The sum of these eight reactions is 2 H2O + ^hv -. O2 + 4 H Due to the action of four quanta, one molecule O2 and four enzyme-bound reduction equivalents are produced from two molecules of water. According to the hypothesis, four quanta are required for the production of one molecule O2. The result shows agreement with Warburg's findings. There is, of course, no relationship whatever between the concept of Schenck and that of Warburg, the former being pure hypothesis and the latter based upon experimental work of the highest precision. Just as Warburg compares photosynthesis and vision with respect to the carotenoids (§31), Schenck (42) considers the excitation of the optical nerve as a process similar to the photochemical part of photosynthesis. He assumes that the reaction of a carotenoid biradical Car^ with O2, i.e., the formation of the compound Car^ O., must be the first dark reaction in the process of vision. In spite of the many hypotheses, there is no definite evidence that the chlorophyll molecule undergoes chemical changes during photosynthesis. Using suspensions of isolated spinach chloroplasts, Witt et al. (68) found that red light flashes of 10"^ sec induce changes in the absorption bands of chloro- phyll; this may be considered to be the result of chemical changes in the molecule. § 13 Interactions Between Chlorophyll and Other Pigments Schenck (42) considers chlorophyll has no specific action in photosynthesis, the whole process being based upon the combination of chlorophyll with specific enzyme systems. Accordingly, the carotenoids should also be able to combine with such specific enzyme systems and produce similar photolytic arrangements. The carotenoids forming free radicals would thus replace chlorophyll if necessary. Stanier el al. (27) assume that the important role of the carotenoids consists in protecting the cells against photodynamic damage caused by photooxidation of chlorophyll. Anderson and Fuller (1) also found carotenoids in chromatophores of the purple sulfur bacteria to be essential for the protection of chlorophyll against photooxidation. Green carotenoid-less plants may indeed produce enough O2 to cause photodynamic death. Claes (13) described carotenoid-deficient mutants of Chlorella which grew only in the dark and were killed by illumination. If carotenoids, in competition with chlorophyll, interfere in the transfer and change of energy, it can be imagined that, in the absence of carotenoids, the formation of peroxide and the consecutive production of O2 will be much more active and may even be too active. Calvin (11) supports this opinion. Kandler and Schotz (28) observed photooxidation of chlorophyll also in higher plants {Oenothera) containing great amounts of carotenoids. It is ol importance to note that in Chlorella blue light is much more active than red SOME PHOTOCHEMICAL CONSIDERATIONS 31 light. This seems incomprehensible insofar as the light absorbed by chloro- phyll should have damaging properties. In certain Chlorella mutants the chlorophyll content — by comparison with the content of carotenoids — is so low that only a small fraction of the blue light could have been absorbed by the chlorophyll. These mutants are, nevertheless, extremely sensitive to illumination. It must therefore be concluded that in this case the photo- oxidation is catalyzed by a substance having an absorption maximum in blue. Thus, the carotenoids and not chlorophyll must be photodynamically active. Kandler assumes that an equilibrium normally exists between both pigments guaranteeing a physiologically optimal use of absorbed radiation energy. Under abnormal conditions the photodynamic action depends either upon chlorophyll or upon the carotenoids. In extreme cases — when one of the pigments is lacking — the remaining" pigment undergoes photo- oxidation. This has been shown by Claes on carotenoid-less mutants of Chlorella and by Stanier on mutants of purple bacteria. From these con- siderations it follows that photosensitization is not necessarily connected with lack of carotenoids. The strong action of blue light shows that carotenoids can also be photooxidized. It seems that energy absorption by various pigments may be of importance in photosynthesis. They all transfer the energy absorbed to chlorophyll a which is the pigment responsible for photosynthesis (4). The other pigments serve to render light of various wave-lengths useful for chlorophyll a (67). Teale (53) succeeded in showing in artificial pigment systems that transfer of energy may equally take place between the molecules of chlorophyll a themselves. Duysens (17), as well as Arnold and Meek (3), reached the same conclusion. Warburg et al. (60) discovered in Chlorella a labile oxygenase system which in the dark transfers molecular Oo to the carotenoids. This reaction is inhibited upon illumination and it disappears after short heating at 65 ° C (see § 66) . It is not inhibited by 1 -•'' A^ HCN and by 1 "^ A^ phenanthroline but it is highly dependent upon the O2 pressures. During this enzymatic oxidation of the carotenoids no oxidation of chlorophyll can be observed. § 14 Some Final Remarks In this chapter the principles of photochemistry and some modern views on electron transport and excited molecules have been discussed. It is self-evident that the scope of this book permits only a few observations and hypotheses dealing with the excitation of chlorophyll molecules to be treated. It may well be that some work of interest has been passed over in silence. The question arises whether the many hypotheses discussed — often of rather a speculative nature notwithstanding some experimental evidence — have seriously contributed to the understanding of the photosynthetical process. The main problems of photosynthesis have always been and still are energetics and chemistry. The great amount of work done on the excita- 32 PROBLEMS IN PHOTOSYNTHESIS tion of chlorophyll does not seem to give any noteworthy contribution to the elucidation of either the energetics or the chemistry of photosynthesis. Broadly speaking, this type of work — however interesting it may be from other points of view — seems to end in a blind alley when we have photosynthesis in mind. Research done with chlorophyll in vitro, in particular, leads to conclusions which may by no means be of any importance to the process of photosyn- thesis in the living plant. In the following chapter we shall deal with quite different trains of thought and research. In contrast to what has been discussed so far, we shall meet with classical experiments based upon classical chemistry and physics worked out with an astonishingly high degree of precision. No hypotheses will be encountered, but only facts forming the basis of a real theory. By means of this theory, the energetical aspects of photosynthesis have been satisfactorily explained and clarified. REFERENCES 1. Anderson, I. C. and Fuller, R. C: Fed. Proc, 77:182, 1958. 2. Arnold, W. and Davidson, J. B.: J. Gen. Physiol, 37:611, 1954. 3. Arnold, W. and Meek, E. S.: Arch. Biochem. Biophys., 60:82, 1956. 4. Arnold, W. and Oppenheimer, J. R.: J. Gen. Physiol., 33:423, 1950. 5. Arnold, W. and Sherwood, H. K. : Proc. Nat. Acad. Sci., 43:105, 1957. 6. Baxendale, J. H., Evans, M. G. and Kilham, J. K. : Trans. Faraday Soc, 42:668, 1946. 7. Baxendale, J. H., Evans, M. G. and P.\rk, G. S.: Trans. Faraday Soc, 42:155, 1946. 8. Brown, A. H. and Frenkel, A. W. : Ann. Rev. Biochem., 22:423, 1953. 9. Brugger, J. E. and Franck, J.: Arch. Bioclxem. Biophys., 75:465, 1958. 10. BiJCHER, Th. and Raspers, J.: Biochim. Biophys. Acta, 7:21, 1947. 11. Calvin, M.: Nature, 776:1215, 1955. 12. Calvin, M. and Sogo, P. B. : Science, 725:499, 1957. 13. Claes, H.: Zschr. Naturf., 9b:A6\, 1954. 14. Commoner, B., Heise, J. J., Lippincott, B. B., Norberg, R. E., Passonneau, J. V. and Townsend, J.: Science, 126:bl, 1957. 15. Commoner, B., Heise, J. J. and Townsend, .!.: Proc. Nat. Acad. Sci., 42:710, 1956. 16. Commoner, B., Townsend, J. and Pake, G. E.: Nature, 174:689, 1954. 17. Duysens, L. N. M. Thesis, Utrecht, 1952. 18. Eley, D. D., Parfitt, G. D., Perry, M. J. and Taysum, D. H.; Trans. Faraday Soc, 49:19, 1953. 19. Evans, M. G. and Gergely, J.: Biochim. Biophys. Acta, 3:\88, 1949. 20. Evans, M. G. and Uri, N.: Nature, 764:404, 1949. 21. FoRSTER, Th.: Fluoreszenz organischer Verbindungen. Vandenhoeck & Ruprecht, Gotdngen, 1951. 22. Franck, J., French, C. S., and Puck, T. T.: J. Pliys. C/iem., 45:1268, 1941 23. Franck, J. and Levi, H.: Zschr. p/iys. C/iem., 527:409, 1934. SOME PHOTOCHEMICAL CONSIDERATIONS 33 24. Franck, J. and Wood, R. W. : ,/. Chem. Phys., 4:551, 1936. 25. Fujimori, E. and Livingston, R.: Xati/re, !80:\036, 1957. 26. Gaffron, H.: Bet. dtsch. chem. Ges., 60:2229, 1927; Biochem. Zschr., 264:25\, 1933. 27. Griffiths, M., Sistrom, VV. R., Cohen-Bazire, G. and Stanier, R. Y. • Xature, 776:\2\\, 1955. 28. Kandler, O. and Schotz, F.: Zschr. Natiirf., nb:708, 1956. 29. Karreman, G. and Steele, R. H.: Biochim. Biophys. Acta, 25:280, 1957. 30. Kasha, M.: Chem. Revs., 4/:401, 1947. 31. Kasha, M.: J. Amer. Chem. Soc., 74:1\, 1952. 32. Katz, E.: Chlorophyll Fluorescence as an Energy Flow Meter for Photosynthe- sis, in Franck, J. and Loomis, W. E.: Photosynthesis in Plants. Iowa State Col- lege Press, Ames, 1949. 33. Kautsky, H.: Zschr. Naturf., f^h -.292, \9S\. 34. Laidler, K. J. : The Chemical Kinetics of Excited States. Clarendon Press, Oxford, 1955. 35. Lewis, G. N. and Kasha, M.: J. Amer. Chem. Soc, 66:2\00, 1944; 67:944, 1945. 36. Michaelis, L.: Fundamentals of Oxidation and Reduction, in Green, D. E.: Currents in Biochemical Research. Interscience, New York, 1946. 37. Michaelis, L. and Schubert, M. P.: Chem. Revs., 22:437, 1938. 38. Perrin, F.: Ann. phys., }2:\69, \929. 39. Pringsheim, P. : Fluorescence and Phosphorescence. Interscience, New York, 1949. 40. Reid, C: Excited States in Chemistry and Biology. Butterworth, London, 1957. 41. SCHENCK, G. O. 42. SCHENCK, G. O. 43. SCHENCK, G. O. Naturw., 40:205, 229, 1953. Naturw., 40:212, 1953. Naturw., 43:1\, 1956. 44. ScHENCK, G. O., GoLLNiCK K. and Neumuller, O. A.: Liebigs Ann. Chem., 603: 46, 1957. 45. -ScHENCK, G. O., and Ziegler, K.: Synthesen zweier cyclischer NaturstofFe mit Briickensauerstoffatomen. in Festschrift Prof . Arthur Stoll, Birkhauser, Basle, 1957. 46. Shian, Y. G. and Franck, J.: Arch. Biochem., 74:253, 1947. 47. Shockley, W. : Amer. Scientist, 42:41, 1954. 48. Strehler, B. L.: Research in Photosynthesis {Gatlinburg Conference 1955). In- terscience, New York, 1957. 49. Strehler, B. L. and Arnold, W.: J. Gen. Physiol., 34:809, 1951. 50. Szent-Gyorgyi, A.: Xature, 748:\57, 1941; 757:875, 1946. 51. Szent-Gyorgyi, A.: The Chemistry of Muscular Contraction. Academic Press, New York, 1947. 52. Szent-Gyorgyi, A.: Bioenergetics. Academic Press, New York, 1957. 53. Teale, F. W. J.: Xature, 757:415, 1958. 54. Uri, N.: Chem. Revs., 50:375, 1952. 55. Uri, N.: Biochim. Biophys. Acta, 78:209, 1955. 56. Vishniac, W. and Rose, I. A.: Xature, 752:1089, 1958. 57. Vladimirov, A. and Konev, S. V.: Biophysics, 2:1, 1957. 58. Warburg, E.: Zschr. Elektrochem., 26:54, 1920. 59. Warburg, O.: Heavy Metal Prosthetic Groups. Clarendon Press, Oxford, 1949. 60. Warburg, O., Krippahl, G., Gewitz, H.-S. and Volker, W.: Zschr. Xaturf., 73b:437, 1958. 34 PROBLEMS IN PHOTOSYNTHESIS 61. Warburg, O. and Negelein, E.: Biochem. Zschr., 202:202, 1928; 27-7:64, 1929; 255:247, 1932. 62. Wassink, E. C: Symposium No. 5. Carbon Dioxide Fixation and Photosynthesis. Cambridge Univ. Press, Cambridge, 1951. 63. Weigl, J. VV. and Livingston, R.: J. Amer. Chem. Soc, 7-7:4160, 1952. 64. Weiss, J.: Naturw., 23:610, 1935. 65. Weiss, J.: Nature, 787:825, 1958. 66. Wertz, J. E.: Chem. Revs., 55:829, 1955. 67. Whittingham, C. P.: Progress in Biopliysics, 7:320, 1957. 68. Witt, H. T., Moraw, R. and Muller, A.: Zschr. phys. Chem., 74:128, 1958; Zschr. Naturf., 7 3b :822, 1958. 69. Zavoisky, E.: J. Phys. USSR, 9:211, 1945. CHAPTER 3 The Energetics of Photosynthesis A. ENERGY TURNOVER § 15 Chlorella and Its Cultivation To study photosynthesis use is made of a unicellular alga Chlorella pyre- noidosa which can be easily cultured in a nutrient solution (Fig. 13). The algal cells are 3 to 5m in diameter and are resistant to mechanical and chem- ical influences. They can be centrifuged off from the nutrient solution and suspended in other solutions. They maintain their power of assimilation in solutions of the most varied composition and with widely differing pH values, and even in distilled water. In carbonate-bicarbonate solution, at pH 9, Chlorella cells remain intact for days, exhibiting constant pho'tosynthetic activity. Organisms such as Chlorella offer the simplest conditions for the diffusion of metabolic gases. Ever since Chlorella has been used in the study of photosynthesis it has been known that there are cells which use light efficiently and cells which do not. One of the most important conditions is the intensity of light at which the cells are cultured. If use is made of artificial light burning constantly at the same degree of brightness, the conditions are too far removed from the natural conditions under which Chlorella has thriven for over 500 million years. Such methods of culture are highly unphysiological, and it is astonishing that some botanists who have cultivated Chlorella for about 40 years still do not take this into consideration. As Warburg (24, 35, 36) pointed out, under these conditions the cells are forced to produce organic substance uninter- ruptedly and even to produce much more than is actually needed for normal anabolic processes. It is clear that the natural defence reaction of the cells is a very inefficient transformation of light energy into chemical energy. When the light intensity is varied so that day and night, dusk and dawn are reproduced, the culture gives rise to cells— named A-cells by Warburg— which use light most efficiently. Figure 14 depicts the equipment used for the cultivation of A-cells in a dark room. Light is proved by a 300 watt, 220 volt metal filament lamp. The resistance is changed automatically so that the voltage increases from 50 to 220 and then decreases to 50 within a 24- hour period. Not only is the light intensity changed but also, as under natural conditions, the spectral composition of the light produced. Although it is, of course, possible to produce any variation in light intensity that may be required, Warburg prefers the method depicted in Figure 15. 35 36 PROBLEMS IN PHOTOSYNTHESIS Fig. 13. Cells of Chlorella pyreiioidosa. Diameter: 3-5 n. % « -\* * ♦ » f #|J^ C9, ^ 3 Fig. 14. Cultivation equipment for Chlorella in Prof. Warburg's institute. The distance between the lamp and the culture flasks is 25 cm. The culture solution* contains the essential trace elements (see §32) and is aerated * The culture solution K contains, per liter of quartz-distilled water, 5 g MgS04.7H20 + 2.5 g KH2PO4 + 2 g NaCl + 2 g KNO.3 + 0.5 g Ca(N03)2.4H20. The pH is approximately 4.2 but rises during the culture period (30, 31 ). Another culture solution is the sah solution S in which the nitrate is replaced by 0.2 g NH4CI. THE ENERGETICS OF PHOTOSYNTHESIS 37 100 Fig. 15. Cultivation of Ch/orclla witli fiuctuating light intensity (Warburg et al., Zschr. Naturf.). with a gas mixture consisting of 5 vol % CO.j, 30 vol % O2 and 65 vol % argon. Each culture flask contains 250 ml Chlorella suspension. Young cells should be used in photosynthetic experiments as older cells, although richer in chlorophyll, contain some photochemically inactive chlorophyll. War- burg (28) distinguishes various cell types in Chlorella, according to the culture method used : A-cells. These are obtained when fluctuating light is used during culture. The growth increases from 60 to 160 jul in 24 hours and to 350 jul in 48 hours The one-day cells contain 4% chlorophyll and two-day cells contain 6% chlorophyll, calculated on a dry weight basis. A-cells contain 1.5 mole glutamic acid for each mole chlorophyll. A-cells are used for the determina- tion of quantum requirements. South cells. These cells are cultured by exposure to south light plus a 300 watt metal filament lamp during the day and a 200 watt metal filament lamp during the night. The growth increase is from 60 to 350 jul in 24 hours and up to 650 /jI in 48 hours. The two-day cells contain 7 to 8% chlorophyll calculated on a dry weight basis. South cells contain almost exactly 1 mole glutamic acid for each mole chlorophyll. North cells. These cells are cultured in a dark room (no daylight) by exposure to two 200 watt lamps. In 48 hours the growth increases from 60 to 1500 }x\. The two-day cells contain 5 to 6% chlorophyll calculated on a dry weight basis. North cells contain 1 to 2 mole glutamic acid for each mole chlorophyll. X-cells. These cells are obtained by strong illumination from four 200 watt lamps situated close to the culture on two sides. Growth increases in 24 hours from 60 to 200 p\. On a dry weight basis, the cells contain 1.2% chlorophyll and 3 to 5 mole glutamic acid for each mole chlorophyll. The X-cells show a much higher degree of lactic acid fermentation than the other cells (see § 62). The optimal temperature for common Chlorella is 25 ° C. Sorokin and Myers (17) succeeded in cultivating a pure strain of Chlorella at an optimal temperature of 39 ° C. Preliminary manometric studies showed that these thermophilic Chlorella produced, photosynthetically, about 100 /xl 02/hour per )ul cells. Burk et al. (8, 9) showed that the energetic behavior of this 38 PROBLEMS IN PHOTOSYNTHESIS thermophilic Chlorella is identical with that of common Chlorella, except that the rate of photosynthesis is 10 times higher and the cell growth is 100 times more rapid (6). Each cell produces 1000 cells within 24 hours. For these reasons, thermophilic Chlorella may be of exceptional value in studying the technical applications of photosynthesis, e.g., air purification in submarines and space ships (6). § 16 Principles of Manometry The manometric methods carefully developed by Warburg (21, 22) are the methods of choice in measuring metabolic processes involving gases (respiration, fermentations and photosynthesis). Measurements are made either with a simple manometer (Fig. 16) or with the differential manometer ,<^^ Fig. 16. Warburg's simple open-arm, fixed-volume manometer. Fig. 17. The differential manometer (Fig. 17). The first differential manometer was constructed in 1900 by E. Warburg (18) who used it for the measurement of ozonisation. It was im- proved for physiological experiments by Barcroft (2) in 1908. Figure 17 shows the differential manometer used by Warburg and Negelein in their experiments on photosynthesis. The simple manometer constructed by Haldane and Barcroft (13) in 1902 for the study of blood gases is the most suitable for physiological experiments. It has been improved by Warburg. However, manometry itself was not new, as de Saussure had already made use of it. The simple manometer is connected to the manometer vessel (Fig. 18) THE ENERGETICS OF PHOTOSYNTHESIS 39 holding the cell suspension at one arm only, whereas the other arm is left open to the atmosphere. In the differential manometer the assimilation vessel contains the cell suspension and the compensation vessel contains the same salt solution but without any cells. t^l- Fig. 18. Manometer vessels. To stopcock Capillary tube For changing culture chemically (by tipping vessel and manometer intact) Fig. 19. Scheme of typical Warburg manometer (Burk and Hobby, Science). 40 PROBLEMS IN PHOTOSYNTHESIS As shown in Figure 18, the manometer vessel has two phases: the cell suspension with the volume Fp- and the gas phase with the volume Vq. The vessel connected with the manometer is suspended in a thermostat and is shaken continuously during the experiment. Figure 19, after Burk (7), shows the typical, open-arm, fixed-volume manometer made classic and universal by Warburg. In the manometer vessel O2 is consumed and CO2 evolved and absorbed by KOH. The change of the amount of gas in the vessel causes an experimentally observed vertical displacement of the fluid in the manom- eter arm open to the atmosphere when the meniscus in the closed arm is maintained at a fixed level by means of the adjustable screw and the fluid- filled sac at the base of the manometer. The narrow, even-bore capillary of the manometer arms has an area of 0.5 to 3 mm-. Obviously, exact calibration of the manometer and sensitivity magnification are of the utmost importance (7, 10). The theory of the simple manometer will be discussed in§18. The manometer fluid may be either ;\yo-capronic acid or Brodie's solution. The latter contains in each 500 ml water 23 g NaCl and 5 g sodium choleinate plus a few drops of thymol solution. As the relative specific gravities are 0.926 and 1 .034, respectively, we find that u "760 X 13.6 ,,,^^ 760 mm Hg = — = 11160 mm ?5^o-capronic acid and 760 mm Hg = ^'"^ ?^ ^^"'^ = 10000 mm Brodie or 1 mm /jo-capronic acid — 0.068 mm Hg and 1 mm Brodie = 0.076 mm Hg § 17 Light Absorption and Its Measurement Figure 20 depicts schematically early, experimental equipment devised by Warburg and Figure 21 shows equipment as used today. The principle is the same in both, the light passing from the source through various filters to give the desired wave-length and entering the thermostat in the horizontal plane. It is then reflected by a mirror inclined at 45 ° and enters the manom- eter vessel from below. The light is then directed on to a bolometer so that its intensity can be measured. This is depicted schematically in Figure 22. It used to be the practice to measure the intensity of the light before it entered the thermostat, as can be seen from Figure 20. However, corrections always had to be made for the difference in permeability the light encountered in its passage through the thermostat. Today, the intensity is measured after the light has passed through the thermostat. The mirror M^ in the thermostat used to be fixed .so that the light was reflected vertically upwards. Now, it THE ENERGETICS OF PHOTOSYNTHESIS 41 ^^ K Fig. 20. Early experimental equipment devised by Warburg. Hg: mercury lamp, b, Ji, J'>: diaphragms. Z-i, L^: lenses. F: cuvettes filled with colored solutions. .S'2.- mirror {S\: position of the mirror for light intensity measurement). B: bolometer with Wheatstone bridge. T: manometer vessel. M: manometer. A'.- cathetometer. " s a a Fig. 21. Newer experimental equipment in Prof. Warburg's institute. For the sake of clarity the thermostat is not shown. is connected to the manometer and moves synchronously with it in the horizontal plane (see Fig. 22). The cross-section of the beam of light is no longer round but has the same shape and surface area as the bottom of the manometer vessel. This provides almost complete illumination of the cell suspension. After removal of the manometer vessels, the beam of light is reflected by the mirror Afg into the horizontal plane and, with the aid of a 42 PROBLEMS IN PHOTOSYNTHESIS Fig. 22. Measurement of the energy of the incident Hght beam. T: thermostat. V: manometer vessel. M \: movable mirror. A/2.- mirror with known refliection power. LB: light beam. B: bolometer (Warburg). lens, concentrated on the bolometer. The permeability of this mirror-lens system is a constant, dependent on the wave-length. For example, it is 0.78 for red light, which has a wave-length of 6440 A. If i' is the intensity measured bolometrically, then the incident intensity i„ of the cell suspension is?V0.78or 1.28r. The determination of the light intensity by means of a bolometer (Fig. 23) is based on the absorption of radiation by thin blackened platinum strips. The absorption increases the temperature and gives rise to a change in the electrical resistance which is then measured in a Wheatstone bridge. Calibra- tion of the bolometer is effected by standard carbon filament lamps supplied by the U.S. Bureau of Standards.* The incident intensity i^ can be measured with the bolometer, taking into consideration the corrections made for permeability. However, the cell suspension absorbs only part of the incident intensity. In early experiments complete absorption was realized by using dense cell suspensions of 10 jul cells or more per ml. This has become unnecessary to-day. With the aid of Ulbricht spheres, it is possible to measure the fraction a of the incident intensity (O absorbed by the cell suspension. For this reason, the density of the cell suspension can be considerably decreased to about 0.5 to 1 fx\ cells/ml. When dense cell suspensions are used, the cells at the bottom of the manom- eter vessel absorb more light than those at higher levels. As the manometer vessels have to be shaken rapidly, cells in such suspensions change continuously from the light to the dark regions, resulting in photochemical inductions and consequent loss of energy. According to Warburg (35), the passage of light through the vessels remains optimal only if the intensity is kept nearly con- stant. This condition can be realized only with thin suspensions which ab- sorb only 3 to 4% of the incident light in the manometer vessels. Ulbricht spheres are hollow vessels impermeable to light, with a diameter * Bolometry, introduced by the American physicist S. P. Langley, was developed and adapted to photochemistry by Lummer and Kurlbaum. It was Lummer's bolometer that played a decisive role in the discovery of light quanta. The bolometer of Lummer and Kurlbaum was used to measure light energy in the experiments of E. Warburg that laid the foundations of quantitative photochem- istry. It is the same bolometer that has now solved the problem of the energetics of photosynthesis (24, reference 9). THE ENERGETICS OF PHOTOSYNTHESIS 43 Fig. 23. The buluincici- (photograph taken in Prol". Warburg's institute). of 50 cm. The inside wall of the sphere is painted in a hght colour and radia- tion is measured with a selenium cell connected to a multiplex galvanometer. More recently, an electron multiplier has been used instead of a selenium cell. This permits the measurement of light absorption accurate to within about 3% of the absorption value. The apparatus used by Warburg (27) is depicted in Figure 24 and the interior of the sphere is shown in Figure 25. Light is provided by a 500 watt metal filament lamp. The spectral area required is isolated with the aid of an interference disc. The manometer vessel contain- 44 PROBLEMS IN PHOTOSYNTHESIS ing the cell suspension and the control vessel containing white Chlorelln cells are placed together in the sphere and are brought alternately into the path of the light.* As in photosynthesis experiments, both vessels are shaken, the light beam moving synchronously. Determination of the degree of absorp- tion depends on three galvanometer recordings made when: 1) the white cells are in the beam of light ; 2) the green cells are in the beam of light, and 3) the white cells are again in the beam of light. If, for example, the record- ings are 100 scale units, 90 scale units and 100 scale units, the permeability a b F Sch Fig. 24. The measurement of light absorption with the Ulbricht sphere shown schemati- cally. A'.- light source. B: diaphragm. W: cuvette with water. /.• interference disc. L: lens. .SV movable mirror, a and b: manometer vessels, one containing green cells and the other white cells. Sch: handle for moving vessel a or b into the light beam (Warburg et al., Zschr. Naturf.). of the cell suspension is 90%, i.e., its absorption is 10%. The problem of light absorption by thin cell suspensions is therefore satisfactorily solved by the use of the Ulbricht sphere. If i^ is the intensity of the incident radiation — measured bolometrically — ■ on the surface area F of the manometer vessel over the time period / sec, the incident radiation energy E^ is = loFt If the fraction a of the incident intensity /„ — measured with the Ulbricht sphere — is absorbed by the cell suspension, then we have for the absorbed radiation energy E E = cxigFt * So-called white Chlorella cells are obtained when all the pigment of normal Chlorella cells is ex- tracted with methanol. The concentration of the white cells in the manometer vessel must be identical with that of the green cells. The use of the white cells results in a more uniform distribu- tion of light within the sphere than when the control vessel contains water only. THE ENERGETICS OF PHOTOSYNTHESIS 45 Fig. 25. The interior of the Ulbricht sphere (photograph taken in Prof. Warburg's institute). If we write «/'„ = /, we findf E = iFt (6) The value of E can be expressed in cal or in /xl quanta. If red light with a wave-length of 6600 A is used, then, according to Table 1, 1 mole quanta = 43300 cal f Henceforth the intensity absorbed will be denoted by i 46 PROBLEMS IN PHOTOSYNTHESIS or . , 43300 _^„.„ 1 Ml quanta = ^^.^n ^ in3 = 0-0019 ca. 22400 X 103 According to equation 2, the general equation is 1 , 12.8 , 1 ^1 quanta = — r — cal A § 18 Theory of Manometry In respiration almost as much COo is produced as O2 disappears, while in photosynthesis nearly as much CO.2 disappears as O2 is produced. Warburg succeeded in overcoming this difficulty by adopting a modified technique (19). As already pointed out, there are two phases in the manometer vessel: the liquid cell suspension and the overlying gas phase. Oo and CO2 are soluble to different extents in the liquid phase, much more CO2 being ab- sorbed than O2. For this reason, it is possible to measure the resulting pres- sure changes with a high degree of precision. Imagine a manometer vessel pardy filled with a liquid which produces a gas. As the volume is kept constant, the pressure increases and the problem now is to calculate the amount of gas produced from the change in pressure. The temperature in the vessel is kept constant. The magnitudes are : P = pressure before the experiment P' = pressure after the experiment h = pressure change IV = volume of the liquid phase in ^1 ]\] = volume of the gas phase in ^1 T = absolute temperature a = absorption coefficient of the gas produced in the liquid phase Before the experiment the amount of gas at ° C and 760 mm Hg in the gas phase is P 273 760 T ValA and in the liquid phase is 760 ^'^-^ ^^ In both phases the amount of gas is ^/27: 760V T ^ (^ Va + f>) Ml and after the experiment 760 '-^{^^V. + V,a) ,\ THE ENERGETICS OF PHOTOSYNTHESIS 47 The increase in the amount of gas is therefore P' - 760 -Tr' 'a + 1 I pa P' - P h 760 760 Ml /ou \ y / Furthermore As the manometer does not contain mercury but another fluid with a normal pressure of P^ we find that (1 G ^ + 1 fo: \ K /^^ ^^^ It follows from equation 7 that the amount of gas produced is proportional to the change of pressure and is independent of the initial pressure P. The expression between parentheses is a constant, called vessel constant A', so that .V = hK (8) Depending on the nature of the gas fOo or CO-j) produced, we distinguish between vessel constants A'o„ and A'^o,; the absorption coefficients of these gases are aQ„ = 0.038 and a^^y,, = 1.182 at 10° C. We therefore obtain the equations xo„ = /zA'(>2 The vessel constants are always positive and are expressed in mm-. The sign of h is positive when gas is produced and negative when the gas is ab- sorbed. The values for v can thus be either positive or negative. In measuring either respiration or photosynthesis we have to consider two gases — O2 and CO., — instead of only one. The pressure change h is equal to the sum of the changes in the partial pressures of both gases, so that h = ho, + //CO, (9) The A- values of both gases are xo, = ho,Ko, (10) >^co2 = /'cojA'coj (11) Further, we know that in photosynthesis 7 = ^^^ (1) xo. From the equations 1, 9, 10 and 1 1 we can derive •^02 = h ^ , ^f. (1-^) AcOa I 7-^02 , Ko^KcOi and xco-, = « Aco, + Ao, (13) 48 PROBLEMS IN PHOTOSYNTHESIS The fundamental equations 12 and 13 are also valid for the differential ma- nometer. Numerical example: The manometer vessel contains 270 ^1 cells. Vp = 7.000 ml Vg = 6.913 ml p^ = 10000 mm Brodie ao, = 0.03 acoj = 0.87 T = 20° C Hence, according to equation 7 273 Ko^= p ""i™' -» and Vg -y + ' FOico^ Kco, = p mm^ SO that and 6913 X 273/293 + 7000 X 0.03 „ . ., ^o. = ^0000 = 0.665 mm- 6913 X 273/293 + 7000 X 0.87 ^ ._ Kca = —^TT^T^T^ = 1.25 J) mm- The values for Aq, and Acq, are calculated from equations 12 and 13. For -y = — 0.8 and h = 60 mm Brodie after 60 min illumination, we find 0.665 X 1.253 , .n . , - ^-^ = ^^ X 1.253 -0.8X0.665 = + ''■''' and ^^ ^ 0.665 X 1.253 ^■^«^ = ^^ >< -1.253/0.8+ 0.66 -5 = " ^^'^ ^^ § 19 The Two-vessel Method When the value of 7 is unknown, equations 12 and 13 do not suffice for calculating Xq, and .Vco,, because they contain three unknowns. Measure- ments with two vessels, however, permit the calculation of both gas amounts (25, 26). The vessels must be different in size (Fig. 18) and therefore have different vessel constants. However, in each vessel the same amount of cells is suspended in the same amount of liquid. Over equal time periods the values of %, and Vco, should be the same for both vessels. But since the vessel THE ENERGETICS OF PHOTOSYNTHESIS 49 constants are different the pressure changes are also different. For vessel I we have ■^02 = ho^Koi -^002 = hco^Kco^ h = /?(>, + hco^ and for vessel II Since h and h' are measured, we can derive the following equations from these 6 equations with their 6 unknowns // K CO, ■" hKco, (14) K CO, -^coj K 02 -^o, and _ h K 02 — /'•^02 J^ O2 _ -^O; (-15) CO, ACO2 From equations 14 and 15 the photosynthetic quotient can easily be calculated (16) _ ^coj _ K'cOiKcOi h K O; — /?Ao., X02 K'o.,Koi hKcot ~ h K C02 By writing K'co^ ^ ^ K'cOi Kco^ K 02 K02 Kco 2 K CO, Aco, K 02 A02 A-'c >2 K 0, K02 K'cOi Aco2 Ko, K 02 K02 A' CO, Ar = c = D CO2 •'^002 we can simplify equations 14 and 15 to the general equations of the two- vessel method xo^_ = h'A - hB (17) XC02 = h'C - hD (18) Fieure 26 shows the arrangement of the vessels in the two-vessel method. Both vessels are illuminated and each contains 7 ml Chlorella suspension. Each non-illuminated vessel contains 7 ml of salt solution and serves as a thermobarometer for the irradiated vessels. The four vessels are connected to manometers, the open arms of which are connected with a two liter flask placed in the same thermostat as the vessels. The four vessels are shaken 50 PROBLEMS IN PHOTOSYNTHESIS Fig. 26. The two-vessel method (photograph taken in Prof. Warburg's institute). in the horizontal plane in the thermostat at 20° C with a frequency of 210 per min and an excursion of 1 cm. The irradiation of the vessels — with which we shall deal in § 30 — must be identical for both vessels because the conditions for the two-vessel method are only met if the chemical processes in both vessels are the same. It is therefore also imperative that the bottoms of the two illuminated vessels are exactly identical in size and form, because only then can light absorption be equal. This can be tested easily by means of the Ulbricht sphere since the absorption values in both vessels should be the same. Numerical example: Large vessel Va = 13.75 ml Vr = 7.00 ml It follows from equation 7 that at 25 ° C A'o, = 1.286 mm- Acq, = 2.087 mm'' THE ENERGETICS OF PHOTOSYNTHESIS 51 Small vessel Va = 9.50 ml I> = 7.00 ml K\,^_ = 0.897 mm- A"co. = 1-698 mm- From the four vessel constants it follows that A = 6.29 mm- C = - 10.31 mm^ B = 7.73 mm- D = - 14.78 mm- According to equations 17 and 1 8 we find xo, = 6.29 h' - 1.11> h xco, = - 10.31 h' + 14.78 h In an experiment the pressure changes after 60 min illumination were for the small vessel: h' = -|-28 mm for the large vessel: h = +12.5 mm so that ;co, - 6.29 X 28 = 7.73 X 12.5 = 80 /il xco, = -10.31 X 28 + 14.78 X 12.5 = -104 ^1 Hence the photosynthetic quotient is 7 = XC0.A0. = -104/80 = -1.30 § 20 The Use of Carbonate Buffer Solutions Instead of aerating with air containing CO., it is also possible to suspend Chlorella in mixtures of carbonate and bicarbonate which furnish the COo necessary for photosynthesis. The amount of COo in such solutions can easily be calculated . The two dissociation reactions of carbonic acid are I H2C03?=^H+ + HCO;r II HCO3 ^ H+ + CO3-- the equilibrium constants being (H+)(HC03-) ki = ki\ = (H.2CO3) (H+)(C032-) (HCO3-) or K = ku (C032-)(H2C03) For (H0CO3) we may write (CO2), so that ^ (HC03-)^ (19) ^ (C03'-)(C02) 52 PROBLEMS IN PHOTOSYNTHESIS When and we find from equation 19 (HCO3-) = ^(NaHCOa) (CO32-) = /^(NaoCOa) _ a^(NaHCO:i)^ /3(Na.C03)(C02) or (NaHCOa)^ - ^ ^ - A- r?m (Na,C03)(C0j - ^^^- ^ ™ Equation 20 has been verified for dilute solutions. The value of K' remains constant if the total Na concentration is kept constant. At 25 ° C the fol- lowing empirical equation has been found K' = 8739 - 1671 log Cn^ (21) The equation is valid for values between 100 and 1000 millimole of the total Na concentration C^^. The constant K' is dependent upon the temperature, but may be calculated approximately from the reaction heat of bicarbonate dissociation. In this type of experiment a mixture of the following composition is used : 85 ml 0.1 molar NaHCOg + 15 ml 0.1 molar NaaCOg.* In this solution C'Na = 85 + 2 X 15 = 115 millimole per liter, so that, according to equation 21, 'the value of A'' at 25 ° C is 5300. When (NaHCOg) = 0.085 mole/liter and (NagCOa) = 0.015 mole/liter, we obtain, according to equation 20, (CO2) = 91 X 10-« mole/liter During the experiment the equilibrium between the salts is disturbed by CO2 assimilation. However, when the CO2 uptake is small by comparison with the salt concentration, the changes in the concentration may be ignored because the carbonate-bicarbonate mixture acts as a buffer. This is shown by the following example. The volume of the cell suspension is 10 ml. We remove 200 /.il CO2, i.e., 0.2/22400 = 9 X 10-« mole/liter. The CO2 concentration, which was 91 X lO"'' mole/liter, will thus be decreased by 10%. The CO2 partial pressure of the carbonate buffer used is 0.20 X IO-2 atm = 20 mm manometer fluid. The removal of 200 m1 CO2 therefore means a change of only about 2 mm manometer fluid in the CO2 partial pressure. The pH of the carbonate buffer can be calculated from the Henderson- Hasselbalch equation ^. , , (NaHCOa) ,., , 1 0-085 .. pH = pK. + log ^-^cO^ = ^-^^ + ^°^ 91 X 10- « = ^-2 * The mixture of 95 ml 0.2 moles NaHCOa + 5 ml 0.2 molar NaiCOj is preferred today. Its pH is 8.8 and the COj partial pressure at 20°C about 2 atm % (see § 29). THE ENERGETICS OF PHOTOSYNTHESIS 53 From equation 10 we find the amount of Oo produced to be Owing to the small CO2 partial pressure of the carbonate buflfer, the gas phase contains very little COo and the manometrically observed pressure change is equal to /?o,, so that Since V^ = 5000 ^1, V, = 10000 p\ and T = 298°, the value of A'o, is 0.5 mm^. When 200 /xl CO, are reduced, ,v„^ = 200 mI i.e. for A',,, = 0.5 the O., partial pressure has been changed by 400 mm. The change of the CO.. partial pressure after removal of 200 ^1 COo was only 2 mm, which is thus only 0.5% of the 0.2 partial pressure. It follows from this that making b = Z?,,, is permissible. § 21 Energy Turnover Warburg and Negelein (20, 30) were the first to study the question con- cerning which fraction of absorbed radiation energy in photosynthesis could be transformed to chemical energy. If the light energy absorbed is E and the Fig. 27. Relationship between the ab- sorbed radiation energy E and the ac- compHshed work U. tga,= lim -^ chemical work performed simultaneously — the increase in total energy — is U, the quotient . = I (5) becomes of special interest. In Figure 27 the abscissa shows the light energy absorbed per sec and the ordinate the chemical work performed per sec. We can see from the curve that r/ changes with the intensity of the radiation ab- sorbed. The more intensive the radiation, the smaller -q becomes, thus mak- ing that portion transformed into chemical energy smaller. With decreasing 54 PROBLEMS IN PHOTOSYNTHESIS intensity dU/dE, i.e., tan a, becomes larger and approaches a limit which can be mathematically expressed as ?7o ~ lim -r^ — constant (22) Warburg set out to determine the highest value of rj at low intensities. Figure 27 shows that U is generally not proportional to E, but is actually dependent upon £■ in a complicated manner. Mathematically, the relation may be expressed as U = a -\- bE -\- cE' a, b and c being constants. Differentiation gives and for E = dU % = b -j- 2cE dE ryo = lim ^j^ = b (23) The constant /; is therefore the limiting value sought. It is shown graphi- cally in Figure 27 by the tangent of the angle ofo at which the curve rises from the zero point of the system of coordinates. If jE" = 0, then U = 0, so that a = 0. Both h and c may be determined by two measurements made at two different intensities such as By eliminating c we find Ui = bEx + cEi^ U2 = bEo + cE-^ E-zEi" - E1E2' ^^ ' The production of 1 mole O2 corresponds to an increase of total energy of 1 12000 cal. Therefore, when .to, /xl O2 develops, then TT 1 12000 _„„, ._. ^ = "«^ 2240(00000 = ^-^^^ "«^ ^'^^ one mole being 22400 X 1000 /xl. The value of Aq, is determined mano- metrically. Warburg and Negelein used a differential manometer in their fundamental studies. It is possible to calculate -q and r?,, from the values of .Vq, and i obtained ex- perimentally. If i is the intensity absorbed per cm^ per sec, the radiation energy E absorbed in the time / of illumination is iFt. Since Xq^_ is determined manometrically, the chemical energy U may be calculated from equation 25 and 77 determined. By two measurements of U and E at different intensities b and consequently 770 may be calculated from equations 23 and 24. THE ENERGETICS OF PHOTOSYNTHESIS 55 Numerical example: I. I = 0.162 X 10"^ cal-cnv-sec-i F = 14 cm- t = 600 sec Ey = iFt = 0.136 cal xo, = 15.4 jA U, = 0.005 X 15.4 = 0.077 ca! rf = U,/Ei = 0.57 caj/cal TI. / = 0.327 X 10-^ cal-cm--.sec-i F = 14 cm- / = 600 sec £., = iFt = 0.275 cal xo, = 23.0 Ml U. = 0.005 X 23.0 = 0.115 cal rj = U-i/E-i = 0.42 cal /cal From these two experiments we find the values of £■), E.2, U] and U,. Equa- tion 24 can then be used to calculate h b = vo = liiTi j-p, = 0.71 cal cal Thus, Warburg and Negelein (30) found in their very first experiments in photosynthesis that an average of 70% of the radiation energy absorbed could be converted into chemical energy. § 22 Application of the Quantum Theory In § 21 77 was expressed in cal/cal. If we express U in ^ul Oo, then r] and Vn can be expressed in jul/cal ; then according to ecjuation 25 1 ^1 O, = 0.005 cal 1 mI cal = 0.005 cal/cal If U is expressed in mole Oo, then rj and rj^. would be expressed in mole/cal whereby 1 Ml/cal = 4.5 X 10-» mole/cal and 1 mole/cal = 1.1 X 10^ cal/cal All these relations may be easily calculated from equation 25. In photosynthesis the value of rj is not constant for any particular wave- length. Its value decreases as the wave-length decreases, as can be shown from experiments made in red, yellow and blue light. Numerical example: a) Red light (6600 A) E = 0.208 cal .vo, = 24.2 m1 U = 24.2 X 4.5 X 10-8 = 108.9 X lO"* mole r, = U/E = 108.9 X lO-VO-208 = 5.23 X lO-^ mole/cal 56 PROBLEMS IN PHOTOSYNTHESIS b) Yellow light (5780 A) E = 0.245 cal xoj = 25.1 m1 U = 25.1 X 4.5 X 10-8 = 113.0 X 10-« mole ■q = U/E = 113.0 X 10-8/0.245 = 4.62 X 10-^ mole/cal c) Blue light (4360 A) E = 0.280 cal xo., = 17.8 m1 U= 17.8 X 4.5 X 10-8 = 80.1 X 10"" mole 77 = U/E = 80.1 X 10-8/0.280 = 2.86 X lO-e mole/cal The dependence of tj upon the wave-length is easily explained by the quantum theory. In § 6 we saw that the quantum yield is If the radiation energy absorbed is equal to E, then E = qhv i.e., the energy £" consists of ^ quanta of /?i^. Since 77 = UE = n'E we have (3) ■n = qhv and therefore (f = rjhv If 7] is expressed in mole/cal, the photochemical equivalent Nhv must be used instead of quanta, N being Avogadro's number. T hus if = rjX/iv (26) The values of -q in the three examples mentioned, together with the values of if and \/ -^ [Fe] + 2CO Iron pentacarbonyl, a compound produced by passing CO over iron filings at about 80 ° C. Light causes the following breakdown Fe(CO)5 + Nhv -* Fe(CO)4 + CO Even though the chemical and optical characteristics of these three iron carbonyl compounds vary greatly, the photochemical cleavage reactions all have the quantum yield ^ = 1 . By contrast, Warburg" (23) found that in cleaving carbon monoxide hemo- globin the quantum yield was 0.25 (quantum requirement 4) [Fe]CO + 4 Xhv ^ [Fe] + CO the quantum number 4 is related to the degree of polymerization of hemio- globin. According to Anderson (1), salts and acidity cleave 4-fold poly- merized hemoglobin to bi-polymerized hemoglobin. Biicher and Negelein (5) found that increasing the salt concentration and decreasing the pH lowered the quantum requirement from 4 to 2. Myoglobin is a non-polymerized heme compound and, by cleaving its CO compound, Biicher and Negelein (5) and Biicher and Kaspers (4) found that ^p = \ [Fe]CO + Xhv ^ [Fe] + CO Warburg (23, 32, 33) also studied the photochemistry of the CO compound of iron oxygenase (cytochrome oxidase). This could not be done with the pure enzyme because it has not yet been isolated and its constitution is still unknown. Instead of direct measurements being made, the inhibition by CO of cell respiration was investigated. Torula yeast and vinegar bacteria were used as test objects. The result of these experiments was (p — 1, so that in all probability cytochrome oxidase is not a polymerized heme compound. * The addition of a ferric salt to a neutral, pure cysteine solution produces a deep-blue ferric cysteine complex which, after some time, becomes colorless. The cysteine reduces the ferric iron to ferrous iron and is thereby oxidized to cystine. If the solution is shaken in air, it turns blue again, producing trivalent iron. This process can be repeated until the iron oxidizes all the cysteine to cystine. THE ENERGETICS OF PHOTOSYNTHESIS 63 In contrast to photosynthesis, the dissociation of iron carbonyl compounds is reversible [FejCO ^ [Fe] + CO In darkness the reaction may proceed from left to right. In light it proceeds only from left to right. Consequently, in illuminated solutions of carbon monoxide hemochromogen and carbon monoxide ferrocysteine there are three parallel reactions — two dark reactions and one photochemical reaction. In the case of carbon monoxide hemoglobin four dark reactions and one photochemical reaction can be differentiated, as the two following dark reactions also occur: [FejCO + 02^ [FeJOo + CO The experiments are carried out with the differential manometer, both vessels containing the solution of the iron carbonyl compound. The gas phases have a certain CO partial pressure. After equilibrium has been reached in the dark, one of the vessels is illuminated with light of known wave-length and intensity. Upon illumination, CO is produced and the pressure rises to constant value. When the light is removed, the pressure of the photochemically liberated CO falls to zero, as, in the dark, the equilib- rium at the beginning of the experiment nmst be restored. From the speed of this return to zero can be calculated how many mole CO per min re-react. These are n mole. Since the number of mole quanta q which are absorbed per min is also known, the quantum yield can be calculated from equation 3. Lautsch et al. (3, 15) made compounds of certain peptides with active groups from the hemin series. These served as oxidase models in their studies. Photochemical cleavage of the CO compounds of these synthetic substances also gave a value of 1 for the quantum requirement. REFERENCES 1. Anderson, K. J.: in Svedberg, T. and Pedersen, K. O. The ultracentrijuge. Clarendon Press, Oxford, 1940. 2. Barcroft, J.: J. Physiol., J7:12, 1908. 3. Broser, W. and Lautsch, W. : Naturw., 42:5X1), 1955. 4. BiJCHER, T. and Kaspers, J.: Biochim. Biophys. Acta, 7:21, 1947. 5. BijCHER, T. and Negelein, E. : Biochem. Zschr., 3/7:163 (1942). 6. BuRK, D. : Personal communication (1957). 7. BuRK, D. and Hobby, G. : Science, 720:640, 1954. 8. BuRK, D., Hobby, G. and Hunter, J.: Science, 727:620, 1955. 9. BuRK, D., Hobby, G. and Hunter, J.: Fed. Proc, 75:227, 1956. 10. BuRK, D., Hobby, G. and Hunter, J.: Arch. Biochem. Biophys., 69:228, 1957. 11. BuRK, D. and Warburg, O.: Zschr. Naturf., 6b:\2, 1951. 12. Gaffron, H.: Ber. dtsch. chern. Ges., 60:755, 1927. 13. Haldane, J. and Barcroft, J.: J. Pliysiol., 25:232, 1902. 14. Haldane, J. and Smith, J. L.: J. Physiol., 20:A91, 1896. 64 PROBLEMS IN PHOTOSYNTHESIS 15. Lautsch, VV. and Schroeder, E. : Zschr. Xatiirf., 9b:277, 1954. 16. Sheppard, S. E.: Phot. ./., 6,5:380, 1925. 17. SoROKiN, C. and Myers, .1.: Science, // 7:330, 1953. 18. Warburg, E. : Sitzungsher . Preuss. Akad. Wiss., 34:712, 1900. 19. W ARBURG , O . : Biochem . Zschr . , / 00 : 2 30 , 1 9 1 9 . 20. Warburg, O.: Nalurw., W-Ml, 1922. 21. Warburg, O.: Ueber die katalytische Wirkimg der lebendigen Substanz. Springer, Berlin, 1928. 22. Warburg, O.: The Metabolism of Tumours. Constable, London, 1930. 23. Warburg, O.: Heavy Metal Prosthetic Groups. Clarendon Press, Oxford, 1949. 24. Warburg, O.: Science, 128:68, 1958. 25. Warburg, O. and Burk, D. : Arch. Biochem., 25:410, 1950. 26. Warburg, O., Burk, D., Schocken, V. and Hendricks, S.: Biochim. Biophys. Acta, 4:335, 1950. 27. Warburg, O. and Krippahl, G. : Zschr. Naturf., ,9^:181, 1954. 28. Warburg, O., Klotzsch, H. and Krippahl, G. : Zschr. Naturf., J2b :622, 1957. 29. Warburg, O., Krippahl, G., Buchholz, W. and Schroder, W. : Zschr. Naturf., 8b:615, 1953. 30. Warburg, O. and Negelein, E. : Zschr. phys. Chem., 702:235, 1922. 31. Warburg, O. and Negelein, E. : Zschr. phys. Chem., 70(5:191, 1923. 32. W^ ARBURG, O. and Negelein, E. : Biochem. Zschr., 200:414, 1928; 20-^:495, 1929. 33. Warburg, O. and Negelein, E. : Biochem. Zschr., 274:64, 1929. 34. Warburg, O. and Schocken, V.: Atch. Biochem., 27:363, 1949. 35. Warburg, O. and Schroder, W. : Zschr. Naturf., /2b:7\6, 1957. 36. Warburg, O., Schroder, W. and Gattung, H.-W.: Zschr. Naturf., 7 7b :6b4, 1956. 37. WiLLSTATTER, R. and Stole, A.: Untersuchungen liber die Assimilation der Kohlen- sdure. Springer, Berlin 1918. B. THE QUANTUM REQUIREMENT § 26 Quantum Requirement and Efficiency The experiments with red and yellow light discussed in § 22 show that the quantum requirement in photosynthesis is not dependent on the wave-length of the light used. Thus, it is the number of quanta and not the energy content which is of importance. This follows from equation 26 moleO., ,, energy mole O. if - rf ?^ . w gj^gj,gy n-ioie quanta mole quanta However, the quantum requirement has been found to be higher in blue than in red or yellow. This discrepancy can be explained by the fact that red and yellow radiation is absorbed only by chlorophyll, whereas blue light is ab- sorbed by chlorophyll and by yellow pigments. Many years ago Warburg and Negelein (61) found that chlorophyll absorbs only 70% of the total ab- THE ENERGETICS OF PHOTOSYNTHESIS 65 sorbed energy in blue (4360 A). The energy absorbed by the other pigments is also photosynthetically active, although the efficiency is much less. This explains why the Cjuantum requirement is higher in blue than in red and vellow. The quantum requirement 4.4 (see Table 4) corresponds to an efficiency value lower than that obtained by Warburg and Negelein in their first experi- ments (see § 21). We find that _ chemical energy _ 112000 _ ^ quantum energy 4.4 X 43300 In later experiments utilizing an improved technique (see § 29), Warburg el al. found considerably lower values for the quantum requirement, i.e., higher values for the efficiency. In experiments lasting several hours (6440 A) they obtained a value as low as 2.91. Such a low quantum requirement corre- sponds to the efficiency 112000 2.91 X 44400 = 87% As the quantum requirement in photosynthesis is not dependent on the wave- length, it is possible to obtain at a wave-length 6800 A (highest v^alue in red) an efficiency of ^ 112000 ^ ' 2.91 X 41400 "^^ If the efficiency t] were 100%, the theoretical quantum requirement would be 112000 \l^ X 42100 1/

' and that of photosynthesis is ^, a relation between both magnitudes and the degree of compensation n can be expressed as follows _ O2 production in light O2 removal in dark or photosynthesis a. = -. -. respiration However respiration = photosynthesis — gain so that photosynthesis a photosynthesis — gain If we introduce cp and tp', we find that (28) a (f — (f' or 1^' = V^^4 (29) a — 1 At complete compensation of respiration (sufficiently weak illumination) the degree of compensation is 1 . It follows from equation 28 that when a = \ the gain is nil, and from equation 29 that the quantum requirement of the gain \/(p' is infinite. If the quantum requirement of photosynthesis is kept constant at 1/^ = 3, it is easy to calculate the quantum requirement of the gain for various values of a. The degree of compensation increases with the intensity. Table 10 shows the quantum requirement of the gain at various degrees of compensa- tion. At 10-fold compensation, 1/^' = 3.3, i.e. the quantum requirement of the gain is 90% of the quantum requirement of photosynthesis. At 40-fold 68 PROBLEMS IN PHOTOSYNTHESIS compensation, l/(^' is 989c of V<^- By increasing the intensity, we arrive at 1/^' = 1 V, which clearly proves that it is really permissible to write gain = photosynthesis In § 26 we found equation 27 for thermodynamic efficiency to be 0.04 A At the quantum requirement 3, this equation gives for red light (6600 A) 0.04 X 6600 (27) If we introduce the degree of compensation a, we find that, according to equation 29, 0.04 X \h ,a-\ a or OL a. 0.04X TABLE 10 Degree of compensation and quantum requirement of the gain at increasing intensities (1/^ = 3) Degree of Compensation 1 2 3 5 10 40 Quantum Requirement of the Gain - 7 6.0 4.5 3.8 3.3 3.07 Warburg and Burk called y\' the economic efficiency. At the compensation point a = 1 we find 77' = 0. At 6-fold compensation we have V = 88^^ = 73% At 40-fold compensation t]' = 86%. With increasing compensation the economic efficiency increases and approaches the value of thermodynamic efficiency. THE ENERGETICS OF PHOTOSYNTHESIS 69 § 28 Some Instructive Experiments In analyzing a few experiments carried out by Warburg and Burk (44) in the National Cancer Institute at Bethesda and by Warburg et al. (47,48) in the Max Planck Institute for Cell Physiology, Berlin-Dahlem, we shall ex- amine the influence of some factors upon the quantum requirement. The intensity (^il quanta/min) of the absorbed compensating light will be indi- cated by / and that of the absorbed measured light hy i' . For experiments in acid culture media the two- vessel method is used. From the manometrically measured pressure changes h and h' the values of .Vq^ and Aco,. are calculated according to equations 17 and 18. The quantum requirem.ent is found by using the equation '

= i'l/xo. = 5.4 X 20/11.0 = 9.8 72 PROBLEMS IN PHOTOSYNTHESIS d. H 9.2 {carbonate bujfer), compensated A'oj = 0.657 mm2 i' = 5.4 /il quanta/min 5 min / 1 5 min / + i' 1 5 min i 15 min / + i' 1 5 min i 30 min / 30 min / + i' Pressurt ' Changes mm + 11. 5 mm mm + 11. 5 mm - 0. 5 mm - 0. 5 mm + 23. mm = +23. 5 mm xo, = hKo^ = 23.5 X 0.657 = 15.4 tx\ 1/95 = i't/xo, = 5.4 X 30/15.4 = 10.5 Though these experiments were pubhshed in 1950, Bassham, Shibata and Calvin (4) subsequently found that the quantum requirement of photo- synthesis increases parallel with compensation. Franck (24), on theoretical grounds only, arrived at the same conclusion. Five years after Warburg and Burk had carried out their conclusive experiments, Emerson and Chalmers (21) had to refute once again these erroneous views (see, however, the remarks at the end of § 29). § 29 The Significance of Carbon Dioxide Pressure The experiments described in the preceding paragraph clearly show that the quantum requirement of photosynthesis in acid medium is 3 to 4, values Warburg obtained nearly 40 years ago. It is of importance to note that in alkaline medium (carbonate buffer) — the medium preferred by many other investigators — much higher values are obtained, the quantum requirement in alkaline medium being three times greater than in acid medium. In order to explain this discrepancy, Warburg et al. (48) studied the influence of the CO2 pressure in both media. They determined, in acid medium (pH 5) using the two-vessel method, the quantum yield at CO2 pressures varying from 0.39 to 50 atm %. The maximum yield (0.342) was found at 5 atm % CO2. At lower and higher pressures the quantum yield decreased (Fig. 29). Similar behavior has been observed in alkaline medium (pH 8.8-9.5). With the one-vessel method the quantum yields were measured at COo pressures varying from 0.22 to 2 atm %. The highest quantum yield (0.304) was obtained at 2.0 atm %. If the ip values calculated are inserted in the curve of Figure 29, we see that — within the limits of experimental error — the same curve is obtained as in acid medium with the two-vessel method. It is remarkable that the measurements in acid medium with the two-vessel method and those in alkaline medium with the one-vessel m^ethod give the same curve representing the dependency of the quantum yield on the CO2 pressure. This result is excellent evidence for the correctness of the two-vessel method Warburg and Burk worked out several years ago. THE ENERGETICS OF PHO lOSYNTHESIS 73 Those workers who find the two-vessel method too comphcated, are now in a position to correctly determine the maximum quantum yield in an alkaline medium. To begin with, the quantum yield in 0.2 molar carbonate buffer at 2 atm % CO2 pressure is measured. Then, the same cell suspension is brought into an acid medium and the increase in the manometrically observed light action is noted when the COo pressure is increased from 2 atm % to 5 atm %. This experiment can be carried on with the one-vessel method, as the photosynthetic quotient does not change between 2 atm % and 5 atm % CO2. We see from the curve of Figure 29 that at 2 atm % CO2 the quantum yield is 0.290 and at 5 atm % CO2 it is 0.345. Thus, when the CO2 pressure is increased from 2 atm % to 5 atm %, the quantum yield 0.36

^ \l,= d.-2y ^ ill iKr\ 1/ l/v = 3.6 =17 1 1 1 1,1=0) / f^ lUU ^l', = A.' % ' ' ' / 50- . ^'/ V'; -9 / Fig. 30. The action of blue-green light. Continuous line : illumination with red meas- ured light only (/ = 16.7 ^ul quanta/min). Dotted line: additional illumination with blue-green light (/ = 0.3 jul quanta/min) (Warburg et al., Angew. Chem.). 60 120 180 240 300 360 420 min — >■ The curve of Figure 30 shows the result of an experiment in which blue- green cadmium light is alternately added to and removed from the red meas- ured light (6440 A). In analyzing the curve of Figure 30 we see that after the addition of blue-green light (dotted line) the quantum requirement is 4.4 after 2 hours, corresponding to an efficiency of 59%. Removal of the blue- green light increases the quantum requirement to infinity (efficiency = 0). Addition of blue-green light again decreases the quantum requirement to 3.6 (efficiency 72%). Removal of blue-green light increases the quantum 76 PROBLEMS IN PHOTOSYNTHESIS requirement to 17 (efficiency 15%). Further addition of blue-green light brings the quantum requirement down to 3.2 (efficiency 81%). Removal again increases the quantum requirement to 12.2 (efficiency 21%). This experiment clearly shows that in the absence of blue-green light efficiency is very low. After its addition, very good efficiencies are immediately obtained. The intensity of the measured red light was 16.7 /^l quanta/min, while the intensity of the added blue-green light amounted to 0.3 ^1 quanta/min only. The values of v were calculated with the aid of equation 27. The results of two experiments are shown in Tables 13 and 14. The figures clearly show that without blue-green light quantum requirement values increase and, in consequence, the efficiency values decrease. TABLE 13 The effect of blue-green light on cuantum requirement and efficiency (measured red light) Incident Intensity in nl quanta/min Time in min Quantum Requirement Efficiency 6440 A blue-green V in % 16.7 0.3 60 9.4 27 16.7 0.3 65 4.4 59 16.7 — 60 /■S-' 16.7 0.3 40 5.8 45 16.7 0.3 30 3.6 72 16.7 — 30 17 15 16.7 0.3 30 4.1 63 16.7 0.3 30 3.2 81 16.7 — 30 7.8 33 16.7 — 30 12.2 21 TABLE 14 The effect of blue-green light on quantum requirement and efficiency (measured green light) Incident Intensity Time Quantum in fj.1 quanta/min in min Requirement Efficiency 5460 A blue-green V in % 93.5 0.3 30 4.66 47 93.5 0.3 30 4.20 52 93.5 — 30 6.6 33 93.5 — 30 8.8 25 93.5 — 30 10.6 21 93.5 0.3 30 5.6 39 93.5 0.3 60 4.7 47 49 . 8 0.3 30 4.0 55 49.8 0.3 30 4.0 55 171 0.3 15 5.5 40 171 0.3 15 5.5 40 171 — 15 10.3 22 171 — 15 25 9 THE ENERGETICS OF PHOTOSYNTHESIS 77 As very small intensities of blue-green light provoke great photochemical activity, Warburg assumes an enzyme mechanism ; a proenzyme is reversibly changed into an enzyme with blue-green proenzyme \ ~ ' enzyme without blue-green The importance of cultivating Chlorella with fluctuating light has been pointed out in § 15. In § 20 we found that the maintenance of an optimal COo pressure is indispensable. The investigations on the effect of blue-green light show a further factor of importance for low quantum requirements. We have seen that good or poor yields can be obtained as desired by adding or removing blue-green light. This may also explain why great'y diff"ering values for the quantum requirement have been obtained in various institutes. Even when manometry and intensity measurements were correctly carried out, neglect of further important experimental conditions (cultivation, CO2 pressure, blue-green light) must have led to discrepancies. However, when all the conditions are met, constant low quantum requirements will be ob- tained at constant illumination in experiments lasting several hours (43). Figure 31 shows the results of an experiment lasting 5 hours where the quantum requirement was always about 3. In an experiment lasting 6 hours (Fig. 32) the quantum requirement was about 4. Warburg et al. (43, 63, 64) carried out 23 experiments lasting 6 hours with green + blue-green. They found an average quantum requirement of 4.07. One of these experiments gave the high value of 7.51 (see Addendum, page 186). The effect of blue-green light is determined by means of the two-vessel method (57). Each vessel is illuminated with red or green measured light and blue-green light. Thus, a total of four light beams are needed each of which is bolometrically measured. Figure 33 depicts schematically the optical equipment with the two ordinary mirrors Al and a semi-transparent mirror M' . The incident intensity of each of the red (or green) beams and of each of the blue-green beams must be exactly identical for both vessels. To begin with, both red (or green) beams are bolometrically adjusted to the same intensity by means of the diaphragms D, the cuvette C being filled with pure water. Afterwards, this cuvette is filled with a solution of HoPtClg which does not absorb red (or green) so that the intensity of the blue-green beams can be adjusted (63). Numerical example: Determination of the quantum requirement after 2 and 5 hours at constant illumina- tion, using the two-vessel method. Cell suspension: 0.5 ^1 cells/ml. A = 8.595 B = 11.05 C = -12.46 D = -18.00 Measured light: green. Incident intensity 24.8 ^1 quanta/min of which 3.3% is absorbed. Blue-green light: 4700 A. Incident intensity 0.95 ^1 quanta/min of which 20% is absorbed. 78 PROBLEMS IN PHOTOSYNTHESIS 'lf= 2.8 12 3 4 Duration of illumination in hours Fig. 31. Oxygen evolution under constant illumination for 5 hours with green measured light and additional blue-green light (Warburg et al., Angew. Chem.). 7«.= 3 59 12 3 4 Duration of illumination in hours Fig. 32. Oxygen evolution under constant illumination for 6 hours with green measured light and additional blue-green light (Warburg et al.. Angew. Chem.). X L, D- ^ X Lj M' zi C -D Fig. 33. Layout of the optical equipment for the produc- tion of two red and two blue-green light beams which are measured separately by means of the bolometer. Continuous line: red light. Dotted line: blue-green light. Li: 500 watt projection lamp. L-i: 300 watt xenon high pressure lamp. M: mirrors. M'- semi-transparent mirror. D: diaphragm. C: cuvette (Warburg et al., Zschr. Naturf.). to bolometer or manometer vessels THE ENERGETICS OF PHOTOSYNTHESIS 79 120 min lie;ht 180 min light 300 min light For the first two hours: For five hours: Small Vessel Pressure Changes + 9 mm + 12 mm Large Vessel Pressure Changes + 4 mm + 5. 5 mm + 21 mm xo, = +33.2 m1 xv.o^ = -40.0 ^1 xo, = +75.5 Ml -vco, = -90.5 ^1 + 9.5 mm To determine the quantum requirement, the number of quanta of blue-green hght absorbed must be multipHed by 0.5, as, according to Warburg and Negelein (61), chlorophyll absorbs only 50% of light with a wave-length of 4700 A (see also § 26). It must also be considered that the Oo consumption due to respiration of the cells at rest (100 /xl O., per 100 ^1 cells per hour) must be added to the calculated value of Aq.,. As each vessel contains 3.5 /xl cells, the respiration at rest is 3.5 ^1 O2 per hour. Thus, we find for the first two hours 120 X 24.8 X 0.033 + 0.5 X 120 X 0.95 X 0.2 \/ip = — — —^ ^^ = 2./2 33.2 + 2 X 3.5 and for five hours 300 X 24.8 X 0.033 + 0.5 X 300 X 0.95 X 0.2 . „, ^'"^ 75.5 + 5 X 3.5 If we had put in all instead of half the absorption of blue-green light, the quantum requirement would have been 3.00 and 3.25, respectively. Cell respiration is in reality greater than respiration at rest, so that in any case the quantum requirement is even lower than the figures obtained. § 31 The Nature of the Photosynthesis Enzyme Warburg (50, 56, 57) determined the catalytic action of eight blue-green wave-lengths. The measured light had the wave-length 6450 A. Figure 34 shows the action spectrum of the photosynthesis enzyme with its sharp maxi- mum at 4600 A. The absorption spectrum of a Chlorella suspension obtained under identical conditions, with the aid of the Ulbricht sphere (Fig. 35), is the absorption spectrum of all the pigments of living Chlorella and has a shape quite different to the action spectrum of the enzyme. The carotenoids and the flavins of Chlorella, like the enzyme, absorb mainly blue-green light, but the spectra of the carotenoids, xanthophylls and flavines extracted from Chlorella do not show the same picture as the action spectrum of the enzyme, especially with respect to the blue part. The unknown pigment acting as an enzyme must be a protein compound. It must be able to undergo a rapid chemical change upon illumination and to return rapidly to its initial state in the dark. Of the known pigments, only visual purple (rhodopsin) may 80 PROBLEMS IN PHOTOSYNTHESIS come into consideration. According to Wald (36) and Morton et al. (3), rhodopsin is a compound of vitamin-A-aldehyde (retinene) with a protein, i.e., a carotenoidoproteid. It fulfils the condition of a required rapid and reversible change upon illumination; this is its physiological function. The reaction consists in hydrogen transfer of the type alcohol ^^ aldehyde retinene reductase vitamin A ^- DPN+ , ~ > retinene + DPNH + H Retinene reductase is probably identical with alcohol dehydrogenase. Another carotenoidoproteid which may be of interest in this connection is 11 \ l( \ sis 10- / \ / \ / /^ \ \ > / \ / \ ^^ Fig. 34. Action spectrum of the photosynthesis enzyme (Warburg et al., Zschr. Naturf.). 4000 4200 4400 4600 4800 5000 6200 ' 1 10- ===i— V J" > V c::::; 0.6- \ \ V V Fig. 35. Absorption spectrum of Hving Chlorella (Warbui-g et at., Zschr. Naturf.). 4000 4200 4400 4600 4800 6000 5200 A ooverdin which has been studied by Kuhn et al. (32). Its prosthetic group is astaxanthine which has hydrogen-transferring properties. Astaxanthine occurs not only in lobster eggs but also in the green alga Hematococcus pluvialis. There is, however, no evidence that this proteid, like visual purple, reacts rapidly and reversibly upon illumination. The action of blue-green light is completely inhibited by traces of HCN (1/350000 A"). Thus, it may be assumed that the reaction caused by blue- green light is a heavy metal catalysis. It is well known that HCN reacts with complex-bound heavy metals (e.g., hemes) and with free heavy metal ions as well. In a-a'-phenanthroline we possess a substance which reacts with free heavy metal ions and loosely bound complexes, but not with com- THE ENERGETICS OF PHOTOSYNTHESIS plex-bound heavy metals. Phenanthroline (1/35000 N) at pH 5 inhibits photosynthesis of Chlorella to about 33%. The remaining 67% photo- synthesis reacts to blue-green light, as in the absence of phenanthroline. It must therefore be concluded that a heavy metal which is loosely bound or is in the state of free ions must be present, in addition to a complex-bound heavy metal. The former reacts with phenanthroline and the latter with HCN. The identity of the second metal has not yet been established, but there is some evidence that it may be vanadium. Warburg (40) observed that m-phenanthroline practically does not react with heavy metal complexes. It is about 200 times less active than o- phenanthroline. Thus, phenanthroline does not act as a narcotic but un- doubtedly influences heavy metal catalysis. § 32 The Significance of Vanadium The investigations of Bertrand (5) have shown that vanadium is found throughout the whole vegetable kingdom. In the animal kingdom it is rarely found; the blood cells of the tunicates contain vanadium instead of iron. Arnon (2) established that vanadium is indispensable to the growth of certain algae. It seems that the element has a specific action and that it cannot be replaced by other heavy metals. Arnon divides the trace elements into two groups. Group A contains iron, boron, manganese, zinc, copper and molybdenum. Group B comprises cobalt, nickel, chromium, tungsten and vanadium. The stock solution of group A has the following composition : 500 mg FeS04.7H,0 + 1000 mg Fe(N03)3'^H,,0 + 300 mg HBO3 + 200 mg MnS04.4HoO + 22 mg ZnS04.7H20 + 8 mg CUSO4.5H0O + 2 mg (NH4)eMo70o4 + 1000 ml 0.005 N H2SO4. Two ml of this stock solution are added to 250 ml of the culture salt solution (see § 15). It appears that of group B only vanadium is necessary for Chlorella cultures. Warburg et al. (54, 57) pointed out that the valency of vanadium is of importance. The blue vanadylsulfate (VOS04.2H.,0) with tetravalent vanadium is inactive, whereas pentavalent vanadium of the yellow sodium metavanadate (NaVOs. 4H2O) is very active. Growth with and without vanadium is identical during cultivation at high light intensities. At low light intensities, as used in measuring the quantum requirement (e.g., 50 ^A quanta/min, wave-length 5460 A), considerable differences have been found. Table 15 shows the results of two experiments. TABLE 15 The influence of vanadium, i = 54.3 jul quanta/min; 5460 A. Blue-green light: i = 0.6 ^1 quanta/min. Time: 30~60 min. Addition of vanadium: 100 /xg /I. 7 \/

1/3 G + V3 Oo In the light reaction one molecule O2 is produced per molecule chlorophyll with the quantum requirement 1 . In the dark, Vs of the O2 produced in the lioht are used for the induced respiration (reaction 2). The over-all reaction THE ENERGETICS OF PHOTOSYNTHESIS 85 shows that apparently 3 quanta are necessary for the reduction of one molecule CO2. In reality, however, only one quantum is involved, in accordance with Einstein's law. The substance obtained during illumination — indicated by C — is partly oxidized in the dark (2/3 according to reaction 2). Thus, the remaining gain (' '3) is not identical with the gain in the light reaction, but is equal to the difference between the gain during illumination and the con- sumption in the dark. The remaining gain cannot be more than \ '3 of the light gain, as the energy of one mole quanta in red is about 43000 cal, i.e., Vsof the energy requirement of photosynthesis (1 12000 cal'mole COo). A higher gain is thermodynami- cally impossible. When - 3 of the O2 produced are used for the induced res- piration — as shown in reaction 2 — the quantum requirement of the over-all reaction is 3. When '' '4 of the Oo react back the quantum requirement is 4. When ^^12 of the Oo are used for the induced respiration, the quantum re- quirement will have the high value of 12, a value Ehrmantraut and Rabino- witch (18) found in their experiments in alkaline medium. When all the O2 is used, the quantum requirement will be infinite. Daniels (33) in 1938, obtained values of nearly 500. These considerations show that the over-all reaction of photosynthesis depends upon relatively small changes in the back-reacting amounts of O2. The photochemical process and the back reaction mask one another. The slower the back reaction, the more apparent the light reaction. In the dark reaction the oxygen of the CO2 molecule is loosened in such a way that the action of one quantum is sufficient to break the bonds and to produce one molecule O2. The CO2 derivative with the loosened oxygen is probably a peroxide, indicated by (ChlGOa) *. Warburg calls it the photolyte of photosynthesis (43, 64). The Oo is developed from the photolyte, inactive CO2 combining with the chlorophyllproteid (reaction 1). Reaction 3 shows how the inactive COo is changed into the photolyte with the aid of the energy of the induced respiration. The formula of the photolyte is written as if it were a simple COo derivative of chlorophyll. This is certainly not the case, as the CO2 is probably bound to the protein part of the chlorophyllproteid. However, the formula correctly shows that the Oo developed from the photolyte under the influence of light is equivalent to the chlorophyll content of the cells. This stoichiometric relationship will be discussed in § 39. To avoid misunderstanding, it may be better to write the formula of the photolyte as CO2* no indication being given of the site of the COo in the chlorophyll- proteid. The process is represented as follows (see also § 60) Reaclion J (lighl) : COo* + Xhv + COo -> CO, + C + O2 Reactiuit 2 {dark) : V3 C + V3 Oo -^ 73 COo + 70000 cal 86 PROBLEMS IN PHOTOSYNTHESIS Reaction 3 (dark) CO2 -^ CO2* - 70000 cal Over-all reaction: Vs CO2 + Nhv -^ 'U C + \l-i O, The light energy can be transferred without notable loss from the chlorophyll molecule to the photolyte molecule. The light acts within the same molecule that absorbs it. The light reaction of photosynthesis is thus nothing but the photodissociation of a pigment. It is comparable to the photodissociation of iron carbonyl compounds (§ 25) [FejCO + Nhv ^ [Fe] + CO and the quantum requirement of 1 is almost self-evident. Numerical example (14): The one-quantum requirement of photosynthesis is studied by means of the two- vessel method. Each vessel contains 49 ^ul cells which absorb 48% of the incident green light (5460 A). The incident intensity is 44 /^l quanta/ min. Photosynthesis is measured with alternate light and dark periods of 1 .5 min. Sm all Vessel Large Vessel A' 1 _ 02 - 1. 321 mm2 A02 = 2 281 mm2 K 'C02 = = 1. 910 mm2 A'cOa = 2 870 mm2 Pressure Changes Pressure Changes 1 . 5 min light + 1.5 mm +0.5 mm 1 . 5 min dark — 2.0 mm -0.5 mm 1 . 5 min light + 2.0 mm mm 1 . 5 min dark — 2.0 mm -0.5 mm 1 . 5 min light + 1.5 mm +0.5 mm 1 . 5 min dark — 2.5 mm -0.5 mm 1 . 5 min light + 2.0 mm + 0.5 mm 1 . 5 min dark — 2.0 mm -0.5 mm 1 . 5 min light + 1.5 mm + 0.5 mm 1 . 5 min dark — 2.0 mm -0.5 mm 1 . 5 min light + 2.0 mm + 0,5 mm 1 . 5 min dark 2.0 mm -0.5 mm 9 min light + 10.5 mm + 2.5 mm 9 min dark — 12.5 mm -3.0 mm h' = +23.0 mm h = +5.5 mm From the values of the vessel constants, it follows that A = 10.10 mm2 B = 15.10 mm^ C = -13.21 mm-' D = -22.81 mm^ According to equations 17 and 18, we find ;co^ = h'A - hB = 23 X 10.10 - 5.5 X 15.10 = +149 m1 THE ENERGETICS OF PHOTOSYNTHESIS 87 and xco, = h'C - hD = -23 X 13.21 + 5.5 X 22.81 = -178 m1 The photosynthetic quotient is -178 7 = + 149 = -1.19 In 9 min the number of quanta absorbed is 9 X 0.48 X 44 = 190 /xl so that the quantum requirement is '^ 149 As can be seen in Table 16, the quantum requirement increases as the fre- quency of intermittency decreases. With increasing frequency, it decreases to a value of about 1 . TABLE 16 Time of intermittency and quantum requirement Incident Time of Duration Small Large Intensity Intermittency of Experi- Vessel Vessel fil quanta /min in min ment in min h ' mm h mm 7 Ih 44 1.5 18 23.0 5.5 -1.19 1.27 44 2.5 25 25.0 4.5 -1.19 1.4 44 5.0 30 16.5 6.0 -1.03 4.1 44 7.5 45 18.5 7.5 -0.97 5.3 24 5.0 30 17.5 6.5 -1.02 2.2 24 10.0 60 21.5 9.5 -0.90 4.6 § 36 Quantum Requirement and Photosynthetic Quotient Burk (9, 12) pointed out that the quantum requirement is low when the photosynthetic quotient is high. This fact has been confirmed by Warburg (44, 57). The quantum yield is good when 7 is —1.3 but poor when 7 is — 0.8. It may be assumed that CO2 is reduced in two steps: firstly, reductive carboxylation to the stage of formic acid COo + 2H + RCHO -> RCHOH-COOH and, secondly, reduction of the carboxylic group to the stage of a carbohydrate R-CHOHCOOH + 2H -> RCHOHCHO + HoO The photosynthetic quotients are for the first stage reduction 7 = —2.0 for the second stage reduction 7 = for the two reactions together 7 = —1.0 88 PROBLEMS IN PHOTOSYNTHESIS For all further reductions, e.g., from carbohydrate to protein, 7 = 0, so that 7 decreases when photosynthesis is accompanied by protein formation. It may well be imagined that some of the energy of the induced respiration is used for secondary metabolic processes. More Oo will react back when carbohydrate acts as a hydrogen acceptor (in the case of protein formation) than when CO., is the hydrogen acceptor. The photosynthetically produced O2 will then be decreased and l/(^ will increase. However, this explanation is not established. In any case, the quantum requirement measured is the correct quantum requirement of photosynthesis only when 7 lies between — 1 .0 and — 2.0, i.e., when growth need not be taken into consideration. For reliable photosynthetic measurements use must be made of cells which produce as litde protein as possible under the experimental conditions. Tolbert and Zill (34, 35) and Calvin et al. (15) showed in 'C-O. experiments that ChloreUa forms glycolic acid in photosynthesis (see § 57) 2COo + 2HoO -> CH.OH-COOH + IV2 O2 According to this reaction, 7 = - 1 .33. It may be concluded that in glycollic acid formation, i.e. when CO2 serves as the only hydrogen acceptor, the quan- tum requirement would have the best value. In the photosynthetic forma- tion of phosphoglyceric acid 3C0.2 + 3H,0 -> CH2OHCHOHCOOH + 2V2O2 the photosynthetic quotient is only — 1 .2. § 37 Splitting of Photosynthesis Into Light and Back Reactions Respiration and back reaction are closely connected processes. It is possible to separate them if respiration is compensated before the beginning of the experiment. Compensadng light must be of low intensity, so that manometric pressure changes will be zero. On additional illumination with green light, the positive pressure changes observed are due to the one-quantum reaction (light reaction) as well as to the back reaction. Negative pressures found after removal of the measured light must be ascribed to the back reaction only. If the quantum requirement for the complete cycle (light reaction + back reaction) is to be determined, the positive pressure changes on intermit- tent illumination at intervals of one minute are read every hour. However, if the quantum requirement of the light reaction alone is to be determined, the measured light is added at intermittent intervals of three minutes. The light reaction occurs almost without back reaction in the first minute of the cycle, but at the end of the third minute the back reaction is nearly complete. The true back reaction is observed after removal of the measured light and has nearly expired after a further period of three minutes. Thus, in the cycle of 6 minutes (3 minutes with and 3 minutes without measured light) it is possible to split photosynthesis into its components. Figure 36 shows the average values of five experiments (49). The quantum yield of the light reaction is 1 and that of the whole cycle 0.268. THE ENERGETICS OF PHOTOSYNTHESIS 89 y / \ y \ 235 V 30 \ X o^ 1 ^ Y^a 1.6 ^ r ^ ^ A ■^ 3 min Fig. 36. Splitting of photosynthesis into a Hght reaction and a back reaction. 22 \A cells per vessel. Respiration compensated. 20° C. Mean of five values, h: compensation light + measured green light (5460 A). /2.- compensation light without measured green light, g: gain. Tan a = 2.35,

1, the value for v is negative, according to equation 33. Then, substance would not be gained in the light reaction, but lost by photooxidation. It follows from this mathematical analysis that when / > 0.624 energy is spoiled. Either more energy-rich substance than necessary will be decomposed in the light reaction or too much energy will be lost as heat when energy-rich substance is built up in the back reaction. § 39 Stoichiometric Oxygen Production Chlorella brought from dark into light immediately develops O2 at constant velocity. However, when the alga is brought from its normal culture medium into a modified, more acid medium, containing" only MgS04, KH2PO4 and NaCl without the usual trace elements, the production of Oj is much higher in the first minutes of illumination than later in the steady state. This initial O2 production is stoichiometrically related to the quantity of illu- minated cells. Thus, the cells initially do not act as catalysers but merely as reaction partners. Warburg (59) calls the stoichiometrically removed O2 the O2 capacity of Chlorella. Depending on the chlorophyll content of the cells, 1 50 to 250 /xl O2 can be removed from 1 ml Chlorella cells. The O2 capacity of Chlorella is of the same magnitude as that of erythrocytes, i.e., about 300 /xl/ml. The difference between Chlorella and erythrocytes is that O2 is firmly bound in Chlorella (42000 cal are required per mole O2) but easily removed from oxyhemoglobin at low O2 partial pressures. To measure the O2 capacity, the cells are suspended in the culture medium mentioned above. Initially, they are illuminated for 30 min with blue-green light at low intensity so that the O2 capacity can be built up by means of the THE ENERGETICS OF PHOTOSYNTHESIS 93 energy of respiration. They are then illuminated with 35 to 60 lA quanta/ min red measured light for 10 min. Within 5 min the O2 capacity appears as a positive pressure. In the next 5 min the steady state of photosynthesis is attained. The much smaller pressure changes have, of course, to be de- ducted from the pressure changes observed during the first 5 min. Thus, the difference between the initial Oo production and the steady O2 production is the Oo capacity. The measurements are carried out with the two-vessel method. Numerical example (59): Small vessel: Vp = 7.0 ml Large vessel: IV = V.O ml A = 8.05 mm- Va = 9.88 ml Vg = 14.35 ml B = 10.24 mm2 C = -11.49 mm- D = -16.57 mm2 200 fil cells per vessel. Chlorophyll content: 3.2 mg = 3.56 /xmole = 79.5 /zl. Blue-green: /' = 1.2 jul quanta/min. Measured light (6440 A): / = 59.8 /xl quanta/min. Small Vessel Pressure Changes h' Large Vessel Pressure Changes h 30 2.5 2.5 5.0 5.0 min i' only min i' -\- i min i' + i min /' + i min i' + i -22.0 mm + 14.5 mm + 10.0 mm + 3.0 mm + 3.0 mm — 11.5 mm + 7.5 mm + 6.5 mm + 2.0 mm + 2.0 mm h' h = 14.5 + 10.0 - 3.0 = 7.5 + 6.5 - 2.0 = = +: + 12 mm mm According to equations 17 and 18, X02 = +48 ^A (O2 capacity A^coj = — 46 /il 7 = -0.96 O2 capacity _ 48 chlorophyll content 79.5 = 0.6 200 /il cells per vessel. Chlorophyll content: 1.17 mg = 1.305 /xmole = 29.2 /il Blue-green: i' = 1.2 yul quanta/min. Measured light (6440 A): /i = 37.5 and i-i = 59.5 ^1 quanta/min. Small Vessel Pressure Changes h' Large Vessel Pressure Changes h 30 min i' only 2. 5 min i' + i\ 2. 5 min i' + ix 5.0 min /' + /i 30 min /' only 2 . 5 min i ' + i-i 2 . 5 min i ' + / 2 5 . min i ' + i^ -10.0 mm + 7.0 mm + 1.0 mm — 3.5 mm — 56. mm + 10.0 mm + 3.5 mm + 1.5 mm — 6.0 mm + 4.0 mm + 0.5 mm — 1.5 mm — 30 . mm + 5.5 mm + 1.0 mm + 0.5 mm 94 PROBLEMS IN PHOTOSYNTHESIS It follows from the first three values that h' = 7.0 + 1.0 + 3.5 = 11.5 mm li = 4.0 + 0.5 + 1.5 = 6.0 mm and hence xo, = +29.6 jul -vro, = -30.0 /xl From the last three values it follows that 7 = -1.0 h' = 10.0 + 3.5 - 1.5 = 12.0 mm h = 5.5 + 1.0 - 0.5 = 6.0 mm and hence Xo, = +33.4 m1 .vco, = -36.0^1 7 = -1.08 Thus, the average value for the O2 capacity is 31 .5 /xl so that O2 capacity _ 31.5 chlorophyll content 29.2 = 1.08 In Example a the cells used contained 6.4% chlorophyll (200 //I cells = 50 mg dry weight) ; in Example b, where the ratio of O2 capacity and chlorophyll content was 1, Chlorella contained only 2.34% chlorophyll. It has been established that the ratio 1 is valid for a chlorophyll content up to 5%t of Ml O2 23.4 min- Eig. 37. Stoichiometric oxygen production (oxygen capacity) from Chlorella. Constant light intensity. End value: 23.4 ^ul = 1.045 iJ.mo\e O2. Chlorophyll content: 22.8 lA = 1.1018 Atmole (Warburg et a I., Naturw.). the cell dry weight. Figure 37 depicts the stoichiometric O2 production in the form of a graph. Oo production is initially rapid but becomes slower and slower and reaches its end value after 5 min. In this experiment the end value is 23.4 lA Oo. Thus, under the influence of light, 23.4 jA = 1.045 /xmole O2 have been produced by 100 jA cells. The chlorophyll content of the cells used is 1.018 /xmole. When the chlorophyll content of the cells is decreased to V2 or Vt — by in- creasing the light intensity during cultivation — O2 capacities decrease in the same proportion. Warburg (58) concludes that the O2 must originate from THE ENERGETICS OF PHOTOSYNTHESIS 95 a carrier substance, one molecule of which is stoichiomctrically linked to one chlorophyll molecule. This chlorophyll-carrier-substance can be denoted by (ChlCOo) * or COo* (see § 35) . § 40 Oxygen Capacity and Quantum Requirement The investigations on Oo capacity have resulted in chlorophyll being in- cluded in the reaction equation of photosynthesis. Thus, the action of chlorophyll is not only physical (absorption of radiation energy) but also chemical. This new concept of the chemical participation of chlorophyll partly rehabilitates the earlier views of Willstatter and Stoll (69). Warburg distinguishes between two kinds of chlorophyll molecule : those which are linked to the carrier substance (bound chlorophyll) and those which are not (free chlorophyll). Only light absorbed by the bound chlorophyll can be photochemically active. Light absorbed by the free chlorophyll is lost to any photochemical activity (62). Let the O2 capacity be A' and equal to the chlorophyll content of the cells. After t min the removed capacity x corresponds to the free chlorophyll. The remaining capacity present after t min is then A' — .v, which is equal to the bound chlorophyll. The incident intensity is i^ and the fraction absorbed a. V Thus, the free chlorophyll molecules absorb the fraction a— and the bound A chlorophyll molecules the fraction a ^— • In the time dt the total absorption is aiodt /xl quanta In the same time dt the Oo produced by the bound chlorophyll is ■ K — X , . ^ ocio Y^ — at Hi O2 A For the quantum requirement we have _ absorbed energy _ ai^dt '

glucose-6-phosphate A TP Mg ions phosphohexo- glucose-6-phosphate ^ ^ fructose-6-phosphate isomerase phosphofrnctokinase fructose-6-phosphate ^ * fructose-l,6-diphosphate A TP Mg ions The second step deals with the formation of Cs-molecules. It begins with the splitting of fructose-l,6-diphosphate into 3-phosphoglyceraldehyde and phosphodihydroxyacetone H2COP03H2 CO HOCH aldolase H2COPO3H2 CO CH20H phosphodihydroxy- acetone + HCO HCOH HCOH HCOH H2COPO3H2 fructose- 1 ,6-diphosphate H.COPOaHs 3-phospho- glyceraldehyde 109 no PROBLEMS OF PHOTOSYNTHESIS An equilibrium exists between the two triose phosphates CH20H CO triosephosphate > HCO HCOH 2COP03H2 ^ isomerase H2COPO,H.2 Due to the triosephosphate isomerase, about 96% of phosphodihydroxy- acetone is present at the equihbrium state so that the reaction practically pro- ceeds from right to left. However, another enzyme, phosphoglyceraldehyde dehydrogenase, drives the 3-phosphoglyceraldehyde so rapidly away that the reaction proceeds from left to right. Warburg and Christian (36), who dis- covered phosphoglyceraldehyde dehydrogenase, called this enzyme the "oxi- dizing fermentation enzyme." With DPN+ and in the presence of inorganic phosphate it catalyses the formation of 1,3-diphosphoglyceric acid, which is afterwards dephosphorylated to produce 3-phosphoglyceric acid. HCO OCO-POsH2 HCOH + H;iPO, + DPN+ ^ HCOH + DPNH + H + H2COPO3H2 H2COPO3H2 3-phospho- 1 ,3-diphospho- glyceraldehyde glyceric acid OCO--PO-,H2 COOH HCOH + ADP ^ HCOH + ATP H2COPO..H2 H2COPO3H2 1,3-diphospho- 3-phospho- glyceric acid glyceric acid In the first of these reactions an energy-rich phosphate bond is produced which, according to the second reaction, is accumulated in ATP. The active group of phosphoglyceraldehyde dehydrogenase has been considered by Racker (24) to be the sulfhydryl group of its glutathion part. However, re- cently, Warburg (38) confirmed the stoichiometric character of the aldehyde oxidation in the presence of phosphate, which he and Christian (36) first reported in 1939. The further oxidation of 3-phosphoglyceric acid is preceded by an intra- molecular transfer of the phosphate group due to the action of phospho- glyceromutase COOH COOH HCOH ^ HCOPO3H2 H2COPO3H2 H2COH 3-phospho- 2-phospho- glyceric acid glyceric acid THE CHEMISTRY OF PHOTOSYNTHESIS 11 In the presence of Mg ions, the enzyme enolase catalyses the conversion of 2-phosphogIyceric acid into phospho-^«o/-pyruvic acid COOH COOH I -HoO I HCOPOsH. ^ > CO~PO:,H.. I enolase II HoCOH CHo 2-phosphoglyceric phospho-enol- acid pyruvic acid The energy-rich phosphate bond produced is transferred by pyruvic phos- phokinase to ADP and pyruvic acid is formed. Mg ions and K ions are in- dispensable to this reaction. COOH COOH I pyruvic phosphokinase \ C0~P0.3Hi + ADP > CO + ATP II K ions Mg ions I CHo CH3 phospho-enol- pyruvic pyruvic acid acid In glycolysis there are two main reactions producing energy-rich phosphate bonds: 1. the conversion of 3-phosphoglyceraldehyde into 3-phosphoglyceric acid. 2. the conversion of 2-phosphoglyceric acid into pyruvic acid. All the reactions discussed are reversible. Under appropriate conditions both pyruvic acid and the intermediate products can be converted back to glucose. The only irreversible reaction is the formation of glucose-6-phos- phate from glucose. However, this reaction can be reversed by a special phos- phatase. Pyruvic acid is reduced to lactic acid by lactic acid dehydrogenase (War- burg's "reducing fermentation enzyme"). CH3.COCOOH -f DPNH + H+-> CHrCHOHCOOH + DPN + It is to be noted that DPNH produced during the oxidation of 3-phospho- glyceraldehyde is oxidized during the reduction of pyruvic acid. Thus, there exists an oxidoreduction reaction between 3-phosphoglyceraldehyde and py- ruvic acid. Glycolysis is therefore neither an oxidation nor a reduction re- action. This is shown by the over-all reaction of glycolysis C6H12O6 «=i 2 CsHeOs glucose lactic acid The whole reaction mechanism can be represented as follows: oxidizing fermen- 1. 3-phosphoglyceraldehyde + DPN+ + H:iP04 ^- — ^ tation enzyme 1,3-diphosphoglyceric acid + DPNH + H + 112 PROBLEMS OF PHOTOSYNTHESIS reducing fermen- 2. pyruvic acid + DPNH + H+ < ^ lactic acid + DPN + tation enzyme § 45 Oxidative Decarboxylation of Pyruvic Acid The opinion used to be held that the further oxidation of carbohydrates was continued immediately after glycolysis. The lactic acid produced was considered to be the primary substance for further breakdown. This is cer- tainly not the case. In muscle cells of some animals some of the lactic acid produced can be used for resynthesizing glycogen as all steps in glycolysis are reversible. However, further oxidation of lactic acid is out of the ques- tion. The primary substance for the continued breakdown of the carbo- hydrates is not lactic acid but pyruvic acid. When lactic acid is produced in cell metabolism, it must be dehydrogenated to pyruvic acid again before any further oxidation is possible. This dehydrogenation reaction is the reversal of the lactic acid dehydrogenase reaction discussed in the preceding paragraph COOH-CHOHCHs + DPN+ -> COOHCOCHs + DPNH + H + It follows from this that lactic acid production is a "blind alley" in the re- action series of carbohydrate breakdown. It only serves for the back oxi- dation of DPNH formed during the oxidation of 3-phosphoglyceraldehyde (see the reaction equation at the end of § 44). Under aerobic conditions, this back oxidation of DPNH is effected in quite a different manner, namely in the normal way via the respiratory chain. Pyruvic acid dehydrogenase catalyses the oxidative decarboxylation of py- ruvic acid CH3COCOOH + HoO + DPN+ <=± CH.3COOH + CO2 + DPNH + H + However, free acetic acid does not occur and, as a matter of fact, this re- action is far more complicated than would be supposed from the above equa- tion. Recent investigations have shown that at least five cofactors are indis- pensable. They are Mg ions, thiamine diphosphate (TPP+), DPN+, co- enzyme A (A — S— H) and lipoic acid.* Gunsalus (9) proposed the following four reactions 1 . Formation of a hypothetical intermediate product with TPP+ CH3COCOOH + TPP+^CH3-CO---TPP + H+ + COo Lipoic or thioctic acid has the formula CH, /\ , /^ H2C CH-(CH,)4-COOH abbreviated as L \i S S For Uterature on lipoic acid, see Reed (28). THE CHEMISTRY OF PHOTOSYNTHESIS 113 2. Forma tW7i of an acetyl-lipoic acid-complex CH3CO---TPP + L< I -> CH3CO— Sx + TPP + ^S >L 3. Transfer of the acetyl group to coenzyme A CH3-CO— S. + A— S— H -* CHsCO-'S— A + L^ _ /^ ace ty [coenzyme A SH 4. Oxidation of the reduced lipoic acid by DPN+ L DPNH + l/| Hence, the over-all reaction is CH3COCOOH + A— S— H + DPN+ > CH3-CO~S— A + CO2 + DPNH + H + According to Reed and De Busk (29), the true coenzyme of decarboxylation is lipothiamide, a compound of lipoic acid with TPP+. The reaction scheme of these investigators corresponds basically with that of Gunsalus. The pos- sible importance of lipoic acid in photosynthesis is discussed in § 55. Hence, the product obtained by oxidative decarboxylation of pyruvic acid is not free acetic acid but acetylcoenzyme A. It is the primary product which enters the tricarboxylic acid cycle where it reacts with oxaloacetic acid. It may be stressed here that Wood, Werkman, Hemingway and Nier (39) found direct carboxylation of pyruvic acid to oxaloacetic acid in bacteria and ani- mal tissues CH3COCOOH + CO>^ COOHCH2COCOOH pyruvic acid oxaloacetic acid It is possible that this reaction may help to supply the oxaloacetic acid neces- sary for the first step in the tricarboxylic acid cycle. § 46 The Tricarboxyb'c AcM Cycle The first step in the tricarboxylic acid cycle is the condensation of acetyl- coenzyme A with oxaloacetic acid to form citric acid (21). Many investi- gators contributed to our present knowledge of this most important cycle ac- cording to which the acetyl group of acetylcoenzyme A is oxidized to CO2 and H2O. Due to the work of Krebs (18), the cycle depicted in Figure 44 is well established and universally recognized. More recently, a dicarboxylic acid cycle has been discovered by Romberg et al. (15, 16, 17), in different microorganisms and mammalian tissues effecting the oxidation of glycolate or glycine via glyoxylate and formate to COo and H.O. The tricarboxylic acid cycle proceeds with the aid of enzymes localized in 114 PROBLEMS OF PHOTOSYNTHESIS hH,0 CH3-C0~S-A + H0OC-CH,-C0^CO0H oxaloacetic acid OH HOOC CHrC-CHrCOOH I COOH Citric acid //- HO0C-CH=C-CHrCOOH COOH cisaconitic acid -2H HOOC-CHOH-CH, COOH malic acid \\ H,0 HOOC-CH=CH-C0OH tumanc aciO + H,0 t 1 ..fl HOOC-CHOH-C-CHrCOOH HOOC-CHrCHrCOOH succinic acid COOH (so-citnc acid ^" HOOC-CO-CH,-CHrCOOH u-keloglulacic acil H HOOC-CO-C-CH,-COOH COOH oxalosuccinic aci Fig. 44. The tricarboxylic acid cycle. the mitochondria and their various cofactors. There are four oxidation steps hnked with the action of particular dehydrogenases. The substrates iso- citric acid, a-ketoglutaric acid, succinic acid and mahc acid are dehydrogen- ated, each giving 2 H atoms to appropriate hydrogen acceptors. The H atoms are finally oxidized to H ions. As depicted in Figure 44, one acetyl group of acetylcoenzyme A is oxidized in one cycle, 3 molecules H2O being used and 2 molecules COo and 8 H atoms being produced. This corresponds to the over-all reaction CH3-CO~S— A + 3 HoO -^ 2 COo + 8 H + H— S— A The molecule of coenzyme A which is produced reacts with another mole- cule of pyruvic acid to form a further molecule of acetylcoenzyme A. The first oxidation step is the conversion of /\yo-citric acid to oxalosuccinic acid catalysed by the iso-citric dehydrogenase. This reaction is followed by the decarboxylation to a-ketoglutaric acid. Thus, the over-all reaction rep- resents an oxidative decarboxylation. COOH • CHOH • CH • CHo • COOH + TPN+ Mn ions COOH COOH • CO CH.CHs- COOH + COo + TPNH + H + In the second oxidation step, a-ketoglutaric acid is converted to succinic acid by oxidative decarboxylation. According to Green (7. 8), Kaufman (14) and Sanadi (30) the following scheme with its three main reactions may be accepted : 1. Formation oj suainylcoenzyme A a-ketoEflutaric acid + A— S— H + DPN + succinvl'-S— A + CO2 + DPNH + H + THE CHEMISTRY OF PHOTOSYNTHESIS H5 This reaction corresponds to the oxidative decarboxylation of pyruvic acid (§ 45). The same 5 cofactors are indispensable. 2. Formation of succinic acid succinyl^S — A + phosphate + GDP -^ succinic acid + A^S — H + GTP Inorganic phosphate is used. The enzyme succinylcoenzyme A-ADP-phos- phorylase discovered by Hift et al. (10) needs guanosine diphosphate (GDP) as an additional cofactor (30). 3. Fort7iaiion of ATP Finally, a GTP-ADP-transphosphorylase transfers the phosphate group from GTP to ADP GTP + ADP ^ GDP + ATP The third oxidation step is the dehydrogenation of succinic acid cataly.sed by the succinic dehydrogenase. The H atoms are not accepted by either DPN+ or TPN + ; they are probably oxidized to H ions by cytochrome b. The enzyme fumarase catalyses the hydration of fumaric acid to malic acid. In the fourth and last oxidation step, malic acid is dehydrogenated to oxalo- acetic acid by malic dehydrogenase and DPN+. This reaction terminates the tricarboxylic acid cycle. Starting from one molecule acetylcoenzyme A, one molecule of oxaloacetic acid is regenerated and can condense with a further molecule of acetylcoenzyme A. The so-called malic enzyme which is closely related to malic dehydrogenase can convert malic acid into pyruvic acid with DPN+ or TPN+. Mn ions COOHCHo-CHOH COOH + TPN+ ^ — ^ CH;rCOGOOH + CO, + TPNH + H + Badin and Calvin (2) consider the reversal of this reaction, i.e., the reductive carboxylation of pyruvic acid, may play a role in photosynthesis (see § 58). The reaction discovered by Ochoa (20, 22, 23) is reversible; the presence o) Mn ions is essential. When it proceeds from right to left and the malic acid produced is converted by the malic dehydrogenase to oxaloacetic acid, the over-all reaction is identical with the Wood-Werkman reaction discussed in §45. pyruvic acid + CO, + TPNH + H+ ^ malic acid + TPN + malic acid + TPN+ -^ oxaloacetic acid + TPNH + H + pyruvic acid + CO 2 -^ oxaloacetic acid In various cells fixation of COo is a most important process.* It is mostly due to reductive carboxylation, i.e., the reversal of oxidative decarboxylation Other reactions of CO2 fixation are the Wood-Werkman reaction and the carboxylation of phospho-^;?o/-pyruvic acid with inosine diphosphate (IDP). For literature on CO2 fixation, see Utter and Wood (32) and Vishniac et al, (33). 116 PROBLEMS OF PHOTOSYNTHESIS The latter reaction discovered by Utter et al. (31) produces oxaloacetic acid COOH COOH CO'-POsH., + GO2 + ID? ^ CO + IT? CHs CH2 COOH It is possible that ADP and not IDP may be the phosphate acceptor. During the breakdown of pyruvic acid 10 H atoms are transferred to the respiratory chain. As the free energy decrease in oxidation of pyruvic acid is about 275000 cal/mole, about 55000 cal/mole are liberated per 2 H atoms. This amount of energy is accumulated in energy-rich phosphate bonds and it is assumed that 3 molecules ATP are produced per 2 H atoms. This oxida- tive phosphorylation is different to anaerobic phosphorylation in glycolysis, the latter being substrate phosphoryladon, the chemistry of which has been clearly defined (see § 44). In § 51 a third form of phosphorylation, due to the action of light, is discussed. § 47 The Pentose Phosphate Pathway In addition to glucose, various carbohydrates — tetroses, pentoses, hexoses, heptoses — have been found to occur in living matter. These may also be sources of energy or fulfil important biological functions ; thus, pentoses are parts of nucleotides. The existence of ways of carbohydrate breakdown other than glycolysis and the tricarboxylic acid cycle might therefore be suspected. In fact, in the most varied types of cell it has been found that glucose-6- phosphate can be oxidized in quite a different way. It had been noted that glucose breakdown continues in various cell extracts even when potent in- hibitors of glycolysis such as iodoacetate or NaF are added. Furthermore, enzymes had been discovered which are TPN-specific whereas the enzymes of glycolysis are mosdy DPN-specific. Warburg et al. {1)1) isolated in 1931 the enzyme glucose-6-phosphate de- hydrogenase from yeast. This enzyme, which they called the "Zwischen- ferment," catalyses, with TPN, the oxidation of glucose-6-phosphate to 6- phosphogluconate. An intermediate product formed is the 5-lactone of 6- phosphogluconic acid, the lactone ring of which is split by a lactonase (5). CHO OC— — I COOH HCOH HOCH HCOH HCOH -2H TPN+ HCOH HOCH O HCOH HC + H.0 H2COPO3H2 glucose-6- phosphate H2COPO3H2 8-lactone HCOH I HOCH HCOH HCOH H2COPO3H2 6-phospho- gluconic acid THE CHEMISTRY OF PHOTOSYNTHESIS 117 By means of 6-phosphogluconic acid dehydrogenase, TPN+ and TPP+, 6-phosphogluconic acid is converted by dehydrogenation and decarboxylation to ribulose-5-phosphate or xylulose-5-phosphate. This is changed into ri- bose-5-phosphate by phosphoriboisomerase. These reactions were also dis- covered by Warburg and Christian (35). According to Horecker (11), the reaction scheme can be written as follows (see also Dickens (6), Racker (25) and Cohen (4)) : COOH I HCOH I HOCH HCOH HCOH I H2COPO3H2 6-phospho- gluconic acid COOH HCOH -2H CO TPN+ HCOH HCOH H2COPO3H2 3-keto-6- phosphoglu- conic acid CHoOH CO, CO TPP+ HCOH *- HCOH I H2COPO3H2 ribulose- 5-phosphate CHO HCOH HCOH HCOH H2COPO3H2 ribose- 5-phosphate From the pentose phosphate produced two C atoms are oxidized to COo, leaving triose phosphate. As pentose phosphate is continuously regenerated, there is a cyclic process using hexose phosphate and producing triose phos- phate and CO2. These reactions do not involve the glycolytic pathway, so that the pentose phosphate pathway is often denoted as the hexose phosphate shunt. Two main reactions are responsible : transketolation and transaldola- tion. The enzyme transketolase (TK) transfers the ketol group ( — CO CH2OH) to aldehydes: CH2OH CH2OH CO I HOCH I HCOH I H2COPO3H2 xylulose- 5-phosphate CHO HCOH I + HCOH I HCOH I H2COPO3H2 ribose- 5-phosphate TK — > CO HOCH HCOH HCOH HCOH H2COPO3H2 sedoheptulose- 7-phosphate CHO + HCOH H2COPO3H2 phosphoglycer- aldehyde The enzyme thus splits xylulose-5-phosphate into a C3 fraction and a C2 frac- tion. However, the C2 fraction is not produced as free glycolaldehyde but reacts with ribose-5-phosphate to give the phosphate of a C7 sugar, sedoheptu- lose-7-phosphate. Transketolase is also involved in the following conversion 118 PROBLEMS OF PHOTOSYNTHESIS where the ketol group is transferred from ribulose-5-phosphate to erythrose- 4-phosphate to produce fructose-6-phosphate and phosphoglyceraldehyde : CH2OH CH20H CO CHO HCOH + HCOH HCOH HCOH CO HOCH CHO HCOH + HCOH HCOH H..COPO3H2 H2COPO3H2 fructose- 6-phosphate phosphoglycer- aldehyde TK — > H2COPO3H2 H2COPO3H2 ribuhse- erythrose- 5-phosphate 4-phosphate The transaldolase (TA) transfers the C3 fraction — CHOH . CO . CH2OH and therefore catalyses the formation of tetrose and hexose from heptose and triose (13, 26). The following reaction shows how this fraction of sedoheptu- lose-7-phosphate is transferred to phosphoglyceraldehyde to produce fructose- 6-phosphate: CH2OH CO HOCH CHO CHO CH2OH CO HCOH + HCOH HCOH H2COPO3H2 HCOH H2COPO3H2 sedoheptulose- 7-phosphate phosphoglycer- aldehyde TA HCOH + HOCH HCOH HCOH H2COPO3H2 HCOH H2COPO3H2 erythrose- 4-phosphate fructose- 6-phosphate Transketolase and transaldolase need TPP+ as a cofactor. It has now been found that transketolation and subsequent transaldolation result in an over-all reaction representing the above-mentioned oxidation of one mole- cule pentose phosphate to one molecule triose phosphate and two molecules CO2. Starting from one molecule glucose-6-phosphate as the primary sub- stance, this oxidation reaction produces one molecule triose phosphate and three molecules CO2. Figure 45 shows the reaction scheme as indicated by Racker (25). The ketol donor in the formation of sedoheptulose-7-phosphate is xylulose-5-phosphate and not ribulose-5-phosphate as was formerly as- sumed. An equilibrium state exists between both pentose phosphates; this is maintained by the enzyme epimerase (12, 27). If the fate of one molecule glucose-6-phosphate in the presence of one mole- cule ribose-5-phosphate is studied, it is seen that the first three C atoms of glu- cose-6-phosphate are oxidized to CO2, whereas the other three C atoms re- THE CHEMISTRY OF PHOTOSYNTHESIS 119 CO 03 .2S ' 0) o ^ — CO 3 O ■C Q. 0) "cO -C Q. CO O CO I 0) CO O o 05 CM o o o c o o _3 O)' o JI Q. CO O JZ D. CD o o CD i CO o Q- ■*-•(/}- O o 4r Q. CD ■ -C -♦-• n o. CO (D o sz -C o D. ■n 1 t^ CO 0) 0) CO CO x: O Q. CO - o o 3 Aci) 0} ■»-• CO JC Q. CO O .C Q. 00 I CO o O O CD 1 fO r CAJ O Q. fO u o 3 r- O) o. c o o CO o JZ Q. CD CO Fig. 45. The pentose phosphate pathway (Racker). main together in a molecule triose phosphate. In Figure 46 I represents the oxidative decarboxylation of glucose-6-phosphate to xylulose-5-phosphate as well as the condensation of the latter with ribose-5-phosphate. This is the transketolase reaction producing sedoheptulose-7-phosphate and triose phos- phate. In II we have the transaldolase reaction in which erythrose-4-phos- phate and fructose-6-phosphate are produced. The latter gives off COo by oxidative decarboxylation, leaving pentose phosphate. Finally, III shows 120 PROBLEMS OF PHOTOSYNTHESIS CO2 CO2 CO2 / / / Ci C, Ci C4 C2 C2 C3 C4 -I— I I I I I Ca C2 C3 Cs C3 C3 Ci Cs J. ^ TK I J. TA I r TK ' '^ C3 + C3 fc.Ci + C6 fc. Ci + C4 ►O2 + Cs III III C4 C4 C2 C« C5 C3 III I I Cs Cs C3 Cs C4 I ' ' A Ce C4 C6 C5 I Cs Fig. 46. The fate of the C atoms of gIucose-6-phosphate (Racker). the other transketolase reaction condensing pentose phosphate with erythrose- 4-phosphate and producing fructose-6-phosphate and triose phosphate. Oxidative decarboxylation of the fructose-6-phosphate produced again gives ribose-5-phosphate which can condense with a further molecule of xylulose- 5-phosphate derived from glucose-6-phosphate. Thus, we have a cyclic proc- ess in which ribose-5-phosphate acts as a catalyst, like oxaloacetic acid in the tricarboxylic acid cycle. In a comparison of the possible pathways of carbohydrate degradation the question arises as to which factors induce the cells to follow a particular path- way. It is probable that the quantitative distribution of the enzymes is of utmost importance. Glycolysis is nearly non-existent in various micro- organisms, but is predominant in muscle cells. Although the enzymes of the pentose phosphate pathway are present in yeast, they barely participate in carbohydrate breakdown, as the enzymes of glycolysis are present in much higher concentrations. In respiration of higher plants the pentose phosphate pathway may play a role since its enzymes have been found to be present. However, at present, no exact information on the importance of the pentose phosphate pathway is available (1, 3). REFERENCES 1. AxELROD, B. and Beevers, H.: Ann. Rev. Plant Physiol., 7:267, 1956. 2. Badin, E. J. and Calvin, M.: J. Amer. Chem. Soc, 72:5266, 1950. 3. Beevers, H. and Gibbs, M. : Plant Physiol., 29:322, 1954. 4. Cohen, S. S. : Other Pathways of Carbohydrate Metabolism. In Chemical Path- ways of Metabolism I, Academic Press, New York, 1954. 5. CoRi, O. and Lipmann, F.: J. Biol. Chem., 194:417, 1952. 6. Dickens, F. : Bnt. Med. Bull., 9:105, 1953. 7. Green, D. E. : Science, 7/5:661, 1952. 8. Green, D. E.: Phosphorus Metabolism, 7:330, 1951. 9. GuNSALUS, I. C: Fed. Proc, 73:715, 1954. 10. HiFT, H., OuELLET, L., LiTTLEFiELD, J. W. and Sanadi, D. R. : J. Biol. Chem.. 204:565, 1953. 11. HoRECKER, B. L.: J. Cell. Comp. Physiol., -^7:Suppl. I, 137, 1953. THE CHEMISTRY OF PHOTOSYNTHESIS 121 12. HoRECKER, B. L., HuRWiTz, J. and Smyrniotis, P. Z.: J. Amer. Chem. Soc, 78: 692, 1956. 13. HoRECKER, B. L. and Smyrniotis, P. Z.: J. Amer. Chem. Soc, 75:1009, 2021, 1953. 14. Kaufman, S.: Fed. Proc, 72:704, 1953. 15. KoRNBERG, H. L. and Beevers, H.: Nature, 780:35, 1957. 16. Kornberg, H. L. and Krebs, H. A.: Nature, 779:988, 1957. 17. Kornberg, H. L. and Sadler, J. R.: Nature, 785:153, 1960. 18. Krebs, H. A. : The Tricarboxylic Acid Cycle. In Cliemical Palliways of Metabo- lism I, Academic Press, New York, 1954. 19. Lepine, R.: Le diabete sucre, Bailliere, Paris, 1909. 20. OcHOA, S.: Pliysiol. Revs., 37:56, 1951. 21. OcHOA, S. et al.: J. Biol. Chem., 793:691, 703, 721, 1951. 22. OcHOA, S., Mehler, A. H. and Kornberg, A.: J. Biol. Cfiem., 774:919, 1948. 23. OcHOA, S., Veiga Salles, J. B. and Ortiz, P. ,T. : J. Biol. Chem., 787:863, 1950. 24. Racker, E.: J. Biol. Chem., 798:13\, 1952. 25. Racker, E. : Adv. EnzymoL, 75:\4\, 1954. 26. Racker, E., de la Haba, G. and Leder, I. G. : J. Amer. Chem. Soc., 75:1010, 1953. 27. Racker, E., Srere, P. A., Cooper, J. R. and Klybas, V. : Arch. Biochem. Bio- pTiys., 59:535, 1955. 28. Reed, L. J.: Adv. EnzymoL, 78:3X9, 1957. 29. Reed, L. J. and De Busk, B. G.: Fed. Proc, 73:113, 1954. 30. Sanadi, D. R., Gibson, D. M. and Ayengar, P.: Bwchim. Biop/iys. Acta, / 7:434, 1954. 31. Utter, M. F. et al.: J. Biol. C/iem., 207:181, 803, 821, 1954. 32. Utter, M. F. and Wood, H. G.: Adv. EnzymoL, 72:41, 1951. 33. VisHNiAC, W., HoRECKER, B. L. and Ochoa, S. : Adv. EnzymoL, 19:\, 1957. 34. Warburg, O.: Biochem. Zschr., 742:311, 1923. 35. Warburg, O. and Christian, W.: Biochem. Zschr., 292:281, 1937. 36. Warburg, O. and Christian, W. : Bwc/iem. Zschr., 307:221, 1939; 303:40, 132 (1939). 37. Warburg, O., Christian, W. and Griese, A.: Bwcfiem. Zschr., 282:151, 1935. 38. Warburg, O., Gawehn, K. and Geissler, A.-W.: Zschr. Naturf., 72b:Al, 1957. 39. Wood, H. G., Werkman, C. H., Hemingway, A. and Nier, A. O. : J. Biol. Cliem., 735:189 (1940); Proc Soc Exp. Biol. Med., 46:313, 1941. B. THE PROBLEM OF WATER PHOTOLYSIS § 48 Introductory Remarks Even the early investigations with isolated chloroplasts (15, 21, 39, 47, 74) showed O2 production upon illumination. The first experiments in vitro were of a qualitative nature only. Engelmann (21) demonstrated O2 pro- duction under the microscope utilizing certain bacteria which move to spots where traces of O2 occur. These bacteria accumulate around the isolated chloroplasts as long as these are illuminated. Beijerinck (15) and Molisch 122 PROBLEMS OF PHOTOSYNTHESIS (39) used bacteria which show luminescence in the presence of Oo traces, a technique that allows the observation of O2 production with the naked eye. At that time, there was no evidence of simultaneous CO2 fixation. It was assumed — without any proof — that Oo was produced by the photolysis of CO2. Later, views were modified. It was admitted that Oo was produced by the photolysis of water and not of COo. The comparative biochemical considerations of Kluyver (34; and van Niel (57), as well as Hill's investi- gations (24, 25) on Oo production of isolated chloroplasts in the absence of COo, provided considerable support for the new hypothesis. In 1946 Nicol (42) wrote that COo photolysis had been accepted as a well-established fact though it was not proven. Stressing this as a typical example of unscientific thinking, he proposed to accept the concept of water photolysis, though at that time the latter was, as it still is to-day, not proven either. We know now that COo photolysis is a fact (see § 49 and § 67). In the presence of HoS, certain green sulfur bacteria reduce, upon illumi- nation, COo to carbohydrate to produce .sulfur. light CO2 + 2H2S > (CH2O) + H2O + 2 S Other pigment-containing bacteria, the purple bacteria, produce sulfuric acid light 2 COo + HoS + 2 H2O ^ 2 (CHoO) + H2SO4 If the over-all reaction of photosynthesis of green plants is written as follows light CO2 + 2 H2O ^ (CH2O) + H2O + 0> a striking resemblance between these reactions is evident. Van Niel therefore proposed to represent photosynthesis by the following general equation light CO2 + 2 HoA > (CH2O) + HoQ + 2 A The compound H2A is a hydrogen donor; it is H2O in the green plant and H2S in the sulfur bacteria. Organic compounds may also act as hydrogen donors in bacterial photosynthesis, as has been shown by Foster (22) for wo-propanol light COo + 2 CHrCHGHCHa ^ (CHoQ) + HoQ + 2 CH3COCH, Comparative biochemistry was found by Kluyver and van Niel to be very useful in microbiology. Its application to photosynthesis does not prove that the Oo produced originates from HoO, as the sulfur originates from H2S, but does provide support for this view. Experiments with labeled O2 and COo are often quoted as evidence for these purely theoretical considerations.* When a plant has at its disposal * See Ruben et at. (48, 49, 50), Kamen and Barker (30), Dole and Jenks (20), Yoshida et at. (77), Winogradow and Teis (75). THE CHEMISTRY OF PHOTOSYNTHESIS 123 water with ^'^O and normal ClOo, the Oo produced contains '-O. By con- trast, when normal water and CIO2 labeled with ^^O are available to a plant normal O2 should be produced. Thus, lighi CO2 + 2 Ho'^O — '- — ^ (CH2O) + H2O + ^O, C"«Oo + 2 H2O light ■^ (CHsi^O) + Ho 1^0 + 0.> However, the experimental results show that neither reaction proceeds in this theoretical way. Neither 100%i''O2 nor 100% Oo has ever been found. A comparison of the work of various investigators revealed marked discrep- ancies. It may be said that 15 to 40% of the O2 produced seems to originate from GO2. This result is not surprising, if one bears in mind that it is not known whether CO2 or H2CO3 is the reaction partner in photosynthesis. It is very probable that H2CO3 is intermediately formed so that the following equilibria have to be taken into consideration H.i^O + CO2 ^ H.>C'sO:i ^ H,0 + C'^Oo According" to Warburg (60, 61, 67), Brown and Frenkel (17) and to van Niel himself (57), the experiments with '''O do not prove that the Oo produced in photosynthesis originates from H2O. Similarly, they do not prove the contrary view, so that they must be considered to be useless. LIGHT ^r DARK ~^ AHjO- 4E CO2 ■4H >4E'H >(CH50) + H20 + E' ► 40H 4H2O + 2A + 4E" BACTERIA 2H,02 + 4E" i 2H2O + OJ GREEN PLANTS Fig. 47. Van NieFs scheme. In Figure 47 van Niel's views are depicted schematically. The symbols E' and E" represent unknown factors which prevent the recombination of H and OH produced by the photolysis of water. The factor E' combines with H and transfers it to COo which is reduced to (CH2O). This is the process occurring in green plants and in photosynthetic bacteria. The fac- tor E" combines with OH and transfers it to the hydrogen donor H2A in bacterial photosynthesis. In green plants OH is transferred to special mechanisms which convert it to H2O and Oo. However, the intermediate for- 124 PROBLEMS OF PHOTOSYNTHESIS mation of H2O2, though accepted by other investigators, is still very doubtful. According to this scheme, light should decompose H2O to H and OH. Other investigators write water photolysis as follows light H2O ^2[H] + [O] where [H] and [O] do not represent molecular hydrogen and oxygen, but re- duced and oxidized hypothetical products. § 49 Hill Reactions Hill (24) was the first to observe that isolated chloroplasts are able to re- duce compounds other than GO2 upon illumination. He proposed the fol- lowing general reaction equation Ught 2 A + 2 H.O ^ 2 HoA + O2 where reducible substances serving as hydrogen acceptors are denoted by A. Thus, the chloroplasts possess a mechanism by which O2 is developed upon illumination and a hydrogen acceptor — but not CO2 — is reduced. Hill replaced the qualitative bacteria method by a quantitative method meas- uring the O2 production spectroscopically by means of the conversion of myo- globin to oxymyoglobin. In his first experiments Hill (24, 25, 27) used ferric oxalate as a hydrogen acceptor light 4 Fe»+ + 2 H2O — > 4 Fe-+ + 4 H+ + O2 However, the ferrous oxalate produced is autoxidizable and as ferric oxalate in light — without chloroplasts — is easily reduced and CO2 is produced, photochemical experiments with this salt lead to complications which Hill was able to avoid by adding potassium ferricyanide. Warburg and Krip- pahl (62) used potassium ferricyanide in their experiments. This salt is much more stable than ferric oxalate and its reduction product is not autoxi- dizable. The correctness of the iron reduction equation has also been con- firmed by Holt and French (28). The Hill reacdon can also be realized with quinone as a hydrogen ac- ceptor. Warburg and Luttgens (59, 64) studied the action of quinone on cell- free leaf extracts, intact and disintegrated chloroplasts and on living Chlorella light 2 quinone + 2 HoQ *■ 2 hydroquinone + O2 As no CO2 occurs in the Hill reaction. Hill concluded that the primary photo- chemical process must be the decomposition of water. However, this con- clusion was over-hasty as nothing was definitely known about the mechanism of this type of reaction. THE CHEMISTRY OF PHOTOSYNTHESIS 125 When Chlorella is suspended in an appropriate nitrate medium, and is illu- minated in the absence of CO2 it produces O2 for several hours, according to the following equation light NO3- + 2H2O ^ NH3 + OH- + 2 O2 This reaction, discovered by Warburg and Negelein (66) in 1920, must be considered to be the over-all reaction of a liffht and a dark reaction dark dark light NO3- + 2 C + 2 HiO -* NH3 + OH- -f 2 CO2 (a) 2 CO2 -^ 2 CO2* (b) 2 CO2* ^ 2 C + 2 O2 (c) over-all: NO3- + 2 HoO -* NH3 + OH" -f 2 O2 (d) In a dark reaction the NO3 ions oxidize C to CO2. After conversion to the loosely bound COo in the photolyte CO2*, this is split in the light. It was tentative to explain the reaction with iron salts in the same way. In 1955 Warburg and Krippahl (62) succeeded in distinguishing the following reactions with livina: Chlorella and ferric ions dark: 4 Fe^+ -1- C + 2 H2O -* 4 Fe2+ + 4 H+ + CO2 (a) dark: CO2 -* CO2* (b) light: CO2* ^ C + O2 (c) overall: 4 Fe3+ + 2 HoO -* 4 Fe2+ + 4 H+ + O2 (d) Thus, the Hill reaction can be considered to be an over-all reaction com- posed of dark reactions and a light reaction. The latter is identical with nor- mal photosynthesis. Schwartz (52) and Good (23) came to similar conclu- sions. It is to be noted that the iron reduction with living Chlorella is a rela- tively powerful reaction. In an experiment lasting 45 hours with 20 )ul cells (5 mg dry weight) to which 61 jumole = 20 mg K3Fe(CN)e have been added, 61 : 4 = 15 /xmole = 0.66 mg CO2, i.e., 13% of the dry weight of the cells are produced. The addition of the iron salt does practically no damage to the cells, and their normal reproduction is not impaired. As already mentioned, Hill used as a reagent ferric oxalate which, after reduction to ferrous oxalate, produces CO2 in the light. Thus, the pres- ence of CO2 was evident so that Hill's original experiments concerned nothing but true photosynthesis. In replacing ferric ions by quinone, we would expect the following reactions dark: 2 quinone + C + 2 H2O ^^ 2 hydroquinone + CO2 (a) dark: CO2 -> CO2* (b) light: CO2* ^ C + O2 (c) over-all: 2 quinone + 2 H2O — >■ 2 hydroquinone + O2 (d) A difficulty arises here insofar as quinone seems to inhibit reactions b and c. The addition of quinone and CO2 stops the production of O2 as soon as the quinone — not the CO2 — is used up. Thus, until now it was im.possible to 126 PROBLEMS OF PHOTOSYNTHESIS assume reactions b and c to be intermediate in the quinone reduction by Chlorella. Two kinds of reactions had to be accepted, one with the hght reaction c and one unknown hght reaction of another type. Such a con- clusion is of course far from satisfactory and very improbable (61, 67). Warburg and Krippahl (63) found that COo is indispensable to the quinone reaction in such small amounts that its necessity had been overlooked in hun- dreds of papers since 1944, but, nevertheless, in amounts still great enough to enable the COo pressures to be measured. However, in his study of the Hill reaction with quinone in 1948, Boyle (16) observed that the presence of very small amounts of CO2 was indispensable. Extracts of young spinach leaves were used. The gas phase contained commercial nitrogen only. In the presence of some KOH in the side-arm of the manometer vessel, traces of COo were removed with the result that Oo production ceased. With- out KOH, the traces of COo in the commercial nitrogen were sufficient to obtain considerable gas pressures due to Oo production. These findings could not be confirmed by either Clendenning and Gorham (19) or Hill (26) so that the necessity for intermediary CO2 in the quinone reaction was re- jected. Today, we know why Boyle's experiments could not be reproduced (63). Spinach grana are very rich in oxalic acid; on illumination this is oxidized to COo by quinone light quinone + oxalic acid -^ hydroquinone + 2CO2 In spite of KOH, illuminated spinach grana normally develop so much CO2 that sufficient CO2 still remains in the gas phase. Thus, spinach grana are certainly not an appropriate test object for proving the necessity of COo. It is highly probable that Boyle's extracts had, by chance, a low oxalic acid content. It is obvious that if extracts with a high oxalic acid content are employed, no difference can be expected, irrespective of whether KOH is used or not (63). The necessity of CO2 means that CO2 is an intermediate product in the quinone reaction as described in the above equations. It is not paradoxical that with the simultaneous addition of quinone and COo only quinone and not CO2 is used up in the over-all reaction. Quinone inhibits O2 respiration but only because it is replaced by "quinone respiration" which energetically permits reaction b. The above equations show that "quinone respiration" produces as much CO2 as disappears in light, so that the total COo turnover does not appear in the over-all reaction. Thus, reaction c ceases when all the quinone is completely reduced, as no more energy is available for reaction b. These conclusions are in good accord with the ferric cyanide experiments. In contrast to quinone, the iron salt does not inhibit respiration of Chlorella. When ferric cyanide and COo are added to Chlorella, the end value of O2 produced in light is equivalent to the sum of ferric cyanide and CO2, as, under THE CHEMISTRY OF PHOTOSYNTHESIS 127 these circumstances O2 respiration + "iron respiration'' produce the neces- sary energy for reaction b. With isolated chloroplasts which do not respire, the end value of the O2 produced is equivalent only to the amount of ferric cyanide, no matter how much CO2 the gas phase may contain. It is not the Hill reagent but the O2 respiration which decides whether CO2 disappears or not in the light re- action, i.e., whether sufficient energy is available for converting GO2 to CO2* in reaction b. Of primary importance for the understanding of the Hill reactions is res- piration — not normal respiration, but induced respiration. The Hill re- agents do not replace CO2, but they do replace molecular O2. The Hill re- actions must therefore be considered to be Oo-free respiration processes; this type of respiration, like O2 respiration, produces the necessary energy for the splitting of CO2 in the light (reactions b and c). These considerations lead to the firm conclusion that the hypothesis of water photolysis has to be abandoned. The explanation of the Hill reactions must be found in respi- ration processes which are directly and inseparably connected with the pho- tolysis of CO2. In photosynthesis and in the Hill reactions the light reaction is the splitting of CO2. In photosynthesis CO2 decreases in the over-all re- action, whereas in the Hill reactions CO2 remains constant and the Hill re- agents decrease. In Section D Warburg's conclusive experiments dealing with the action of quinone on living Chlorella, isolated chloroplasts and lyophilized cells will be further discussed. These experiments provide definite evidence that the hypothesis of water photolysis can no longer be maintained (63). This is embarrassing insofar as many important contributions to the chemistry of photosynthesis are based upon this hypothesis. Phenylurethane and ethylurethane inhibit true photosynthesis (65) as well as the Hill reactions (27). This was considered by Hill and Scarisbrick (26, 27) to be a very valuable argument in favor of the mechanism of O2 pro- duction in vitro being identical with that in true photosynthesis. According to Hill (24), the chloroplast reaction is not influenced by HCN. Phenanthroline inhibits the Hill reactions by about 60% (62). The addi- tion of Zn ions, which are bound by phenanthroline, cancels this inhibitory action (65). Schwartz (52) arrived at similar results with lyophilized cells. According to Arnon and Whatley (9), other metal ions exert this eflfect. Thus, phenanthroline (0.002 molar solution) specifically and reversibly in- hibits photosynthesis, O2 production of isolated chloroplasts with quinone, the ferric cyanide reaction with living Chlorella and the Oo capacity. Respiration is not inhibited by phenanthroline (see Table 18). § 50 Phosphorylations Ochoa and Vishniac (43, 44) found that, under appropriate conditions, illuminated isolated chloroplasts can reduce DPN+ and TPN+. These re- 128 PROBLEMS OF PHOTOSYNTHESIS actions were confirmed by Arnon (3), Tolmach (55) and San Pietro and Lang (51), although Jagendorf (29) using highly purified chloroplasts observed the Hill reaction with TPN+ only. The reactions proceed as follows light 2 DPN+(TPN+) + 2 H2O ^ 2 DPNH(TPNH) + 2 H+ + O. With the DPNH(TPNH) produced, reductive carboxylations may occur. In § 46 some examples of reductive carboxylations were discussed. As these are reacdons of CO2 fixation, it was reasonable to examine whether such re- actions were responsible for CO2 fixation in photosynthesis. Ochoa showed that illuminated chloroplasts are able in the presence of malic enzyme, TPN+, Mn ions and pyruvic acid, to produce appreciable amounts of malic acid. When a-ketoglutaric acid and iso-citric dehydrogenase were used, much z^o-citric acid was obtained. Malic enzyme occurs in the cytoplasm of plant cells but not in the chloroplasts; this may explain why isolated chloroplasts, which cannot catalyze reductive carboxylations, are not capable of exhibidng true photosynthesis. However, the question arises whether reductive car- boxylations are really the main reactions of COo fixation in photosynthesis. In sections C and D of this chapter other important reactions of COo fixation will be discussed. It is doubtful if the reduction of DPN+ (TPN+) must be considered to be a primary reaction. Although this has been postulated by Arnon in the case of TPN+, other authors, for reasons to be discussed later, assume that a pri- marily acting hydrogen acceptor has to be inserted. Calvin (18) considers this primary hydrogen acceptor to be lipoic acid but Wessels (70) supposes it to be vitamin K. If the primary hydrogen acceptor is indicated by X, the reduction of TPN+ is X + H.>0 -^ XH2 + V2 O2 XH2 + TPN+ -> X + TPNH + H + The reduction of phosphopyridine nucleotides in the Hill reaction links the latter with enzymatic processes and with the reaction of oxidative phosphoryl- ation found by Lehninger (37) O2 2 DPNH + 2 H+ + 6 ADP + 6 ph — > 2 DPN+ + 2 HoO + 6 ATP The enzymes responsible for oxidative phosphorylation are localized in the mitochondria so that a relationship between chloroplasts and mitochondria should exist. Vishniac and Ochoa (58) proved in their experiments with chloroplast-mitochondria systems that formation of ATP takes place with simultaneous oxidation of reduced phosphopyridine nucleotides by molecular O2. Investigations by Arnon (4, 6, 7, 10, 73), to be discussed in § 51, showed, however, that both intact chloroplasts and chloroplast fragments without any external enzyme systems — i.e., without mitochondria — are able to pro- duce ATP without being able to fix CO2. It is also to be noted that cells THE CHEMISTRY OF PHOTOSYNTHESIS 129 rich in chloroplasts are generally poor in mitochondria. Wassink (68, 69), Wintermans (76) and Kandler (31, 32) also observed the uptake of inorganic phosphate by illuminated chloroplasts. According to Wassink, it is possible that not ATP but another polyphosphate with a sulfhydryl group is produced. The existence of such a still unidentified organic phosphate ester is also claimed by Krall (35). Allen et al. (1) showed that the rate of ATP production by chloroplasts, anaerobically, upon illumination is far higher than observed with mito- chondria in oxidative phosphorylation. In § 57 it will be shown that, ac- cording to Kandler (33), the rate of phosphorylation in photosynthesis of whole cells is of the same magnitude as that of oxidative phosphorylation. It must be pointed out however that Ohmura (45) observed oxidative phosphorylation with chloroplast fragments in the dark: these fragments showed the Hill reaction in the light when intermediate products of the tri- carboxylic acid cycle were added. Thus, it is possible to prepare chloroplast fragments which show oxidative phosphorylation and photosynthetic phos- phorylation (see § 51). Ohmura concludes that the latter is nothing but a combination of water photolysis and oxidative phosphorylation. This as- sumption would accord with the views of Ochoa and Vishniac. § 51 Light Phosphorylation Arnon (4-14, 56, 71, 72, 73) called ATP formation by illuminated intact chloroplasts photosynthetic phosphorylation. With the aid of light energy and under appropriate conditions, chloroplasts can synthesize ATP light ADP + ph ^ ATP (a) According to Anderson and Fuller (2) and Newton and Kamen (41), iso- lated chromatophores of purple sulfur bacteria are also capable of light- dependent ATP formation. The most important difference between photosynthetic phosphorylation and oxidative phosphorylation is that the former does not need molecular O2. In earlier work, Arnon (4) was not able to establish the role of DPN + or TPN+ in light phosphorylation. Later, he found that TPN+ — but not DPN+ — acts as a catalyst, a further fact demonstrating the difference be- tween the two types of phosphorylation, DPN+ being indispensable to oxida- tive phosphorylation. Arnon's photoreduction of TPN+ is expressed as follows light 2 TPN+ + 2 H2O > 2 TPNH + 2 H+ + O2 (b) This reaction needs a TPN+-reducing factor which occurs in chloroplast extracts It was found that when 1 mole O2 is produced 2 mole TPN + are reduced and 2 mole inorganic phosphate are esterified, as the following equation shows 130 PROBLEMS OF PHOTOSYNTHESIS light ■* 2 ATP + 2 TPNH + 2 H+ + Oo 2 ADP + 2 ph + 2 TPN+ + 2 H.O This reaction, which is the over-all reaction of both the light reactions indi- cated above, shows that only a part of the light energy would be used for ATP production (conversion into phosphate bond energy) and the remaining part would be used for the reduction of TPN^. It is to be noted that in Lehninger's reaction of oxidative phosphorylation (§ 50) ATP formation is accompanied by oxidation of reduced phosphopyri- dine nucleotide. In Arnon's reaction ATP formation is accompanied by reduction of oxidized phosphopyridine nucleotide. However, Marre and Forti (38) found that reduction of TPN+ in illuminated chloroplasts is not dependent on the production or presence of ATP. Thus, light energy provides ATP and TPNH. Both substances form what Arnon calls the assimilatory power. He considers them to be the true "first products of photosynthesis," a term usually applied to products formed during CO2 fixation (see §53). More recently, Arnon (5) succeeded in combining his various findings into an interesting hypothesis of light phosphorylation. Photosynthetic phos- phorylation as shown in reaction a, in which ATP is the sole product of the light action, is designated as cyclic photophosphorylaiion. The process shown in the over-all reaction of a and b, in which light energy is used for the produc- tion of ATP and TPNH is termed non-cyclic photophosphorylation. This process, which provides Oo and assimilatory power for the reduction of COo, must be considered to be the light phase of photosynthesis in green plants. In cyclic photophosphorylation light energy is also converted into chemical energy, but it does not seem to be related to true photosynthesis in green plants as neither Oo production nor CO^ fixation is possible. This type of photophos- phorylation is peculiar to the photosynthetic bacteria. Their chromatophores cannot carry out non-cyclic photophosphorylation. They follow the vitamin K pathway of cyclic photophosphorylation (see Fig. 48). After absorption NON-CYCLIC ELECTRON TRANSPORT CO3 ASSIMILATION CYCLIC ELECTRON TRANSPORT REDUCTASE VIT.K -►TPN* I. FMN i ► TPNH q: ' ' LU o, H^ ^ \ DC H2O o CHL-4 ML ^ y t..X- CYT«^ CYT„ V^^ OH" LIGHT ph. ADP ► ATP CD < C02 -► (CH20) Fig. 48. Arnon's scheme for photosynthesis THE CHEMIS'IRV OF PHOTOSYNTHESIS 131 of light quanta, chlorophyll molecules are brought into the excited state and expel electrons which are transported in a cyclic system as follows: light 2Chl — > 2Chl* + 2e oxidized vitamin K + 2e > reduced vitamin K reduced vitamin K + 2Fe^+-cvti > oxidized vitamin K + 2Fc''+-cyti 2Fe2+-cyti + 2Chl* + ADP + ph > 2Fe3+-cyt, + 2Chl + ATP light ADP + ph > ATP A modification of the vitamin K pathway is the FMN pathway occurring in green plants. In this pathway the electrons also return to chlorophyll in a cyclic system. However, this cycle includes TPN+ and a second cytochrome cytn- Both pathways of cyclic electron transport as well as the non-cyclic pathway for the generation of assimilatory power are shown schematically in Figure 48. Arnon's general hypothesis of photosynthesis embracing green plants as well as photosynthetic bacteria does not necessitate water photolysis. In green plants the Oo production results from an "open" non-cyclic electron transport. The electrons expelled from the excited chlorophyll molecules do not return to chlorophyll, as in the cyclic pathways, but are removed by TPN+, together with H ions originating from water in the presence of a light-dependent reductase. TPN+ + 2e + H+ > TPNH The electrons removed must be continuously replenished. This is made possible by the interaction of the remaining" OH ions from water and a cyto- chrome (cytii) peculiar to the green plant and not found in photosynthetic bacteria. Thus, the role of water is to produce H ions for TPNH formation and OH ions to yield molecular Oo and donate electrons to a cytochrome chain. According to this hypothesis water is still the source of O2 though the process of photolysis is not involved : 2H2O > O2 + 4H+ + 4e In bacterial photosynthesis external hydrogen donors are still needed. However, their function is not the reduction of a precursor of O2 but merely the production of H to reduce phosphopyridine nucleotides (PN), i.e. produce one of the components of assimilatory power. The other component of as- similatory power is ATP which photosynthetic bacteria are able to produce with light energy by cyclic photophosphorylation. Figure 49 shows, ac- cording to Arnon, the reaction schemes of photosynthesis in green plants and photosynthetic bacteria. Warburg (59) discovered that CI ions are essential for photosynthesis in green plants. Arnon's experiments confirm this finding, CI ions being re- quired for reaction c as well as for the FMN pathway. They are not re- 132 PROBLEMS OF PHOTOSYNTHESIS quired for the vitamin K pathway which is the only type of bacterial photo- synthesis. Thus, in the absence of CI ions chloroplasts behave like chromato- phores in being able only to follow the vitamin K pathway. GREEN PLANTS PHOTOSYNTHETIC BACTERIA H X o CYCLIC PHOSPHORYLATION: ADP + ph ►ATP NON-CYCLIC PHOSPHORYLATION; 2 TPN- + 2 H2O + 2 ADP + 2 ph ► 2TPNH + 2H +O2 + 2 ATP CYCLIC PHOSPHORYLATION: ADP + ph ►ATP < ASSIMILATION OF CO2: C02 + 2TPNH + 2H'+ nATP ► (CH2O) + H2O + 2TPN"+ n ADP + n ph REDUCTION OF PN : 2 PN' + 2 H2 ► 2 PN H + 2 H' ASSIMILATION OF CO2: CO2 + 2 PNH + 2 H^+ nATP ► (CH20) + H20 + 2PN^+nADP + nph _i _J < a: LU > O CD + H n '-'^^^ ^ /pi_i ni i D 1 H n CO2 + 2 H2 ""^"^ (CH2O) + H2O Fig. 49. Similarities and difierences in photosynthesis of green plants and bacteria (Arnon) Arnon (5, 13) considers that the Hill reactions are non-physiological vari- ants of the over-all reaction, e.g.: 4Fe3+ + 2H2O + 2ADP + 2ph > 4Fe2+ + O2 + 2ATP + 4H + TPN+ does not participate. It is replaced by the Hill reagents, e.g. ferric ions. This non-physiological non-cyclic photophosphorylation also requires CI ions. According to Arnon's recent investigations, the reduction of TPN"*" does not result from water photolysis. In his earlier work it was the mysteri- ous product [H] which elicited the reduction of TPN+. Today he claims that H ions from water, together with electrons expelled from excited chloro- phyll molecules are sufficient for this reduction in the presence of a light- dependent reductase. Thus, water is split into H ions and OH ions, the latter being converted into Oo. In photosynthetic bacteria the hydrogen donor which is still needed does not reduce COo, as had been claimed by van Niel, but gives off hydrogen to reduce phosphopyridine nucleotides. In terms of comparative biochemistry, the common denominator of photosynthesis in green plants and in bacteria is, according to Arnon (5), the conversion of light energy absorbed by chlorophyll into phosphate bond energy. It is not the photochemical formation of a COo reductant. Thomas et al. (54) succeeded in preparing chloroplast fragments of Spiro- gyra which show evolution of Oo as well as uptake of CO2 without the addi- THE CHEMISTRY OF PHOTOSYNTHESIS 133 tion of enzymes or cofactors to the suspension. The rates of both reactions were about the same as those observed in intact cells. These findings are in contrast to those of Arnon, who stated that the addition of certain enzymes and cofactors is indispensable for obtaining" photosynthetic phosphorylation and CO2 uptake in isolated chloroplasts and chloroplast fragments. Thomas et al. explain the discrepancy as follows. Arnon used spinach chloroplasts which are of the granulated type, whereas Spirogyra chloroplasts are lamellated, spiral-shaped chloroplasts that are grana-free. In the latter thin layers of stroma occur between bundles of lamellae (see § 3). This layered structure may be capable of protecting the stroma against damage far more effectively than may be possible in granulated chloroplasts. The lamellae are highly resistant to experimental conditions (crushing of the cells), whereas the granu- lated chloroplasts are easily damaged. The presence of intact stroma seems to be necessary if complete photosynthesis is to be obtained. Either the stroma contains important enzymes and cofactors or it protects enzymes lo- cated on the surface of the lamellae. According to Arnon, the light reaction in photosynthesis only serves for the production of ATP and TPNH, these substances being necessary for the dark CO2 fixation reaction : dark CO2 + 2 TPNH + 2 H+ + 2 ATP ^ (CH.O) + H2O + 2 ADP + 2 ph Arnon (5, 8, 56) found that the assimilatory power, i.e., ATP and TPNH, generated in the light in the absence of COo in the green water-insoluble parts of the chloroplasts (grana) is able to carry out CO2 fixation in the dark in chlorophyll-free extracts (stroma) obtained by suitable centrifugation of the chloroplasts. These extracts contain the assimilatory power produced in the previous light reaction together with soluble enzymes. Small amounts of glucose- 1 -phosphate and ^^C02 were added. The dark fixation of '^COo by the chlorophyll-free extract to which assimilatory power was added was of the same order of magnitude as CO2 fixation in the light in the presence of ^^G02 since the commencement of illumination. The products of CO2 fixa- tion were identical in both cases, as was shown by paper chromatography and autoradiography. Instead of using ATP and TPNH generated by a light reaction, both substances were supplied from external, chemical or enzymati- cal sources. They were as effective in producing CO2 fixation as the assimi- latory power generated by illuminated chloroplasts. Thus, Arnon found a physical separation of the light and the dark phase through the identification of ATP and TPNH produced in the light and the presence of CO2 fixing enzymes in aqueous extracts of chloroplasts. The light phase is localized in the grana and the dark phase in the stroma (5, 8, 56). However, the necessity of the TPN+-reducing factor shows that, in the light, some stroma factors must be involved. It follows from these im- portant investigations that CO2 fixation of isolated chloroplasts must be con- sidered to be a process independent of the photosynthetic pigment system. 134 PROBLEMS OF PHOTOSYNTHESIS It does not matter whether assimilatory power is produced by the action of Hght or is suppHed to the system from outside sources. In vivo, however, photochemical generation of assimilatory power remains closely connected with COo fixation. More recently, Krogmann (36) provided evidence of a third type of light- dependent ATP synthesis — a type associated with the oxidation of the dye trichlorophenol indophenol. Incubation of spinach chloroplasts with this dye in its oxidized form, phosphate and ADP results in ATP synthesis only in the presence of light and Oo. The oxidized dye is reduced by the action of light and reoxidized by O.,. When the photoreduction of the dye is in- hibited by certain chemicals (e.g. dichlorophenyl dimethylurea), no reoxida- tion by O2 is possible and ATP synthesis stops. Upon addition of gluta- thione, cysteine or ascorbic acid, the dye can be reduced. This chemical reduction cannot be inhibited by dichlorophenyl dimethylurea. Light- dependent reoxidation does occur but, under these circumstances, light must have another site of action, as it cannot participate in a photoreduction process. Nakamoto et al. (40) described the eff"ect of O2 on other chloroplastic photophosphorylation systems. All these experiments clearly show how complex phosphorylation must be in photosynthesis and support the finding that photosynthetic phosphorylation may also be of the oxidative type. In § 69 we shall further examine the problem of light induced phosphoryla- tion in connection with Warburg's findings concerning quinone catalysis. According to Warburg's experiments light induced phosphorylation does not exist. It is nothing but oxidative phosphorylation in the dark. § 52 Some Final Remarks In this section as well as in Section C of Chapter 4 the hypothesis of water photolysis forms the basis of many tentative approaches to the chemistry of photosynthesis. Recent investigations of Warburg have shown that the mechanism of the Hill reaction can be brought into accord with his findings in living ChloreUa. The action of ferric ions and of quinone must be con- sidered to be the same in isolated chloroplasts as in living ChloreUa. The photodecomposition of water can thus be replaced by the photodecomposition of the photolyte COo*. As a matter of fact, there is no evidence whatsoever that light quanta of low energy can decompose water (46), though efforts have been made to render this acceptable with the aid of excited states of chlorophyll, free radicals, etc. It seems extremely abnormal that water, which plays a most important but nevertheless more or less passive role in liv- ing Nature, should exert such a highly "explosive" action in photosynthesis. It must be admitted that nothing is impossible in Nature; nevertheless, it is hardly permissible to accept this adage as the only evidence of reactions we would like to see proceeding becauss they fit in well with our hypotheses. Part of the O2 of the photolyte certainly originates from water, due to the intermediary formation of H2CO3. The li igin cl the photosynthetically pro- THE CHEMISIRY OF PHOTOSYNTHESIS 135 duced O2 is of no importance to those in favor of CO2 photolysis. However, for those who accept water photolysis, all the Oo produced must of necessity originate from water. Comparative biochemistry contributed greatly to the general acceptance of water photolysis. However, the question arises whether it would not be possible to apply CO2 photolysis to the photosynthetic sulfur bacteria. Light: CO2* ^ C + O, Dark: 2 HoS + Oo ^ 2 H.O + 2 S Dark: CO. -> CO2* Over-all: 2 H2S + CO2 -> C + 2 H2O + 2 S In the green plant a part of Oo is visibly produced. The remaining part is used in the induced respiration providing additional energy. In the anaero- bic sulfur bacteria it is, of course, impossible that the Oo produced is freely developed. It must be used entirely for a reaction, specific and indispensable to this organism, i.e., the oxidation of HoS. A comparison of this reaction with that of induced respiration in plant photosynthesis shows some similarity insofar as both reactions provide additional energy for the conversion of CO2 to CO2*. It is evident that these considerations are pure speculation. There is no proof whatever that the reactions indicated above would proceed in photosynthetic sulfur bacteria. The question is whether these reactions are less probable than those discussed in § 48 and whether experimental evidence instead of theoretical considerations will one day bring enlightenment. It is doubtful whether comparative biochemistry, i.e. the thesis of unity in biochemistry, can serve as a reliable guide in all the fields of biochemical research. In any case, Cohen (19a), Stanier (53) and others warn against exaggerations and, thus, question the general validity of this thesis. Never- theless, Bassham and Calvin (14) still take it for granted that Ruben's isotope studies and van Niel's suggestion clearly indicate that the photosynthetic O2 has its ultimate origin in the O atoms of H2O. In Section D of this chapter the important role which must be imputed to COo in the chlorophyll-polypeptide is discussed. It is becoming increasingly apparent that the photosynthetic process centers around C-Oo. COo is not merely a reaction partner but must be considered to be the primary substance. In the green plant, CO2 is loosened in the photolyte so that it can be split by the relatively weak light energy. The energy needed for the formation of the photolyte is provided by the induced respiration. It is very probable that a peroxide may be intermediately produced and that phosphorylations take place (§ 69). It must be borne in mind that phosphorylation always occurs in respiration or, in other words, no phosphorylation, no respiration. The converse, no respiration, no phosphorylation, seems incorrect as Arnon found phosphoryl- ation upon illumination independent of O2. Arnon does not take into con- sideration the induced respiration which, being respiration, must be con- 136 PROBLEMS OF PHOTOSYNTHESIS nected with phosphorylation. He considers back reactions unlikely since chloroplasts would not be able to carry out oxidative phosphorylation when supplied with O2 in the dark with appropriate substrates and TPNH. It has already been pointed out that there is no photosynthesis without respira- tion. As there is no respiration without phosphorylation, we logically arrive at the conclusion that there is no photosynthesis without phosphorylation, i.e., oxidative phosphorylation. As a matter of fact, the phosphate turnover in photosynthesis is still a sub- ject of many contradictory investigations. As yet, anaerobic phosphoryla- tion is the only kind of phosphoryladon the chemistry of which is known (see the reaction with the oxidizing fermentation enzyme discovered by Warburg and Christian discussed in § 44) . This is still not the case with oxidative phos- phorylation and it is far less the case with phosphorylations proceeding upon illumination. The hypothesis of ATP formation by means of light energy may be an interesting oudet, but, for the time being, it belongs to the same category as water photolysis which was at the time an equally interesting out- let for which no evidence was available. On the other hand there is evidence that reduction of TPN+ takes place under the influence of light. However, we may readily admit here the same mechanism as found in the Hill reaction with ferric ions. Dark- 2 TPN+ + C + 2 H.O -^ 2 TPNH + 2 H+ + CO2 Dark: CO2 - CO.3* Ught: CO.,* - C + O2 Over-all- 2 TPN+ + 2 H2O -^ 2 TPNH -f 2 H+ + O2 Traces of reactive C may be sufficient to drive the reaction. In the over-all reaction only TPN+ determines the amount of O2 produced. Until now this mechanism has not been proved. Parts of Arnon's work are of importance, though the reduction of TPN + with simultaneous formation of ATP is rather unconventional. It does not seem impossible to bring Arnon's findings into accord with Warburg's theory. 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Willstatter, R. and Stoll, A. : Untersucfmngen iiber die Assimilation der Kohlen- sdure. Springer, Berlin, 1918. THE CHEMISTRY OF PHOTOSYNTHESIS 139 75. WiNOGRADOw, A. p. and Teis, R. V. : C. R. Acad. Sci. URSS, 33:490, 1941 ; 56-59 1947. 76. WiNTERM.ANS, .1. F. G. M.: Thesis, Wageningen, 1955. 77. YosHiD.^, T. et al.: Acta Phytochim. {Japan), /J:13, 1942. C. INTERMEDIATE PRODUCTS OF PHOTOSYNTHESIS § 53 The Carbon Cycle of Photosynthesis To establish the intermediate products of photosynthesis, Calvin et al. (8, 9, 10, 19, 22) carried out a series of basic experiments with labeled CO2, opening up new interesting ways of research. The results of similar experi- ments have been published by Fager et al. (24). The test object Chlorella is illuminated during 10 to 60 sec in an atmosphere of i^CO.. Then, the alga is killed in hot 80% alcohol and the alcoholic extract examined by means of paper chromatography and autoradiography. Even the first experiments showed that the first products to appear were carbohydrates; and after longer illumination the labeled carbon was also found in fats, amino-acids and pro- teins. As will be discussed later, other investigators found different se- quences, e.g., amino-acids before sugars. By steadily decreasing the time of illumination, Calvin tried to trap the first CO2 fixadon product to appear; this was considered to be 3-phosphoglyceric acid. The further reduction of this substance to sugars was represented as follows: H2COPO3H, HCOH *COOH 3-phosphogly- ceric acid + 2H — ■ — ■ — ■ *■ dehydrogenases H2COPO3H0 HCOH *CHO 3-phosphogly- ceraldehyde triose phosphate isomerase H2COPO3H2 CO H2COPO3H2 I HCOH *CHO 3-phosphogly- ceraldehyde H2COPO3H 3^.2 + CO *CH20H phosphodihy- droxyacetone aldolase >■ *CH20H phosphodihy- droxyacetone H2*COP03Ho I CO HOCH I HCOH HCOH H2*COP03H2 fructose-l ,6- diphosphate The condensation of two molecules triose phosphate is the reversal of the anaerobic degradation of fructose-l, 6-diphosphate (§ 44). The distribution of the radioactive isotope *C among the C atoms of 3-phosphoglyceric acid and of fructose-l, 6-diphosphate shows that the CHO group of 3-phospho- 140 PROBLEMS OF PHOTOSYNTHESIS glyceraldehyde joins with the CHoOH group of phosphodihydroxyacetone. Therefore, the labeled C atoms in fructose-l,6-diphosphate should be sym- metrically distributed (see § 57). Further research (18) showed, however, that ribulose-1, 5-diphosphate was a precursor of 3-phosphoglyceric acid. This pentose diphosphate is car- boxylated, producing 3-phosphoglyceric acid. H2COP03H2 COH COH + CO, HCOH H2COPO3H2 H2COPO3H2 HCOH 1 H.O COOH ) 1 COOH HCOH H2COPO3H2 ribulose-1 ,5- diphosphate {enol-form) 3-phospho- glyceric acid A labile ketoacid is intermediately produced (40). With this carboxylation reaction proceeding much faster than the carboxylation reactions discussed in § 46, the cycle represented in Figure 50 is started. The ribulose-1, 5-diphos- phate is regenerated, according to the Horecker cycle, thus enabling renewed uptake of COo. Ribulose-1, 5-diphosphate is produced from ribulose-5-phosphate. Calvin assumed that one molecule 3-phosphoglyceraldehyde and one molecule sedo- heptulose-7-phosphate are converted to two molecules ribose-5-phosphate. However, more recent investigations (7) have shown that in this conversion only one molecule ribose-5-phosphate and one molecule xylulose-5-phosphate are produced instead of two molecules ribose-5-phosphate. By the action of phosphoketopentose epimerase, xylulose-5-phosphate is changed into ri- bulose-5-phosphate. In the conversion of fructose- 1,6-diphosphate + 3- phosphoglyceraldehyde to erythrose-4-phosphate + ribulose-5-phosphate, xylulose-5-phosphate occurs as an intermediate (see Fig. 50). The enzymes indicated in Figure 50 are needed for the various reactions of the carbon cycle. Their presence in cell-free extracts of green plants has been determined by Racker (47). When DPN+, ATP, labeled bicarbonate and small amounts of ribose-5-phosphate are added to such extracts, paper chromatography shows, after some time, the presence of labeled sugars and sugar phosphates. The carbon balance of Calvin's cycle can be written as follows : 3 A TP 3 C5 + 3 CO2 > 6 3-phosphoglyceric acid 12 H 6 3-pho?phogivceric acid > 6 Cn 6 ATP THE CHEMISTRY OF PHOTOSYNTHESIS 141 D- o s:. Ifl (X (U 03 o "D >, s:. O :^ ^ ^ H CO O LTl p3 U u 3 o o H o bb tn "3 -a ex i3 S 2 -^ O en en o i; .S .2 o i-H c; „ a CO O a m tn ii bJD 00 o v; t^ OJ 4J 6 o en (-■ M OJ CX o en CL O ^ Oh H CN r^ T3 >. O U 142 PROBLEMS OF PHOTOSYNTHESIS 2 C3 —*■ Ce Ce + 2 C3 -^ C5 + C7 Cy -\- C3 —*■ 2 C5 9 ATP 12 H + 3 CO2 ^ C3 + 3 HoO According to Figure 50, the following reactions must also be considered Ce + C3 — »- C4 + C5 C4 -|- C3 — *■ C7 § 54 Is Photosynthesis the Reversal of Respiration? The main pathway of respiration is constituted by the sequence glycoly- sis, tricarboxylic acid cycle and biological oxidation. Ochoa (43) assumed that in photosynthesis the tricarboxylic acid cycle may proceed in the oppo- site direction. The reductive carboxylation of a-ketoglutaric acid to iso- citric acid, which is assumed to occur in photosynthesis, is indeed the rever- sal of the oxidative decarboxylation of /50-citric acid to a-ketoglutaric acid occurring in respiration. The same could be said for the reductive carboxyl- ation of pyruvic acid and the oxidative decarboxylation of malic acid (44, 45), all the reactions of the tricarboxylic acid cycle being reversible. With the exception of the conversion of glucose to glucose-6-phosphate, all the re- actions of glycolysis are also reversible. Ochoa and Vishniac (46) reduced 3-phosphoglyceric acid to fructose-1,6- diphosphate upon illumination in vivo. This process goes as follows: light 1. 2 DPN+ + 2 H2O ^ 2 DPNH + 2 H+ + O2 Mg ions 2. 2 3-phosphoglyceric acid + 2 ATP ^ * 2 1,3-diphosphoglyceric acid + 2 ADP 3. 2 1,3-diphosphoglyceric acid + 2 DPNH + 2H+ ^ 2 3-phosphoglyceraldehyde + 2 DPN+ + 2 ph 4. 2 3-phosphoglyceraldehyde ;i^ fructose-l,6-diphosphate In these experiments the chloroplasts were suspended in a solution of 3- phosphoglyceric acid containing ATP, DPN+, Mg ions and the necessary enzymes. Only reaction 1 is photochemical. No fructose- 1,6-diphosphate was obtained without illumination. The over-all reaction light Mg ions DPN+ 2 H2O + 2 3-phosphoglyceric acid + 2 ATP ^ fructose-l,6-diphosphate + 2 ADP + 2 ph + O2 is the reversal of the degradation reaction of hexose phosphate to triose phos- phate, in which reaction ATP is produced and DPN+ reduced (see § 44). It was assumed that in the photochemical reaction light energy produces water photolysis. ATP serves as an additional energy source. The investigations with labeled CO2 carried out by Calvin (§ 53), Kandler THE CHEMISTRY OF PHOTOSYNTHESIS 143 (§ 57) and Warburg (§61) show, however, that citric acid and other inter- mediary products of the tricarboxyhc acid cycle do not belong to the pri- mary products of CO2 fixation. Moyse and Jolchine (29, 41, 42) observed that in certain leaves the tricarboxylic acid cycle proceeds in the light as freely as in the dark. Besides the carboxylation of ribulo.se- 1,5-diphosphate proposed by Calvin, they found irreversible carboxylation of phospho-enol- pyruvic acid to produce oxaloacetic acid which is then reduced to mahc acid (6, 55) (see §46). COOH CO'^POsHa + CO2 CHo COOH H.O ^ CO + H3P04 phospho-enol- pyruvic acid COOH I CO CH2 I COOH oxaloacetic acid -\- 2H *■ CH2 COOH oxaloacetic acid COOH CHOH CH2 COOH malic acid With the use of ^^C02, the production of labeled malic acid was established. Moreover, labeled citric acid, wo-citric acid and fumaric acid as well as amino acids were found. These findings are in contrast to Calvin's views regarding the blocking of the tricarboxylic acid cycle by the photoreduction of lipoic acid (see § 55). Warburg, also, does not admit photosynthesis to be the simple reversal of respiration. There is no definite evidence that the occurrence of the products of the carbon cycle cannot be due to the induced respiration. In Figure 51 some relationships between the carbon cycle and respiration are depicted schematically. § 55 The Significance of Lipoic Acid When lipoic acid is added to a system of illuininated algae and quinone, the rate of O2 production is considerably increased (15). It seems as if lipoic acid is intercalated between the light activation reaction and the quinone reduction. Calvin (20) originally assumed that lipoic acid acts as both a hydrogen acceptor and an oxygen acceptor 2[H]+ [0] + L<'l / SH SOH 144 PROBLEMS OF PHOTOSYNTHESIS a "(3 O o + O CO — CO >, »- -Q O 3: rN O 0) CO ■ o o D3 CO CO I CO CO -C Q. CO n rs < -C <4 Q. rf + + O ^ ^ r Q. iw. Q. W 0) CO o X 0) CO O 3: 1 (D 1- 1 1 o o Q. CO o Q. CO O CO O > Q. 4J IS +-> >^ D, 1) -M O a; C a T3 c (^ c o u V o J3 U C o S-. CJ ■M o c V J2 '-J-J bb THE CHEMISTRY OF PHOTOSYNTHESIS 145 Later, Calvin (19) regarded lipoic acid as a hydrogen acceptor only /S /SH 2[H] + L< I ^L< \S \SH The electron transfer of the process is best represented by the following re- actions HoO ^ 2 H+ + 2 e + \/. 0-, (a) {AG = +37500 cal) /S /SH L/ I + 2H+ + 2e^L< (b) \S \SH (AG = +13800 cal) Calvin and Barltrop (20) assume that lipoic acid reacts with chlorophyll as follows S S L<{\ + Chi* -^ h(^ ' + Chi \s \s. The biradical produced accepts, according to Bickel and Kooymans (12), the H atoms of a hydrogen donor HD ,^S. /SH L< + 2 HD -> L< + 2 D. \S. \SH 2 D. ^ D> It is evident that another compound must serve as the oxygen acceptor, i.e., the acceptor of electrons from O2. According to these views, illumination creates an excited state of chlorophyll (oxidation) and the energy of this state (electron energy) must be converted to chemical energy by means of the acceptor. Levitt (36) postulates the formation of the oxidized chlorophyll as follows : light Chl-Mg — ^ Chl-Mg+ + e Thus, the acceptor molecule removes an electron from the chlorophyll so that the remaining positive chlorophyll molecule can accept another electron from another source. This is the process represented in Figure 52. Herisset (28), comparing chlorophyll and Mg-free pheophytin, considers the Mg atom should play an important role in photosynthesis. In discussing the oxidative decarboxylation of pyruvic acid (§ 45) we learnt that lipoic acid acts as an indispensable cofactor. Lipoic acid is considered to be a part of the pyruvic acid dehydrogenase, so that it is supposed that this enzyme also plays an important role in photosynthesis. Calvin (18) ob- served that upon illumination practically no labeled carbon is found in inter- mediate products of the tricarboxylic acid cycle, in contrast to what happens 146 PROBLEMS OF PHOTOSYNTHESIS V 2e + 2H" + H202 Y 1 /SH \SH 1 2H2O Fig. 52. Electron transfer of excited chlorophyll. photosynthetic carbon cycle pyruvic acid Fig. 53. The status of lipoic acid. Acetyl CoA I tricarboxylic acid cycle in the dark. Thus, it seems that the action of light blocks the link between the photosynthetic carbon cycle and the tricarboxylic acid cycle. The photo- reduction of lipoic acid would strongly inhibit the conversion of pyruvic acid to acetylcoenzyme A. Figure 53 shows the position of lipoic acid, schemati- cally. This means that respiration proceeding in the dark through the tri- carboxylic acid cycle — a process producing CO2 — would be diverted in light to proceed through another cycle — the carbon cycle — showing uptake of CO2. According to Steward and Thomp.son (49), it is physiologically im- probable that the tricarboxylic acid cycle is suppressed in the light. First of all, there is no proof that respiration proceeds only through this cycle. As there is no photosynthesis without respiration (see § 42), it is impossible to admit that green cells in the light do not respire. Secondly, Steward and Thompson assume that the tricarboxylic acid cycle may well proceed in the light with substrates other than those used in the dark, and utilize carbon sources drawn from amino-acids (e.g., glutamic acid) and synthesize pro- teins. THE CHEMISTRY OF PHOTOSYNTHESIS 147 Chlorophyll Fig. 54. The chlorophyll aggregate as a semiconductor. According to a further hypothesis, chlorophyll acts photochemically not as a single molecule but as an organized, orientated aggregate. The lamellae structure of the grana discussed in § 3 support the view that the chlorophyll aggregate can be considered to be a kind of photoelectric battery and the absorbed light energy its driving force. On one side of the aggregate a fat or lipoproteid layer and on the other side a hydrophilic layer act as "elec- trodes." The former would be composed of the lipoid soluble lipoic acid and the latter would be water (Fig. 54). Commoner's experiments discussed in § 9 lead to the assumption that the chloroplast acts as a semiconductor. Katz (35) and Calvin (9) compare the chlorophyll aggregate to a semiconductor. As in the transistor (Fig. 6), the electrons set free in the chlorophyll layer migrate to the lipoic acid layer where they are accepted by lipoic acid. The "holes" produced are occupied by electrons originating from water (Fig. 54). As sufficient water is present, no back reaction can take place. The lipoic acid molecules are continuously regenerated so that the electrons produced are continuously accepted. According to Levitt (36), the chlorophyll excited by one quantum would give only one electron to lipoic acid and, after absorption of a second quantum, a second electron. Bearing this in mind, we can imagine what would happen at the "electrodes" of the chlorophyll aggregate. By the absorption of one quantum about 42000 cal/mole would be taken up. Assuming a loss of 5000 cal/mole, only 37000 cal/mole of the light energy would be available. Con- sidering the relationship between potential and change of free energy AG = -23074 nE (44) we have for n = 1 a potential difference between the aforementioned layers of the chlorophyll aggregate ("electrodes") of 1.6 volt. It is now possible to determine the potential of each of the "electrodes." According to reaction b, the increase of free energy during the transfer of one electron to lipoic acid is about 7000 cal/mole, corresponding to a potential of 0.3 volt. The remain- ing energy is 37000 — 7000 = 30000 cal/mole, corresponding to a potential of 1.3 volt. Thus, the potential difference of 1.6 volt is distributed over the photoelectric battery in such a way that the potential is 0.3 volt on the nega- tive side (lipoic acid side) and 1.3 volt on the positive side (water side). 148 PROBLEMS OF PHOTOSYNTHESIS + + O t OJ (f)-(D T. X CO (f) X Sr /••-Q o o (X)- ■ — \ CO / o o > Q. ■a -I-' o 3 c _o Si C 03 (U C o c o o a; C >- c XJ Q. 15 C _o in LTl The following reaction is supposed to proceed on the positive side 2 H.O -^ H2O2 + 2 H+ + 2 e In fact, this reaction corresponds to a potential of 1 .2 volt. The H2O2 pro- duced may react with cytochrome f 2Fe-'+ + HoOo -^ 2 Yc'+ + HoO + 0-- The Calvin scheme and its connections with respiration are shown in Figure 55. Calvin's views are intriguing but they are based upon quite a number of pure hypotheses needing further experimental evidence. THE CHEMISTRY OF PHOTOSYNTHESIS 149 The significance of lipoic acid as the primary hydrogen acceptor still re- mains doubtful. Lipoic acid occurs in chloroplasts in low concentrations only. The chlorophyll concentration is about 1000 times that of lipoic acid, so that the chlorophyll aggregate should be composed of at least 1000 chloro- phyll molecules. Arnon et al. (2, 4) and Geller (25) point out that water photolysis with isolated chloroplasts is not influenced by inhibitors of sulf- hydryl groups (e.g., arsenite), findings which are certainly not in favor of the lipoic acid hypothesis. Biswas and Sen (13) incubated cells o{ Scenedesmus and Chlorella with labeled NaHCOa and various concentrations of lipoic acid. They observed practically no stimulation of ClOo uptake upon illumination. By contrast, with concentrations higher than 0.25 mg/ml, a marked inhibi- tory effect of lipoic acid upon CG-y uptake was noted. Similar experiments with simultaneous addition of quinone showed a slight increase of CO2 up- take with low concentrations of lipoic acid. Paper chromatography revealed that, with low concentrations of lipoic acid, intermediate products of the tricarboxylic acid cycle and related amino-acids were more radioactive, and sugars and sugar phosphates less radioactive than without lipoic acid. Ac- cording to Calvin, if lipoic acid were reduced to its thiol form, a greater in- corporation of '^C in sugars and sugar phosphates would be expected. It follows from these investigations that it is very doubtful whether lipoic acid is of significance in photosynthesis. Reed (48) arrives at the same conclu- sion. S 5^ The Significance of Vitamin K Wessels and Havinga (59, 60, 61) examined the Hill reaction with various quinones and dyes as hydrogen acceptors. They found that the reduction power of these substances is strongly dependent upon the redox potential. This is not astonishing", as the redox potential of a system is a measure of its reducing or oxidizing power. It was found that reduction does not occur with quinone derivatives and dyes having a redox potential lower than + 0.040 volt. It would therefore be questionable whether DPN+ and TPN+, which have a redox potential of —0.300 volt, could serve as a Hill reagent. Wessels and Havinga suggest vitamin Ki instead of lipoic acid as the pri- mary hydrogen acceptor, i.e., reduced vitamin Ki as the primary hydrogen donor to TPN+. The redox potential of vitamin K] is about —0.030 volt, whereas that of lipoic acid is about —0.300 volt. For a better understand- ing of the energetic situation, the various reactions are indicated below, the equations a and b of § 55 being repeated HoO^ 2 H+ + 2e 4- V2O. (a) (AG = +37500 cal) yS xSH L< I + 2H+ + 2.^ L< (b) (AG = +13800 cal) 150 PROBLEMS OF PHOTOSYNTHESIS or in the over-all reaction ,S /SH L<^ I + H2O -^ L< + V2 O2 (c) S \SH (AG = +51300 cal) In the case of vitamin Ki HoO ^2H+ + 2e + 1/2 O. (a) (AG = +37500 cal) vitamin Ki + 2H+ + 2e -»► reduced vitamin Ki (d) (AG = +1500 cal) or in the over-all reaction vitamin Ki + HoO -^ reduced vitamin Ki + V2 O2 (e) (AG = +39000 cal) For reaction b (lipoic acid) the redox potential is, according to equation 44, 13800 „ „„^ , E = — , . , ■ „ = -0.300 volt 46148 and for reaction d (vitamin Kj) we find 1500 „„.^ , ^^ -46148= -0-032 volt It follows from these figures, which are not standard values, that, according to the findings of Wessels and Havinga, vitamin Ki should have the preference over lipoic acid. The latter has the same low potential as TPN+ and could therefore not function as the primary hydrogen acceptor. Accepting the requirement of one quantum per two electrons in reactions c and e, we find for the efficiencies in red ,. . .^ 43000 o,^ hpoicacid: -^y^ = ^^/o . . ., 43000 ...^ vitamm Ki: ^qqoO = ^^^/o Of course, an efficiency of more than 1 00% is impossible as has been pointed out by Bassham and Calvin (9). However, Wessels thinks that the reduc- tion of TPN+ by reduced vitamin Ki proceeds with the aid of energy-rich phosphate bonds produced in a partially back oxidation of reduced vitamin Ki. The denominator will therefore be higher, and give more reasonable figures. Figure 57 shows the various conversions in the vitamin Ki cycle. Generally speaking, there are some arguments in favor of the importance of this vitamin, as can be seen from its role in oxidative phosphorylation and its possible function in the respiratory chain. It is a striking fact that vitamin Ki occurs in chloroplasts only and is not present in the cytoplasm. Anti- vitamin-K substances inhibit Hill reactions. The light-sensitive vitamin Ki is protected by CI ions. As Warburg and Luttgens (56) and Arnon and What- THE CHEMISTRY OF PHOTOSYNTHESIS 151 Fig. 56. Vitamin Ki (2-methyI-3-phytyl-l,4- naphthaquinone) . E O ij 3 U V en JJ o c > be Q. 152 PROBLEMS OF PHOTOSYNTHESIS ley (3) have shown, CI ions exert a protective action upon chloroplasts in vitro. The fact that the molecules of vitamin K, and chlorophyll possess a phytol chain (Fig. 56) may also be of interest. Both substances probably occur in the monomolecular layers between the lipoid and the protein-water lamellae of the grana. In spinach leaves the vitamin Ki content amounts to about 0.04 mg per g dry weight, so that the ratio chlorophyll/vitamin Ki is of the magnitude 200. The ratio chlorophyll lipoic acid is about 1000. According to Marre and Servettaz (38), the reduction of TPN+ does not follow that of a compound with a more positive redox potential, such as vita- min K or FMN {E = -0.06). They found a distinct effect of TPN+ on the reduction of cytochrome c (or f) in illuminated chloroplasts and on the rate of photosynthetic phosphoryladon, indicating that reduction of TPN + precedes and conditions the production of ATP. This is in agreement with the findings of Marre and Forti (37) mentioned in § 51. Thus, it seems that the role of TPNH in photosynthesis would be not only that of a reducing agent of some product of CO2 fixation, but also that of an electron carrier in photo- synthetic phosphorylation, as Arnon (4) postulated. § 57 Some Other Investigations According to Calvin's hypothesis, the essential reduction process in photo- synthesis is the reduction of 3-phosphoglyceric acid TPNH A TP 3-phosphoglyceric acid 7 ^ triose phosphate This reaction is the reversal of the oxidation of triose phosphate discussed in § 44. According to Bassham et al. (11), the concentration of 3-phosphogly- ceric acid in the light is about 30% lower and that of triose phosphate about 70% higher than in the dark. Nevertheless, it is assumed that in the light the reaction distinctly proceeds from left to right. Kandler (30) points out that this can only be possible when the concentrations of the necessary cofac- tors are changed accordingly and when ATP is present in particularly high concentrations. We learnt in § 53 that 3 mole ATP would be used per mole CO2 in photosynthesis. Thus, Calvin postulates a stoichiometric relation- ship between CO2 reduction and ATP turnover. In respiration, 6 mole ATP are produced per mole CO2 (3 mol ATP per V2 mole O. used). When res- piration of Chlorella is overcompensated 40-fold, 40 mole COo are reduced in photosynthesis against one mole CO2 produced in respiration. This means that the ATP turnover is increased 20-fold. However, the investigations of Kandler (30), Strehler (50) and Bradley (14) clearly show that the ATP content of the cells does not increase upon illumination. A stoichiometric relationship between CO2 reduction and ATP turnover is therefore improb- able. These results make the conversion of 3-phosphogIyceric acid to triose phosphate questionable. THE CHEMISTRY OF PHOTOSYNTHESIS 153 The symmetric distribution of radioactivity in fructose- 1,6-diphosphate is due to the condensation of two similar trioses. Investigations by Kandler and Gibbs (27, 34) show a distinct asymmetrical distribution as can be seen from the following reaction scheme : H2COPO3H2 H2COPO:iH2 H2COPO3H2 COH |l COH HCOH H2COPO3H2 ribi/lose-7,5- diphosphate COH + *C02 ^ COH HOO*C— COH light hama- light *■ melonic — ^ TPNH ? acid H2COPO3H2 labile intermediate compound CO -HOCH + O2 1 H*COH I HCOH H2COPO3H2 fructose-7,6- diphosphate The C4 atom of fructose-l,6-diphosphate shows the highest radioactivity. The labile intermediate compound — a carboxylation product of ribulose-1,5- diphosphate (cf. Calvin's intermediate ketoacid) — is reduced in the light, and a polyhydroxyacid, probably hamamelonic acid, is produced as an inter- mediate (31, 32). It may be that, instead of the carboxyl group of the la- bile compound, a peroxide group is formed being able to split off O2 exo- thermically. This splitting would be associated with its reduction to alde- hyde. C / O c / o + o, ^O— OH ^H It is to be noted that according to these views Oo is produced from COo. Kandler suggested that Warburg's labile CO2 would thus more or less find its place in this reaction scheme, a somewhat bold assumption indeed (see § 59). Furthermore, Kandler assumes that the labile intermediate compound may partially produce 3-phosphoglyceric acid providing the cells with other important constituents such as amino-acids. This is represented in Figure 58. By means of transaldolation and transketolation, a part of the fructose- 1,6-diphosphate produced is converted to ribulose-l,5-diphosphate again, so that Kandler's cycle may be written as follows light 6 ribulose-l,5-diphosphate + 6 CO2 *■ 6-fructose-l,6-diphosphate + 6 O2 dark 6 fructose-l,6-diphosphate + ATP glucose + 6 ribulose-l,5-diphosphate + ADP + ph 6 CO2 + ATP -»► glucose + ADP + ph + 6 O2 According to the over-all reaction, the minimal requirement of ATP is only 1 mole per 6 mole CO2. The mole ATP used in the cycle compensates 154 PROBLEMS OF PHOTOSYNTHESIS SUCROSE STARCH RIBULOSE-1,5- _ DIPHOSPHATE "*" -CO, LABILE » INTERMEDIARY COMPOUND FRUCTOSE-1,6- ■ DIPHOSPHATE HAMAMELONIC ACID OXALOACETIC ACID ^CO, 3-PHOSPHOGLYCERIC ACID PHOSPHO-ENOL- PYRUVIC ACID ASPARAGINIC ACID MALIC ACID ALANINE TRICARBOXYLIC ACID CYCLE Fig. 58. Kandler's scheme. Separation of photosynthesis and respiration. for the loss of inorganic phosphate due to the conversion of fructose- 1,6-di- phosphate to saccharides. The ATP turnover in the hght at 40-fold over- compensation is thus not increased 20-fold but remains practically constant. In the light, the labile intermediate compound is reduced, via hamamelonic acid, to fructose-l,6-diphosphate when sufficient reducing power is avail- able. If not, the labile compound is slowly split to 3-phosphoglyceric acid (respiration). Kandler compares this clear separation of photosynthesis and respiration with Warburg's induced respiration. It is difficult however to find anything common to the mechanisms. Thus, according to Kandler, the production of 3-phosphogiyceric acid from ribulose-l,5-diphosphate is a step in the direction not of photosynthesis, but of respiration. The decisive, photochemical reduction mechanism in the direction of photosynthesis starts with the conversion of the labile compound to hamamelonic acid. Bassham et al. (8) found, after extrapolation to very small times, the ac- tivity of 3-phosphoglyceric acid to be only 75% of the theoretical value of 100%. The sugar phosphates showed an activity not of 0% but of about 17%. These figures justify the assumption that 3-phosphogiyceric acid and sugar phosphates are simultaneously produced from a common, not yet iden- tified precursor substance. There are two possible explanations for this devi- ation from the theoretical values of 100% 3-phosphoglyceric acid and 0% sugar phosphates. Either the alcohol added to the extracts stops carboxyla- tion more rapidly than reduction, or the enzymatic processes occurring after the production of 3-phosphoglyceric acid are much faster than the formation of 3-phosphoglyceric acid itself. The latter explanation would imply the dependence of the activity distribution upon the interactions of substrates and enzymes, so that it would be independent of the sequences in the carbon cycle. THE CHEMISTRY OF PHOTOSYNTHESIS 155 For this reason, only the first explanation is worthy of consideration. Kan- dler (33) found that alcohol poisoning blocks the reduction reactions which lead to the formation of sugar phosphates somewhat more rapidly than the carboxylation to 3-phosphoglyceric acid. Thus, the statement made by Ba.ssham et al. that the reduction of 3-phosphoglyceric acid may continue to a certain extent in the alcoholic extract seems unjustified. By contrast, the alcoholic extraction may give rise to some splitting of labile intermediate products produced in the photosynthetic process to 3-phosphoglyceric acid and cause further deviations from the real activity values. Though Kandler's investigations may upset the correctness of Calvin's carbon cycle, many questions remain unanswered. The hypothetical labile intermediate compound is difficult to visualize chemically. Tolbert and Zill (51, 52, 53) observed that a part of the fixed ^'*C is excreted in the culture medium as glycolate. Calvin et al. (21) also found that glyco- late appears among the first labeled products. Warburg (58) obtained, in experiments with the addition of blue-green light, 7-values approaching 1.3 which corresponds to the formation of glycolic acid (CHiOH-COOH) from CO2 and H2O (see § 36). As has been discussed in § 54, Moyse and Jol- chine obtained labeled malic acid and other acids of the tricarboxylic acid cycle from certain leaves. In § 61 we shall discuss important investigations by Warburg from which it follows that ^^C-labeled amino-acids appear prior to 3-phosphoglyceric acid. Gibbs (26) postulated that during photosynthesis COo is reduced first to formate and then to the formaldehyde level and transferred to either a C5 or a C2 acceptor to form a hexose or a triose respectively. ^^C-labeled formate was added to Chlorella and after 4 min of photosynthesis the tracer in the glucose was located in the C4 atom. This primary reduction to formate is reminiscent of the early hypothesis of Willstatter and Stoll (62) according to which formate bound to the Mg atom of chlorophyll should be intermediately produced. § 58 Some Final Remarks The use of heavy and radioactive isotopes as tracers has considerably en- riched our knowledge of metabolic processes. However, it must be kept in mind that the tracer methods may give rise to erroneous conclusions if the interpretations of experimental results are incorrect or when certain neces- sary experimental conditions are not fulfilled. We have met examples of this in § 42 and § 48. The various investigations with labeled carbon for determining substances produced in photosynthesis show relatively little agreement. In principle, it is possible that labeled carbon appears in all the products of intermediate metabolism when cells are exposed to an atmosphere containing ^^COo. There is no irrefutable evidence that the labeled products found concern photosynthesis only. Furthermore, there is still considerable ignorance of and disagreement on the fate of ^^C02 in the dark. It is ex- 156 PROBLEMS OF PHOTOSYNTHESIS tremely difficult to separate COo fixation upon illumination from that which normally takes place in the dark. Investigations by Miyachi et al. (39) clearly show how slight our knowledge is with respect to CO2 fixation in the dark aero- bically and anaerobically and how careful one has to be in concluding whether CO2 fixation observed in a given experiment is really related to photosynthe- sis or to other metabolic conversions. Wood (63) and Utter and Wood (54) point out that it must be far from easy to determine the first product of photosynthesis in which labeled carbon is incorporated. The sequence of the products which occur in the carbon cycle after short illumination is deduced from the measured activities of these products. When, for instance, CO2 is reduced to (CH2O) via the compounds Al, Ao etc. Al must attain the same specific activity as CO2 before Ao does, A2 must reach it before As, etc. This has been correctly observed by Calvin (21). Figure 59 shows a theoretical example by Utter and Wood. CO2 can be reduced to (CH2O) in three ways, according to the sequences Ai, A^ etc., -► Bi < ► B2 ■* *■ B3 < 1 i r CO2 •*-- Al ■< *■ A2 •« ► A3 •* A4 < ►(CH2O) t ► Ci •< *■ C2 < »■ Cs ••--' I Fig. 59. Different ways of '^C incorporation in photosynthesis (Wood). ^1, B'l etc. and Ci, d etc. The reaction times are represented by the lengths of the arrows. It is supposed that by decreasing the time of illumination the sequences B and C will become more and more improbable so that finally only sequence A will come into consideration. This, however, does not mean that sequence A is the most important. It may be possible that in se- quence A the reaction A^ -^ A^ proceeds so slowly that the over-all speed of the reaction CO2 -^ (CH2O) in sequence A might be of the same magnitude as that of the sequences B and C. \i Ai represents 3-phosphoglyceric acid, labeled carbon certainly appears very rapidly in the carboxyl group of this acid, but this provides no evidence that 3-phosphoglyceric acid is the primary product on the way to (CHoO) and that all other ways leading to this end product are excluded. As a matter of fact, Badin and Calvin (5) observed, at low light intensities, carboxylation to malic acid as the first reacdon (see § 46). Thus, in any case CO2 fixation can occur in two ways: at high in- tensities via 3-phosphoglyceric acid and at low intensities via carboxylation to a dicarboxylic acid. There is no reason to believe that other reactions may not be involved if the experimental conditions are changed. In investigations with radioactive isotopes measurement is made not of the specific activities but of the counts per minute (cpm). However, the rela- tionship between the specific activity of a substance produced and that of ^•*C02 is of much more importance than the cpm values (63). When, for instance, 1 /xmole yfj gives 2000 cpm and 0.1 ^mole A^ 1000 cpm, the latter THE CHEMISTRY OF PHO lOSYNTHESIS 157 substance has a far higher concentration of labeled carbon than the former. Thus, A2 should have its place in the sequence before Ai and not after A^, even though A2 has a lower cpm value. For analysis, the labeled products are extracted from the whole cell, though the reactions involved occur in certain areas of the cell (grana, mitochondria). Only the specific activity at the site of photosynthesis is of importance. The shorter the exposures to light, the less the likelihood that the decisive equi- libria have been reached throughout the whole cell. The extracted com- pounds therefore do not give a true picture of what happens at the site of photosynthesis. As will be discussed in § 61 , Warburg in similar experiments found glutamic acid to be one of the primary products in photosynthesis. Although Calvin observed that Chlorella extracts are very rich in glutamic acid, he did not find any noteworthy incorporation of labeled carbon in this amino-acid. It could be concluded from this finding that the tricarboxylic acid cycle does not participate in photosynthesis, as one of its intermediate products, a-ketoglu- taric acid, is in a state of equilibrium with glutamic acid. It is highly prob- able that the tricarboxylic acid cycle is not involved in photosynthesis, but the exclusion of glutamic acid is certainly not justified. We shall see later that glutamic acid, which is part of the chlorophyllproteid, is of the greatest importance in photosynthesis (see § 64). There is some morphological evidence that chlorophyll occurs in the chloro- plast in units each containing several hundreds of molecules. In some hypoth- eses a physiological significance has been attributed to such units. Accord- ing to Calvin and others, the chlorophyll aggregate having semiconductive properties would be composed of about 1000 molecules. Arnold and Meek (1) believe that one quantum absorbed deals with several hundreds of chloro- phyll molecules. For the time being, the assumption of chlorophyll units with physiological functions is still speculation, though such units may per- haps exist from a morphological point of view. Emerson and Arnold (23) determined with very short-lasting light flashes (10~-^ sec) of high intensities and long dark intervals the maximal O2 production during one light flash. They compared the amount of Oo observed with the chlorophyll content of the cells, and calculated that 2500 chlorophyll molecules are involved in the production of one molecule Oo. Warburg et al. (57) directly measured the light reaction without intermittent light and found, per one molecule chloro- phyll, the production of one molecule O2 (see § 39). Thus, a comparison of the ratio chlorophyll/02 with intermittent and with continuous illumination reveals a discrepancy of three powers of 10. Burk (16, 17) pointed out that Emerson's calculations are based upon the assumption that enough light is absorbed per flash to permit complete photosynthesis. In reality, however, it is impossible to imagine light saturation during such short illumination times. The light intensity of the flashes is some 1000 times too low, i.e., absolutely insufficient to decompose the photolyte in 10~^ sec. Burk regrets with good 158 PROBLEMS OF PHOTOSYNTHESIS reason that investigations of this kind, which, lacking physical significance, contributed so much to the stoichiometric relationship between chlorophyll and O2 not having been discovered much earlier. The photosynthetic carbon cycle as proposed by Calvin and his group is based upon a series of hypotheses. Like many other contributions to the chemistry of photosynthesis, it is based upon the photolysis of water, a hy- pothesis which seems to be improbable today. The participation of primary hydrogen acceptors such as lipoic acid or vitamin Ki lacks support. Further, there is not sufficient evidence to assume a chlorophyll aggregate and its semi- conductive properties. There is no doubt that the work done with '^C02 has opened new and interesting perspectives in the field of photosynthesis. However, too hasty conclusions and too much combination of experimental results with unproven theoretical considerations are extremely dangerous. They may certainly lead to intriguing schemes which suggest the whole prob- lem is solved, but as long as no definite evidence is provided, there is no rea- son whatever to accept them as true and irrefutable. REFERENCES 1. Arnold, W. and Meek, E. S.: Arch. Biochem. Biophys., 60:82, 1956. 2. Arnon, D. I., Allen, M. B., Whatley, F. R., Capindale, J. B. and Rosenberg, L. L. : Proc. 3rd Internal. Congr. Biochem., Brussels, 1955. 3. Arnon, D. I. and Whatley, F. R. : Science, nO:bSA, 1949. 4. Arnon, D. I., Whatley, F. R. and Allen, M. B.: Science, 727:1026, 1958. 5. B.'\DiN, E. J. and Calvin, M. : J. Amer. Chem. Soc, 72:5266, 1950. 6. Bandurski, R. S.: J. Biol. Chem., 217 A^l, 1955. 7. Bassham, J. A., Barker, S. A., Calvin, M. and Quarck, U. C. : Biochim. Bio- phys. Acta, 21:l>l(i, 1956. 8. Bassham, J. A., Benson, A. A., Kay, L. D., Harris, A. Z., Wilson, A. T. and Calvin, M.: J. Amer. Chem. Soc, 76:1760, 1954. 9. Bassham, .T. A. and Calvin, M.: Photosynthesis. In Green, D. E., Currents in Biochemical Research, Interscience, New York, 1956. 10. Bassham, J. A. and Calvin, M.: The Path of Carbon in Photosynthesis. Prentice- Hall, Inc., Englewood Cliffs, N. J., 1957. 11. Bassham, J. A., Shibata, K., Steenberg, K., Bourdon, J. and Calvin, M.: J. Amer. Chem. Soc, 75:4120, 1956. 12. Bickel, a. F. and Kooymans, E. C: Nature, 770:211, 1952. 13. Biswas, B. B. and Sen, S. P.: Nature, 181 -.nX"), 1958. 14. Bradley, D. F. : Arch. Biochem. Biophys., 68:172, 1957. 15. Bradley, D. F. and Calvin, M.: Arch. Biochem. Biophys., 53:99, 1954. 16. Burk, D.: Fed. Proc, 72:611, 1953. 17. Burk, D., Cornfield, J. and Schwartz, M. : The Scientific Monthly, 7,3:213, 1951. 18. Calvin, M.: Fed. Proc, 13:697, 1954. 19. Calvin, M.: Proc. .3rd Internal. Congr. Biochem., Brussels, 1955. 20. Calvin, M. and Barltrop, J. A.: J. Amer. Chem. Soc, 74:6153, 1952. 21. C.^LViN, M., Bassham, J. A. and Benson, A. A.: Fed. Proc, 9:524, 1950. THE CHEMISTRY OF PHOTOSYNTHESIS 159 22. Calvin, M. and Benson, A. A.: Science, lOlAie, 1948. 23. Emerson, R. and Arnold, VV. : J. Gen. Physiol. , 76:191, 1932. 24. Fager, E. W., Rosenberg, J. L. and Gaffron, H. : Fed. Proc, P:535, 1950. 25. Geller, D. M.: Thesis, Harvard Univ., 1957. 26. GiBBS, M.: Fed. Proc, 77:228, 1958. 27. GiBBS, M. and Kandler, O.: Proc. Nat. Acad. Sci., 4.3:446, 1957. 28. Herisset, a.: Thesis, Paris, 1952; Bull. Soc. Chim. Biol., 34:S67, 1952. 29. JoLCHiNE, G.: Bull. Soc. Chim. Biol., 35:481, 1956. 30. Kandler, O.: Zschr. Naturf., 726:271, 1957. 31. Kandler, O.: Naturw., 44:562, 1957. 32. Kandler, O.: Arch. Biochem. Biophys., 7J:38, 1958. 33. Kandler, O.: Zschr. Naturf., 73b :2\9, 1958. 34. Kandler, O. and Gibbs, M.: Plant Physiol., .37:411, 1956. 35. Katz, E. : Chlorophyll Fluorescence as an Energy Flow for Photosynthesis. In Franck, J. and Loomis, W. E., Photosynthesis in Plants, Iowa State College Press, Ames, 1949. 36. Levitt, L. S.: Science, 120:33, 1954. 37. Marre, E. and Forti, G. : Science, 726:976, 1957. 38. Marre, E. and Servettaz, O.: Arch. Biochem. Biophys., 75:309, 1958. 39. MiYACHi, S., Hirokawa, T. and Tamiya, H.: Research in Pliotosyntliesis (Gatlin- burg Conference 79.5.5). Interscience, New York, 1957. 40. Moses, V. and Calvin, M.: Proc. Nat. Acad. Sci., 44:260, 1958. 41. MoYSE, A. and Jolchine, G. : Bull. Soc. Chim. Biol., 38:76\, 1956. 42. MoYSE, A. and Jolchine, G.: Bull. Soc. Chim. Biol., 39:725, 1957. 43. OcHOA, S. : Enzymatic Mechanisms of Carbon Dioxide Assimilation, In Green, D. E., Currents in Biochemical Research, Interscience, New York, 1946. 44. OcHOA, S.: Physiol. Revs., .37:56, 1951. 45. OcHOA, S., Mehler, A. H. and Kornberg, A.: J. Biol. Chem., 774:979, 1948. 46. OcHOA, S. and Vishniac, W. : Science, 775:297, 1952. 47. Racker, E.: Nature, 77.5:249, 1955. 48. Reed, L. T: Adv. EnzymoL, 75:319, 1957. 49. Steward, F. C. and Thompson, J. F.: Nature, 766:593, 1950. 50. Strehler, B. L.: Arch. Biochem. Biophys., 43:67, 1953. 51. ToLBERT, N. E.: Fed. Proc, 74:292, 1955. 52. ToLBERT, N. E. and Zill, L. P.: J. Biol. Chem., 222:895, 1956. 53. ToLBERT, N. E. and Zill, L. P. : Research in Photosynthesis {Gatlinburg Conference 1955). Interscience, New York, 1957. 54. Utter, M. F. and Wood, H. G.: Adv. EnzymoL, 72:41, 1951. 55. Walker, D. A.: Nature, 778:593, 1956. 56. Warburg, O.: Heavy Metal Prosthetic Groups, Clarendon Press, Oxford, 1949. 57. Warburg, O. : Science, 728:68, 1958. 58. Warburg, O., Krippahl, G. and Schroder, W. : Zschr. Naturf., 70b :63\, 1955. 59. Wessels, J. S. C: Rec. trav. chim., 73:529, 1954. 60. Wessels, J. S. C. and Havinga,'' E. : Rec. trav. chim., 77:809, 1952. 61. Wessels, J. S. C. and Havinga, E. : Rec. trav. chim., 72:1076, 1953. 62. Willstatter, R. and Stoll, A.: Untersuchungen iiber die Assimilation der Kohlen- sdure, Springer, Berlin, 1918. 63. Wood, H. G.: Fed. Proc, 9:553, 1950. 160 PROBLEMS OF PHOTOSYNTHESIS D. CARBON DIOXIDE § 59 The Fluoride Reaction If stoichiometric O2 production is to take place after a dark period, the presence not only of O2 but also of CO2 is necessary. Without CO2, re-illu- mination would not yield any O2 (14, 22). Warburg el al. (4, 16) found that Chlorella contains chemically bound CO2 which is stoichiometrically connected with chlorophyll. Warburg calls this CO2 fraction the labile CO2 of photo- synthesis. Fig. 60. Manometer vessel for the determination of labile carbon dioxide. .S".- Chlorella suspension. F: NaF solution. 53.0 A Fig. 61. Anaerobic removal of labile carbon dioxide in the dark by NaF from 200 m1 Chlorella (pH 3.8, 20° C). Chlorophyll content: 3.83 yumole. Curve I: with- out NaF. Curve H with Vso ^V NaF. Pressure difference: 49.5 mm. KcOi — 1.73 mm''. CO, removed = 49.5 X 1-73 = 85.7 fi\ = 3.86 yumole (Warburg et al., Zschr. Naturf.). 25 5 75 10 12.5 15 17.5 20 22 5 25 mm The addition of NaF to Chlorella removes all the labile CO2. A conical manometer vessel (Fig. 60) contains a Chlorella suspension (e.g., 200 ix\ cells in 3 ml salt solution at pH 3.8). The gas phase contains pure, COs-free argon and the side-arm a solution of NaF (e.g., 0.2 ml 0.2 N NaF at pH 3.8). The fluoride is given to the cells in the dark and CO2 is developed. A clear- cut end value is reached after about 20 min. As Figure 61 shows, the amount of CO2 removed in the dark from 200 jul cells in 25 min is 85.7 ^1 = 3.83 )umole, i.e., about 43% of the cell volume (more than the amount of O2 erythrocytes can bind) . In the experiment shown in Figure 61 , the chlorophyll content is 3.83 )umole* so that the stoichiometric relationship between chloro- phyll and labile CO2 is obvious. It is possible to vary the chlorophyll con- tent by cultivating at various light intensities. The end value of the CO2 re- moved will, however, vary accordingly. These experiments prove that the * This high value corresponds, at 22.5% dry weight, to a chlorophyll content of 7.7%. THE CHEMISTRY OF PHOTOSYNTHESIS 161 CO2 removed by NaF must be linked in some way or other with chlorophyll. If the NaF concentrations are varied, the end values remain the same but the lower the NaF concentration, the slower the rate of CO2 removal. The COo removal by NaF is not a simple reaction. It is inhibited by small amounts of HCN and by heating for 5 min at 65 ° C. Thus, it seems that NaF does not have a direct action. Its addition starts an enzyme reaction responsible for the COo removal by means of a heavy metal compound. The addition of phenanthroline does not inhibit the action of NaF, so that the enzyme does not seem to be a dissociating heavy metal compound (see § 31). As phenylurethane does not influence the CO2 removal, it may be concluded that the enzyme is not structure bound. The fluoride reaction is reversible if the concentration of NaF is not too high. When the NaF is washed out after CO2 removal, the cells are again able to take up CO2. This renewed uptake is only possible in the presence of O2. Therefore, the binding of labile CO2 in the cells is not a reaction by itself but needs respiration, i.e., energy. Warburg and Krippahl (15) established that the addition of a drop of octa- nol to Chlorella removes 1 mole CO2 from 1 mole chlorophyll. This CO2 re- moval is irreversible because of the cytolytic properties of octanol. It fol- lows from this that chlorophyll isolated by means of organic solvents does not contain labile CO2. As a matter of fact, there is no place for CO2 in the chlo- rophyll formula. We must therefore distinguish between isolated and func- tional chlorophyll, i.e., between "dead" and "living" chlorophyll (see § 2). Numerical example (15): Vessel I: I',- = 3.2 ml V,, = 15.48 ml A'co, = 1.725 mm- Vessel II: Vp = 3.2 ml \\, = 15.53 ml /Ceo, = 1.732 mm^ Per vessel 200 n\ cells in 3.0 ml salt solution, pH 3.8. Chlorophyll content 7.7%, i.e., at 22.5% dry weight: 3.83 /xmole. In the side-arm of vessel I: 0.2 ml H2O. In the side-arm of vessel II: 0.2 ml 0.2 .YNaF (pH 3.8). Gasphase: argon. Ex- periment carried out in the dark. Temperature: 20° C. Vessel I Vessel II Pressure Changes Pressure Changes After Tipping After Tipping + 0.5 mm +32.0 mm mm +11.5 mm + 1.0 mm + 7.0 mm + 0.5 mm + 1.0 mm + 1.0 mm + 1.5 mm + 0. 5 mm mm 2. 5 min 2. 5 min 5 min 5 min 10 min 20 min 45 min +3.5 mm +53.0 mm Vessel I: xco, = 3.5 X 1.725 - 6.0 yul. Vessel II: xco, = 53.0 X 1.732 = 91.8 Mi- Labile CO2: 91.8 - 6.0 = 85.8 n\ = 3.86 /imole labile CO2 in ^mole _ 3.86 _ chlorophyll in jumole 3.83 162 PROBLEMS OF PHOTOSYNTHESIS § 60 The Reaction Equations of Photosynthesis The stoichiometric relationships between chlorophyll and O2 and between chlorophyll and CO2 show that the O2 produced in the light originates from the CO2 bound to chlorophyll. Warburg (22) shows this clearly in the fol- lowing experiment: a cell suspension is divided into three samples. All CO2 is removed from the first sample by NaF, i.e., 1 mole CO2 per 1 mole chlorophyll. From the second sample only 0.5 mole COo per mole chloro- phyll is removed and from the third sample no CO2 is removed. After- wards the three samples are saturated with 10 vol % CO2. Illumination shows that no O2 can be produced in the first sample, only 0.5 mole O2 is produced in the second sample and 1 mole O2 per mole chlorophyll in the third sample. When NaF is washed out of the first sample, so that CO2 can combine again with chlorophyll, 1 mole O2 is produced per mole chlorophyll upon illumination. The conclusion of this very instructive experiment is that upon illumination the O2 produced originates only from the labile CO2, i.e., from the CO2 which can be removed from chlorophyll by the addition of NaF. All other forms of CO2 present in the cells (dissolved CO2, bicarbon- ates) are not influenced by the action of light. As already shown in § 35, O2 capacity and labile CO2 and their connections with chlorophyll can be represented by the following reaction equations: Reaction 7 {light) : CO2* + Nhp + CO2 -* CO2 + C + O2 Reaction 2 (dark) : Vs C + Vs O2 ^ Vs CO2 + 70000 cal Reaction 3 {dark) : CO2 -* CO2* - 70000 cal Over-all reaction: V3 CO2 + Nhv -^ 1/3 C + 1/3 O2 The production of the photolyte (reaction 3) is not a simple reaction in it- self. It needs about 70000 cal/mole, an amount of energy produced by in- duced respiration (reaction 2). Of the 110000 cal necessary to split one mole CO2, 70000 cal are supplied by a respiratory process and the remaining 40000 cal are provided by one mole quanta. All difficulties with respect to the quanta are eliminated by this scheme. As Warburg (6) pointed out, all has been found experimentally and measured in living Chlorella. Reaction 1 is measured by O2 production and CO2 consumption in the light. Reaction 2 is measured by O2 consump- tion and CO2 production in the dark. Reaction 3 is measured by the time of recovery that elapses before the light is again able to produce as much O2 as in reaction 1. This recovery period lasts about 20 min in Warburg's experiments (see § 41). THE CHEMIS'IRY OF PHOTOSYNTHESIS 163 § 61 Amino-acids in Chlorella Warburg et al. (10, 11) found that Chlorella contains a very active glutamic acid decarboxylase which anaerobically splits glutamic acid into 7-amino- butyric acid and CO2. Under aerobic conditions the reaction proceeds in the opposite direction. anaerobic rnOR ■ r^H, • THo • r.HNHo • rooH -, > nooH • CH, • P.H, • nHoNH, + CO2 glutamic acid aerobic y-amitwbutyric acid With respect to HCN, this reaction behaves like the fluoride reaction, so that it was supposed that the latter is nothing but decarboxylation of glutamic acid. It has indeed been established that the COo removed by NaF origi- nates fiom glutamic acid. It has been possible to show by means of paper chromatography that during the fluoride reaction glutamic acid disappears and 7-aminobutyric acid is produced. Chlorella cultivated under special conditions is able to develop CO2 anaerobically without the addition of NaF (see § 62). Paper chromatography with aspartic acid, glutamic acid, ala- nine and 7-aminobutyric acid as test substances showed, according to War- burg (6), that Chlorella under its normal living conditions contains little aspartic acid, much glutamic acid, very much alanine but no 7-aminobutyric acid. Warburg et al. (6, 12) used labeled CO2 to study the behavior of glutamic acid in photosynthesis. A manometer vessel contains a Chlorella suspension. The side-arm of the vessel is divided into two compartments, one containing ^^C^arbonate and the other an excess of lactic acid. When the lactic acid is given to the carbonate, a pressure of radioactive CO2 develops in the ves- sel. The vessels prepared in this way are illuminated for 0.5, 1 and 5 min. Afterwards, they are, together with control vessels kept in the dark, immersed for 5 min in water at 75 ° C to stop enzymatic reactions and, at the same time, to extract the soluble substances from the cells. The extracts obtained are analyzed by two-dimensional paper chromatography and the activities of the spots measured with a Geiger counter. It follows from these experiments that amino-acids become radioactive much more rapidly than 3-phosphoglyceric acid, which was generally thought to have priority in similar investigations. Furthermore, it was found that aspartic acid and alanine became radioactive more quickly than glutamic acid. From this it may be concluded that a chain of amino-acids acts as a catalyst in Chlorella. In this peptide chain glutamic acid does not seem to occupy the first place. In his investigations on chloroplastin, Kaufmann (1) observed that the grana proteins of various higher plants contain a series of loosely bound amino- acids, glutamic acid being quantitatively predominant. The grana were washed by the method of Warburg and Liittgens (3) to remove the greater part of the glutamic acid. Thus, living chlorophyll must be considered to be 164 PROBLEMS OF PHOTOSYNTHESIS a chlorophyll peptide composed of aspartic acid, alanine and glutamic acid the last-mentioned being of particular importance in photosynthesis. Chlorella contains glutamic acid in concentrations varying from 0.5 to 1.3% of the cells' dry weight, depending on the method of cultivation em- ployed. In chlorophyll-rich cells (5 to 8%) 1 to 2 mole glutamic acid is present per mole chlorophyll. It is possible that in such cells all the glutamic acid is bound to chlorophyll. In chlorophyll-poor cells only a part of the glutamic acid is bound to chlorophyll and the remaining part exists in a free state. It is an important fact that Oo inhibits the breakdown of glutamic acid. The addition of NaF increases respiration and the energy derived from the increased respiration resynthesizes the decomposed glutamic acid until an equilibrium state is reached between decomposition due to NaF and resyn- thesis due to respiration. Anaerobically, 0.001 A^ NaF completely decom- poses glutamic acid. In the presence of O2 the decomposition is incomplete because of the above-mentioned resynthesis. The action of light decreases as the glutamic acid decomposes, and increases as it is resynthesized. Thus, glutamic acid must be closely connected with the light action. Two different NaF concentrations are added, in the pres- ence of O2, to a Chlorella suspension. The decomposition of glutamic acid and the decrease in the light action are measured when the steady state is reached. Table 19 clearly shows the close relationship mentioned (6, 13). TABLE 19 Glutamic acid and light action NaF Concentration Remaining Glutamic Acid in % Remaining Light Action in % V640 A^ V320 A^ 79 36 82 36 The objection could be raised that NaF inhibits the light action not because of the decomposition of glutamic acid, but because of some other, unknown reasons. A comparison of the inhibition of the light action by 0.001 A^ NaF under anaerobic and aerobic conditions shows — as already mentioned — that all the glutamic acid is decomposed under anaerobic conditions and that little glutamic acid is decomposed under aerobic conditions. With the same NaF concentration, the inhibition of the light action is very pronounced under anaerobic conditions and very weak under aerobic conditions. Thus, under all conditions the decomposition of glutamic acid runs parallel to the de- crease in the light acdon; it must be concluded that the former is the cause of the latter. The inhibitory action of long-lasting withdrawal of Oo upon photosynthesis must be attributed to the decomposition of glutamic acid. THE CHEMISTRY OF PHOTOSYNTHESIS 165 The addition of O2 results in glutamic acid being resynthesized and photosyn- thesis starting again. This is further evidence of the absolute necessity of Oo in photosynthesis (see § 42). Under appropriate cultivation conditions, the ratio between the chloro- phyll content, the Oo capacity and the CO2 removed in the dark after the addition of NaF is 1:1:1. This very important observation has been con- firmed by Vishniac and Fuller (2) with Chlorella, Scenedesmus and Euglena, using continuous and fluctuating illumination and cultivation at pH 6.8 in- stead of 3.8. After removing the COo with NaF, the algae were washed and allowed to recover ^^COo in the presence of Oo. After further addition of NaF the COo produced was radioactive : it was concluded that the CO2 fixed during the recovery is located at the same site as the COo removed by NaF. Paper chromatography showed 90% of the radioactivity in the a-carboxyl group of glutamic acid. Chlorophylls a and b did not exhibit any radio- activity. § 62 Lactic Acid Fermentation in Chlorella As has been stated in § 15 and § 61, the glutamic acid content of the cells depends upon the kind of cultivation. The behavior of the X-cells is particu- larly atypical (§ 15). They are produced in very strongly illuminated cul- tures — without day light — with four 200 watt metal filament lamps. The action of light in the X-cells is at least 10 times smaller than in other types of cells, probably to counteract over-illumination during cultivation. Anaero- bically, the X-cells produce lactic acid in far greater amounts and at a higher rate than ordinary cells (13). Indeed, it has been found that anaerobic lactic acid fermentation depends upon cultivation conditions (8). The immediate inhibition of photosynthesis upon withdrawal of Oo has, however, no con- nection whatever with this lactic acid formation. This inhibition is solely the result of the fact that induced respiration cannot occur in the absence of Oo. In 1936 Warburg and C'hristian (7) demonstrated that lactic acid fermen- tation proceeds as follows (see also § 44) CH;rCOCOOH + DPNH + H+ ;=^ CH3CHOHCOOH -f DPN + pyruvic acid lactic acid It should be noted that the active enzyme of Chlorella, lactic acid dehydrogen- ase, is a D-enzyme and the lactic acid produced is d-( — )-lactic acid. As yet, no other D-enzyme has been observed in Chlorella. Glutamic acid decar- boxylase and malic acid dehydrogenase, which also occurs in Chlorella, are L-enzymes. The alanine present in large amounts in the alga is L-alanine. The significance of lactic acid in photosynthesis probab.'y lies in its regu- lation of the pH in Chlorella to assure the optimal value for the activation of glutamic acid decarboxylase. Figure 62 shows that D-lactic acid — but not L-lactic acid — exerts an influence upon light absorption. Both isomers of 166 PROBLEMS OF PHOTOSYNTHESIS lactic acid are added to a solution containing Chlorella extract and DPN+. The light absorption at 3400 A increases only when D-lactic acid is added, as only this isomer can be dehydrogenated to pyruvic acid by D-lactic acid dehydrogenase. Fig. 62. The lactic acid dehydrogenase of Chlorella and d- and L-lactic acid. Curve I: L-( + )-lactic acid. Curve H: D-( — )-lactic acid. Only curve H shows light absorption at 3400 A indicating that only D-lactic acid reduces DPN+ (War- burg et al., Zschr. Naturf.). The X-cells exhibiting high lactic acid production are particularly useful when the glutamic acid of Chlorella is to be decomposed without adding NaF. Owing to lactic acid formation in these cells, glutamic acid is decomposed anaerobically without NaF and aerobically resynthesized. § 63 Breakdown and Resynthesis of Glutamic Acid Lyophilized cells of Chlorella cannot divide and cannot reduce CO2 upon illumination. When suspended in water or in salt solutions, they are able to decarboxylate L-glutamic acid but not other amino-acids or pyruvic acid. They are thus particularly useful for determining glutamic acid. The side- arm of a conical manometer vessel contains 20 mg lyophilized cells. The vessel itself contains 3 ml of the glutamic acid solution to be analysed (pH 3.8). After tipping the contents of the side-arm into the vessel, the pH in- creases to about 5. The temperature is 20 ° C and the gas phase contains argon only. Figure 63 shows that after 280 min 4.49 jumole CO2 are pro- duced by decarboxylation of the 4.8 Mmole glutamic acid added (curve II). Curve I represents a control experiment without the addition of glutamic acid. The CO2 of curve I originates from the glutamic acid present in the lyophilized cells where it is so tightly bound that extraction is very difficult even at 100° C. The breakdown and resynthesis of glutamic acid can also be demonstrated by means of paper chromatography. Various concentrations of NaF are added under anaerobic and aerobic conditions to a suspension of living Chlo- rella (100 jul cells in 3 ml salt solution, pH 3.8, temperature 20 ° C). When the IHE CHEMISTRY OF PHOTOSYNTHESIS 167 o o 130 )20 100 Fig. 63. Decarboxylation of L-glutamic acid under anaerobic conditions. 20 mg Chlorella powder. pH 3.8, 20° C. Curve I: 3 ml salt solution in the manometer vessel. Curve II: 3 ml salt solution + 4.8 yumole L-glutamic acid in the manometer vessel. a = 4.49 nmole COo; h = 0.96 yumole CO2 (Warburg et al., Zschr. Naturf.). 10 15 20 25 30 35 Duration of illumination in mm 40 Fig. 64. Influence of NaF on photosynthesis under aerobic conditions. 100 ^1 cells sus- pended in 7 ml salt soludon in each manometer vessel. pH 3.8, 20° C. Gas phase: 10 vol % CO.>, 30 vol % O.-, 60 vol % argon. Curve I.- without NaF; Vq = 10.808 ml, Kq, = 1.031 mm2, Kcoj = 1-624 mm^. Curve II: with 0.001 .V NaF; Vq = 10.624 ml, Kq, = 1.016 mm2, Kco2 = 1-609 mm-'. Incident light intensity: 200 yul quanta/min. Result: pracucally no inhibition of photosynthesis (Warburg et al., Zschr. Naturf.). 168 PROBLEMS OF PHOTOSYNTHESIS Steady state is reached, the suspension is centrifuged, the cells are again sus- pended in 3 ml water and heated to 90° C for 5 min. The liquid is centri- fuged again, dried in vacuo and examined. The chromatograms show the presence of aspartic acid, glutamic acid, alanine and 7-aminobutyric acid. There are no variations in the concentrations of aspartic acid and alanine. Table 20 shows the expected changes, which have already been discussed, with regard to glutamic acid and 7-aminobutyric acid. The experiments with lyophilized cells and those using paper chromatog- raphy clearly show that the labile CO2 of Chlorella developed when NaF is added or when other measures are applied originates from decarboxylation of glutamic acid. By adding NaF to living cells we are able to elicit the breakdown and resynthesis of glutamic acid in life and to study the function of this amino-acid in living Chlorella. TABLE 20 Results of chromatography studies 1 Glutamic ■y-Aminobutvric Acid Acid Breakdown Formation anaerobic i/,6oo A' NaF + + + + + + anaerobic Vso A^ NaF + + + + + + + + anaerobic Vso N NaF + 10-3 A' HCN aerobic Vieno A' NaF + + aerobic 'Ao A^ NaF + + + + aerobic Vso A^ NaF + 10-3A^HCN It follows from Table 20 that noteworthy differences exist between high and low NaF concentrations. The concentration Vso N is very high and de- composes glutamic acid irreversibly, under anaerobic and aerobic conditions. With low concentrations (Viooo to V 300 N NaF) glutamic acid is reversibly decomposed and respiration is increased. Thus, it is not possible to deter- mine the presence of glutamic acid by means of manometric pressure changes when low NaF concentrations are used under aerobic conditions. To meas- ure aerobic glutamic acid breakdown manometrically, Warburg (13) uses initially a low concentration of NaF and afterwards a high concentration. If, for instance, 40 m1 CO2 are directly produced aerobically with Vso N NaF and only 30 m1 CO2 when previously Viooo N NaF had been given, it may be concluded that the low NaF concentration decomposed 25% of the glutamic acid. THE CHEMISTRY OF PHOTOSYNTHESIS 169 The aerobic resynthesis of glutamic acid should not be considered to be a simple reversal of the anaerobic decarboxylation reaction. As the resynthe- sis occurs only under aerobic conditions. O2, i.e., respiration, must be indis- pensable. In fact, when small amounts of NaF are added to Chlorella under aerobic conditions, respiration increases. § 64 The Necessity for Glutamic Acid in Photosynthesis The foregoing paragraphs have shown that, upon addition of NaF, the glutamic acid of Chlorella can be decomposed and resynthesized by alternat- ing anerobic conditions and aerobic conditions. It has been found that 0.001 tV NaF inhibits photosynthesis anaerobically but not aerobically. The decomposition of glutamic acid and the inhibition of photosynthesis on the >A=157mr 20 30 40 50 60 Duration of illumination in mm fig. 65. Influence of NaF on photosynthesis under anaerobic conditions changing into aerobic conditions. This experiment is the continuation of the experiment in Fig. 64, at the end of which the composition of the gas phase was changed to 10 vol % COo and 90 vol % argon. After 50 min anaerobiosis in the dark glutamic acid is decarboxylated. At / = illumination with 200 jx\ quanta^min. pH, temperature and vessel constants as in Fig. 64. Result: initial marked inhibition of photosynthesis steadily decreasing with increasing O2 pressure and resynthesis of glutamic acid (curve II) (Warburg et al., Zschr. Xaturf.). one hand, and the resynthesis of glutamic acid and the reincrease of photo- synthesis, on the other, are simultaneous processes. Figure 64 shows an aerobic experiment without NaF and with 0.001 A^ NaF described by War- burg (17). The gas phase contains, initially, 10 vol % CO2, 30 vol % O2 and 60 vol % argon. Under these aerobic conditions, photosynthesis is not inhibited by 0.001 A^ NaF. After changing the content of the gas phase to 10 vol % CO2 and 90 vol % argon, anaerobic conditions set in. The ves- sels are shaken for one hour in the dark so that all the glutamic acid is decom- posed. Illumination with the same intensity as in the experiment depicted 170 PROBLEMS OF PHOTOSYNTHESIS in Figure 64, results in the curves shown in Figure 65. It follows from curve II that photosynthesis with 0.001 A^ NaF is initially inhibited, but reincreases when Oo production changes the initial anaerobic conditions to aerobic con- ditions. Respiration of the cells sets in and this energy resynthesizes glutamic acid, so that photosynthesis can increase again. By ascertaining the glu- tamic acid content at the beginning and at the end of the anaerobic experi- ment in NaF-free controls, the constant value of 1.7 /xmole/100 ^J.\ cells is found. In the presence of 0.001 A^ NaF the glutamic acid content at the beginning of the experiment is zero. At the end it is 1.47 ^mole/lOO ^1 cells. Therefore, because of the autocatalytic Oo production the glutamic acid content increases from to 86% of the normal value (1.7 /xmoleTOO /xl cells). During this time photosynthesis increases from a very low value to 82% of its normal value, as follows from the difTerence of the slopes of curves I and II in Figure 65. This quantitative parallelism between the re- synthesis of glutamic acid and the increase of photosynthesis (see also Table 19) proves most convincingly the close connection between photosynthesis and glutamic acid. For these experiments illumination with high intensity is necessary so that the Oo saturation pressure is obtained within about 10 min in the NaF-free controls. If the intensity is too low, the saturation pressure would not be obtained for one hour, and six hours would be required for the resynthesis of glutamic acid in 0.001 A^ NaF. In this type of experiment it is sufficient to know the ratio of the pressure changes in NaF and in the control. However, if the exact amount of the O2 produced is to be ascertained, equadon 12 can be used. Owing to the high light intensity, it is permissible to call 7 = —1, so that X,,., = /. -/^°^-V =2.8 A Ml With regard to the ratio of the pressure changes, it must be borne in mind that in the experiment depicted in Figure 65 COo is bound when glutamic acid is resynthesized. To this amount of COo we have to add the so-called aerobically dissociating COo which will be discussed in § 65. With 10 vol % CO2 in the gas phase, this amounts to about half of the other COo. As 1.47 //mole = 1.47 X 22.4 ijl\ glutamic acid is resynthesized, xeo, = 1.47 X 22.4 X 1.5 = 49^1 Hence, the binding of CO2 in NaF elicits the negative pressure change _49 _49 //CO, = -r — = yrr = -30.5 mm It follows from Figure 65 that the difference A in the pressure changes of both curves is 157 mm. The CO2 binding is thus 20% of A. The remain- ing 80% must be attributed to the decreased O2 production, i.e., indicate the inhibition of photosynthesis. THE CHEMISTRY OF PHOTOSYNTHESIS 171 It must be emphasized that the reaction CO2 + 7-ainin(jl)ut\ ric acid s^lutamic acid does not represent CO2 hxation in photosynthesis. The more 7-aminobutyric acid produced, the greater the inhii^ition of photosynthesis. § 65 shows that the aerobically dissociating COo — a great amount of COo fixed in the pres- ence of O2 and removed in the absence of O2 — must be considered to be of utmost importance. This fraction of CO2 is also quantitadvely connected with glutamic acid. If half of the glutamic acid is decomposed by the addi- tion of NaF, photosynthesis and also aerobically dissociating CO2 are halved. § 65 Dissociating Carbon Dioxide When Chlorella is saturated at various COo pressures and NaF is added, not only glutamic acid but also bicarbonates develop CO2, e.g., H2CO3 + HPO4-- ^ HCO:r + H2PO4- H2CO3 + protein- :^ HCO3- + H-protein Furthermore, it may be possible that CO2 originates from carbamino-acids produced in the cells by the action of CO2 upon amino-acids (e.g. glutamic acid). COOHCH2CH2CHNHCOOH^COOHCH2CH2CHNH2COOH + CO2 COOH N-carboxyl-glutamic acid • glutamic acid The CO2 derived from such carbamino-acids is dependent on CO2 pressure, whereas the CO2 derived from glutamic acid is not dependent on the CO2 pressure. Warburg (13) found that more CO2 is bound under aerobic than under anaerobic conditions. When, for instance, 100 ^1 cells containing 1.84 /zmole chlorophyll and suspended in 3 ml salt solution at pH 3.8 are satu- rated with CO2 pressures varying from 5 to 50 vol %, the amount of CO2 removed by Vso A^ NaF in 20 min at 20 °C is as shown in Table 21 . TABLE 21 Dissociating CO2, anaerobically and aerobically CO2 Pressure in Vol % 5 10 20 50 anaerobically (CO-, + argon) ^1 COo aerobically (25 vol % COo) ix\ COo 38.8 38.0 44.7 51.5 48.2 63.0 51.8 79.5 56.3 97.0 If the CO2 removed from glutamic acid at zero CO2 pressure is subtracted from the anaerobic values, the anaerobically dissociating COo is obtained. If the anaerobic values are subtracted from the aerobic values, we find the 172 PROBLEMS OF PHOTOSYNTHESIS aerobically dissociating CO-2, i.e., the CO2 which is bound under the influence of O2 only. Table 22 and Figure 66 show the results of these subtractions. TABLE 22 Dissociating CO2, anaerobically and aerobically CO 2 Pressure in Vol % 5 10 20 50 anaerobically dissociating CO2 in ^1 aerobically dissociating CO2 in fx\ 5.9 6.8 9.4 14.8 13.0 27.7 17.5 40.7 It follows from Figure 66 that, both anaerobically and aerobically, the degree of saturation is reached at a CO2 pressure of 50 vol %. The degree of satu- ration is about 2.5 times greater for aerobically dissociating CO2 than for anaerobically dissociating CO2. 40 50 volume % CO? Fig. 66. Evolution of dissociating COo from Chlorella with '/so A' NaF. pH 3.8, 20° C. Curve I: anaerobically dissociating COi. Curve H: only aerobically dissociating CO2 (Warburg et al., Zschr. Naturf.). Curve II for aerobically dissociating CO2 is strongly dependent upon the method of Chlorella cultivation. Cells cultivated with fluctuating light (A- cells) give the best quantum yields and bind COo aerobically much more strongly than south cells which give poor quantum yields (see § 15). It is also of interest to note that the saturation value for aerobically dissociating COo is equal to the glutamic acid content of the cells. In the experiment shown in Figure 66, the latter was 38.4 ix\ and the saturation value for aero- bically dissociating COo was 41 jjX. As the chlorophyll content was 41.2 /xl, it follows that, within the limits of experimental error, chlorophyll content, glutamic acid content and aerobically dissociating CO2 are identical. THE CHEMISTRY OF PHOTOSYNTHESIS 173 In X-cells containing 5 times as much glutamic acid as chlorophyll War- burg found that 100 fx\ cells contained 1.9 /xiTiole glutamic acid and 0.418 fxmole chlorophyll and that the saturation value for aerobically dissociating CO2 was 1.9 ^tmole. Thus, in these cells, too, the saturation value for aerobi- cally dissociating CO2 is equal to the glutamic acid content, but not to that of the chlorophyll content. To summarize these most interesting findings, Chlorella contains three types of COo: 1 . COo^ of glutamic acid which is not dependent on CO2 pressure. 2. COo , originating from bicarbonates, which is dependent on COo pres- sure. 3. COo , i.e., the dissociating COo, originating in the presence of Oo, which is dependent on the COo pressure and occurs in a concentration of the same magnitude as that of glutamic acid. It is possible to establish the three COo values by means of the fluoride method (5): saturation of the cells with argon : .y ^ul CO2 saturation of the cells with argon + 1 vol % COo : y ix\ COo saturation of the cells with argon + 10 vol % COo + Oo: z m1 CO2 We then have COo' = .V m1 COo" = (v - .v) Ml CO,'" = u -V) Ml In the light glutamic acid does not decrease, but aerobically dissociating COo decreases markedly. After 5 min illumination at high intensity (800 ix\ quanta/min) the aerobically dissociating COo decreased in one experiment (100 n\ A-cells) from 77 /xl to 49 /xl. When the cells were put in the dark afterwards ^/so A^ NaF developed 75 m1 CO2 after 20 min. Thus, the light value (49 ^1) increased within 20 min in the dark to its initial value. On illu- mination the O2 capacity of cells which were previously in the dark sets in, and the dissociating COo decreases markedly during the stoichiometric O2 production. However, in the dark, when induced respiration restores Oo capacity, the dissociating CO2 increases to its initial value (12). § 66 The Carotenoid Oxygenase of Chlorella When a small amount of octanol or quinone is added to Chlorella (south cells suspended in salt solution K or S) in the dark, respiration ceases. Neverthe- less, there is a very pronounced Oo uptake without production of COo. The O2 consumption of 200 ^1 cells at 20° C and at pH 6.5 is about 500 jA per hour, i.e., 5 times greater than the Oo uptake during respiration. Warburg et al. (20), who discovered this reaction, showed that lyophilized cells oi Chlo- rella — without addition of quinone — behave in the same way. Brief heating to 65 ° C stops the reaction, which is therefore assumed to be of an enzymatic 174 PROBLEMS OF PHOTOSYNTHESIS nature. The reaction is not inhibited by either lO"'' A^ phenanthroHne or lO""^ A^ HCN. It was found that the carotenoid concentration decreases during the reaction so that, according to Warburg, the reaction is an enzy- matic oxidation of carotenoids by molecular O2. The enzyme responsible, carotenoid oxygenase, is activated by the addition of small amounts of qui- none. In lyophilized cells the reaction proceeds spontaneously. From ex- periments with fresh cells it follows that the rate of O2 uptake depends upon the amount of quinone and upon the pH (optimal 6.5). The rate rises with increasing Oo pressure. 4000 4200 Fig. 67. Oxygenase reaction and carotenoid concentration. 200 yul Chlorella in the n.an- ometer vessel suspended in salt solution S. pH 3.8. In the side-arms no quinone or 2 mg quinone. Air in the gas phase. Curve I : control without quinone, normal respiration. Curve II: 2 mg quinone, incident intensity of white light 730 yul quanta/min. After 225 min 60 ix\ O2 used. Curve III : 2 mg quinone in the dark. After 225 min 370 yul Oo used (War- burg et al., Zschr. Naturf.). The action of the oxygenase is light-sensitive. Less O2 is consumed in the light than in the dark and the carotenoid concentration is not decreased as much in the light as in the dark. The simplest explanation is that the oxy- genase transfers O2 in the dark and removes it in the light. Figure 67 shows the behavior of the carotenoids. A Chlorella suspension of 200 /xl cells in salt solution S is divided into three samples. Sample I, which serves as a con- trol, is shaken with air in the dark for 225 min and no quinone is added. Sample II is shaken with air and strongly illuminated for the same time with THE CHEMISTRY OF PHOTOSYNTHESIS 175 2 mg quinone added. Sample III is shaken with air in the dark for the same time with 2 mg quinone added. The cells are afterwards washed with water, centrifuged and extracted with methanol. Using Willstatter's method, the chlorophylls are removed by saponification and the remaining carotenoid ether solution filled up with ethanol to 100 ml. P'or 1 cm layer thickness In iji is plotted against the wave-length. It follows from Figure 67 that the activation of the oxygenase by means of quinone reduces the carotenoid con- centration in the dark to about 50% of the initial value, but reduces it in the light to about 25% of the initial value. 160 — I 1 1 \ I 80 100 120 140 160 180 200 Fig. 68. O.xygenase reaction with lyophilized cells of Chlorella. In the manometer vessel 3 ml Vso M phosphate, 0.1% KCl (pH 6.5). In the side-arms 25 mg lyophilized cells. Air in the gas phase. KOH in the center well. At time t = 0, the lyophilized cells are tipped into the vessel. Curve I: dark. Curve II: incident intensity of white light 200 yul quanta/ min. Curve HI: incident intensity of white light 750 ^ul quanta/min (Warburg et al., Zsc/ir. Naturf.). Figure 68 illustrates the behavior of lyophilized cells in the dark and upon illumination with two light intensities. A suspension of 25 mg lyophilized cells corresponding to 100 tx\ fresh Chlorella consumes 160 /xl O2 in the dark after 190 min (curve I). On illumination the uptake decreases more mark- edly at high light intensities than at lower light intensities (curves II and III). This experiment shows some similarity to the compensation of respi- ration by photosynthesis. However, it should be kept in mind that lyophilized cells produce neither CO2 due to respiration nor Oo from CO2 (see § 63). In the manometric experiments discussed certain precautions have to be observed. Lyophilized cells must be brought into the side-arm of the ma- nometer vessel and only at time t = are they tipped into the salt solution of the main compartment, where Oo uptake immediately sets in. If the lyo- philized cells were suspended in the salt solution before the manometric reading, the greater part of the oxidation process would pass unobserved. Precautions are also necessary for the activation with quinone. The center 176 PROBLEMS OF PHOTOSYNTHESIS well of the manometer vessel should contain KOH, as is shown, for instance, in Figure 19. The addition of quinone produces CO2 from glutamic acid and this CO2 must, of course, be absorbed by KOH. However, the quinone added may distil into the KOH where it may be oxidized to tetrahydroxy- quinone. This reaction also consumes Oo. For this reason, it is necessary to carry out control studies without cells and to subtract the pressure changes obtained in the controls from those obtained in the experiment. There is a further complication: on illumination quinone produces Oo (Hill reaction). To avoid this, quinone should be completely reduced : this can be done by means of brief illumination with very high light intensity. Otherwise, the O2 production due to quinone would prevent the correct measurement of Oo uptake due to the carotenoid oxygenase. Thus, there are so many sources of error in the activation with quinone that it is preferable to use lyophilized cells to measure oxygenase activity. § ^1 Experiments with Quinone When quinone is added to Chlorella in the dark and under anaerobic con- ditions, a considerable amount of CO2 is developed in a short time, and qui- none is reduced to hydroquinone. The amount of quinone reduced is not equivalent to the amount of CO2 produced. The major part of the CO2 pro- duced originates from decarboxylation of glutamic acid, a reaction which is not an oxidation process. Warburg and Krippahl (18) found that addition of 0.1 mg quinone gives the greatest CO2 production, whereas addition of 2.0 mg quinone elicits the smallest production of CO2. Whereas 0.1 mg quinone added to 100 jul cells in 3 ml salt solution K has no poisoning effect, 0.2 mg quinone inhibits O2 respiration and, therefore, photosynthesis com- pletely and irreversibly. It has been found that, for instance, after addition of 0.1 mg quinone the end value of 57 /xl CO2 developed from 100 /xl cells was attained after 30 min. Of this amount, 40 /xl CO2 were due to glutamic acid breakdown; the origin of the remaining 17 /xl CO2 was unknown. Similar experiments in the dark under aerobic conditions also show that 0.1 mg quinone does not inhibit O2 respiration but that 0.2 mg quinone does. However, under these conditions O2 is used to a great extent without CO2 pro- duction, due to the action of the carotenoid oxygenase. The light reaction of Chlorella with quinone has been discovered by ma- nometry with KOH in the center well of the vessels (3), so that the influence of CO2 could only have been observed with very low CO2 pressures. In these experiments the pH should be low and constant (3.8) to decrease the bicar- bonate content of the medium as much as possible. The suspension of 100 lA cells in 3 ml salt solution K is previously treated with 0.1 mg quinone in the dark to drive out the major part of the labile COo. The suspension is first aerated with argon and a little CO2 for 20 min. After closing the stop cocks, the vessels are shaken — also in the dark — for 40 min. Afterwards, 2 mg quinone are tipped from the side-arm and strong illumination (725 /jlI quanta/ THE CHEMISTRY OF PHOTOSYNTHESIS 177 min) sets in. It was found that at ClOo pressures between 0.125 vol % and 1.49 vol % the end values for the O- produced are practically idendcal. They are equivalent to the amounts of quinone added. However, the rates of O.. production increase with the CO2 pressures. Under 0.015 vol % CO2 the rates are very low (Fig. 69). These experiments can only be explained if we admit that CO2 is an intermediate product of the Hill reacdons, as has been proved in other ways for the Hill reagents: NO3 ions and ferric ions (see §49). 10 15 20 Duration of illumination in mm 25 Fig. 69. Influence of CO2 pressure on the quinone light reaction. In the main com- partment, 100 yul Chlorella (south cells) suspended in 3 ml salt solution K + 0.1 mg qui- none. In the side-arm 2 mg quinone dissolved in 0.2 ml water. In the gas phase argon with various contents of CO2. Illumination; 725 /il quanta/min (Warburg et al. Zschr. Naturf.) . In isolated grana and in lyophilized cells of Chlorella the Oo evolution in the light upon addition of quinone proceeds twice as quickly when the gas phase contains 1 .4 vol % COo in addition to argon. In the grana the end value for the Oo produced is the same whether the gas phase contains, besides argon, CO2 or not. It corresponds to about 83% of the amount of quinone added. The end values for O2 production in lyophilized cells are also independent of the presence of CO2 in the gas phase. They are about 50% of the values calculated from the quinone turnover (17). The investigations discussed in this paragraph are still being carried out in Warburg's institute. The results already obtained clearly show the prime importance of CO2 in the Hill reactions. 178 PROBLEMS OF PHOTOSYNTHESIS § 68 Quinone as a Catalyzer In §49 we discussed the necessity of COo in the quinone reaction of grana: lighl 2 quinone + 2H2O > 2 hydroquinone + O2 The necessary half-value pressure of COo should be 10 mm HoO. At a half- value pressure of 1 mm H2O no O2 is produced. However, the grana do not consume CO2, so that the action of COo must be that of a catalyzer. Inter- mediate reactions must therefore take place and it seems quite logical to as- sume the formation of a peroxide. As a matter of fact, the O2 developed is molecular O2 and therefore has to be formed from a peroxide. From a chemical and energetical point of view evolution of Oo in the atomic state must be excluded. Warburg (21) proposes the following reaction scheme: quinone + COo + 2HoO » hydroquinone + HO.COOOH HO.COOOH > H.COOH + O2 quinone + H.COOfI > hydroquinone + CO2 2 quinone + 2H2O > 2 hydroquinone + O2 Thus, quinone reduces CO2 to percarbonic acid, a well-known substance with the formula O HO— C \ O— OH It is reduced to formic acid giving off molecular Oo. The formic acid pro- duced in statu nascendi reacts with a second molecule quinone to produce CO2. The intermediate products do not appear in the over-all reaction. It must be assumed that they are all bound to chlorophyll. These reactions do not take place in solutions and are inhibited by 0.001 N phenylurethane. It is of course possible to advance a similar scheme assuming that quinone does not oxidize CO2 but water to produce H0O2 : 2 quinone + 4HoO > 2 hydroquinone + 2H2O2 2Hobo > 2H2O + O2 2 quinone + 2H2O > 2 hydroquinone + Oo The over-all reaction is the same as before. However, the percarbonic acid pathway seems a prion to be more probable as the experiments show that COo pressure is absolutely indispensable. On the other hand, a formic acid stage has never been established. An important argument against the H2O0 path- way lies in the fact that catalase is present. This enzyme is inhibited by HCN but the O2 evolution in the quinone reaction is not inhibited by HCN. An important condition for the quinone reaction is a sufficiently acid me- dium so that the hydroquinone produced cannot react back with O2. War- burg (21) pointed out how interesting it is to study conditions for obtaining THE CHEMISTRY OF PHOTOSYNTHESIS 179 this back reaction in which quinone would act as a catalyzer. If the pH of the grana suspension is increased from 6.5 to 8 quinone reaction changes from the stoichiometric state to the catalytic state and we find an over-all reaction of zero : 2 quinone + 2H2O > 2 hydroquinonc + O2 2 hydroquinone + O2 > 2 quinone + 2H2O The back reaction of hydroquinone may be either enzymatic by means of the copper of the phenoloxidase or directly chemical by autoxidation. The en- zymatic reoxidation takes place rapidly at low O2 pressures; it is inhibited by HCN. By contrast, the autoxidation proceeds only at higher O2 pressures and — in contrast to the enzymatic reoxidation — results in formation of H2O2: 2 hydroquinone + 2O2 > 2 quinone + 2H2O2 The ratio H2O2/ O2 equals 1 . The less quinone added in these catalytic quinone reactions, the closer conditions are to the physiological. According to Warburg, the optimal amount of quinone to be added is equal to the chlorophyll content of the grana, e.g., 1 /xmole benzoquinone or 1 /^mole /3-naphthaquinone sulfonic acid for a quantity of grana containing 1 ^mole chlorophyll. In stoichiometric experiments 20 /^moles quinone are added to 1 ^mole chlorophyll. As already mentioned, the enzymatic back reaction is inhibited by HCN. However, the strange thing is that the forward reaction of O2 cannot be in- hibited by HCN. This can be shown in the stoichiometric quinone reaction — i.e. under the proviso that no back reaction of O2 can take place — when HCN is added. The O2 evolution is then not inhibited by 0.01 A^HCN and only slightly inhibited by 0.1 A^ HCN. Thus, as the O2 production is not inhibited by HCN and the enzymatic back reaction of O2 is inhibited by HCN, quinone catalysis at low O2 pressures must always be inhibited by HCN. If the Oo pressure is increased to such an extent that autoxidative back reaction proceeds rapidly enough, neither the forward reaction nor the back reaction of O2 will be inhibited by HCN. Thus, in 0.01 A^ HCN we have complete quinone catalysis; however, H2O2 is produced instead of water, HCN inhibiting the catalase of the grana. In 0.01 A^ HCN we there- fore have the following reactions : 2 quinone + 2H2O > 2 hydroquinone + O2 2 hydroquinone + 2O2 > 2 quinone + 2H2O2 2H2O + o~ > 2H2O2 The over-all reaction is no longer zero. O2 is consumed and H2O2 produced. The addition of HCN thus gives, manometrically, an alteration in pressure changes from zero to markedly negative values. In contrast to simple autoxi- dation, the ratio H2O2/O2 now equals 2 (see Table 23). 180 PROBLEMS OF PHOTOSYNTHESIS TABLE 23 O2 consumption and H2O2 formation in grana (about 1 ^mole chlorophyll) in air, in 0.01 N HCN. 0.5 to 1 yumole benzoquinone or /3-naphthaquincne sulfonic acid added. pH 8.3. Incident energy about 36 /imoles quanta/min. 0-2 //2O2 fi moles /i moles //2O2/O2 -6.2 + 12.5 2.04 -7.9 + 17 2.16 -7.2 + 13 1.80 -5.6 + 11 1 .96 -6.7 + 13 1.94 -8.2 + 15.7 1.92 average values —6.97 + 13.7 1.97 Warburg (21) compared these findings obtained with grana in the presence of quinone with those obtained with Chlorella without quinone. When Chlorella in C02-containing air is illuminated normal photosynthesis is ob- tained. Addition of HCN up to 0.01 A' elicits complete inhibition and no pressure changes are noted; no H2O2 is produced. However, when the O2 pressure is increased from 0.2 to 1 atm, considerable negative pressures and H2O2 production are obtained upon illumination but not in the dark. Addi- tion of CO2 accelerates the H2O2 production which can be completely in- hibited by 0.0001 N phenanthroHne. In these experiments the ratio H2O2/O2 is not equal to 1. as is the case in simple autoxidation, but has the value 2 (Table 24). This means that the reaction equations found for quinone catalysis in grana in 0.01 A^ HCN are the same for Chlorella without quinone in 0.01 A' HCN. Warburg assumes the presence of an oxidant E, which he considers to be an enzyme, in Chlorella. The following reaction scheme can then be applied to Chlorella: 2E + 2H2O 2EH2 + 20 2 -^ 2EH2 + O2 ^ 2E + 2H2O2 2H2O + O. 2H2O2 These experiments prove that Chlorella contains a natural catalyzer which is reduced upon illumination evolving O2. The catalyzer is steadily re- oxidized in the dark. It behaves in the same way as quinone added to grana or Chlorella (see Tables 23 and 24). TABLE 24 O2 consumption and H2O2 formation in 100 yX Chlorella per hour, in 0.01 A' HCN, in 20 vol % CO2-O2. pH 4. Incident energy about 36 ^moles quanta/min. 02 //2O2 nmoles fimoles HiOi/Oi -x.-ii + 2.40 1.82 -1.26 + 2.60 2.04 -1.97 + 4.95 2.50 -2.04 + 4.12 2.02 -1.36 +2.72 2.00 -2.14 +4.12 1.92 average values —1 .68 + 3.48 2.06 THE CHEMISTRY OF PHOTOSYNTHESIS 181 It should be borne in mind that quinone reactions involve heavy metal catalysis. The forward reaction of O2 is catalyzed by the unknown heavy metal which is inhibited by 0.0001 A' phenanthroline but not by 0.01 A^ HCN. The enzymatic back reaction of Oo is catalyzed by the copper of the phenol- oxidase. It is still doubtful whether a third heavy metal — the iron of the very active catalase of green cells and grana — plays a role in the quinone reac- tions. B Fig. 70. Manometer vessel for determining the CO., partial pressure We come now to the question how COo fixation is related to the experi- mental results described. To study this, the partial pressure of CO2 must be established. For this purpose, Warburg worked out a new type of manom- eter vessel (Fig. 70). The main compartment contains the suspension of grana or cells. The side-arm A connected with the main compartment contains H2SO4. The trough contains KMn04 and the side-arm B connected with the trough K4Fe(CN)e. When H2SO4 is tipped into the main compart- ment CO2 evolves from any bicarbonate present in the suspension. When K4Fe(CN)6 is tipped from the side-arm B to the trough containing KMn04 the contents of the trough become strongly alkaline and very rapidly absorb all the CO2 present: KMn04 + 3K4Fe(CN)6 + 2H2O -> MnO. + 3K3Fe(CN)6 + 4KOH In this way it is possible to determine the CO2 partial pressures at times to and t and to establish the exact change in COo partial pressure in the time t — to. Any decrease in the CO2 partial pressure means CO2 fixation. In spite of many variations in his experiments, Warburg always found that the O2 evolutions in the presence of quinone and HCN were never accompanied by changes in the CO., partial pressure. The explanation of these results lies in the fact that there is no respiration. It was, indeed, found that no CO2 is fixed in grana as they do not respire. Similarly, Chlorella cells do not respire in the presence of quinone and no CO2 fixation is ob.served. However, when Chlorella cells are treated with 0.01 A' HCN in the dark, they show respi- 182 PROBLEMS OF PHOTOSYNTHESIS ration as well as CO2 fixation, but under the same conditions upon illumina- don they exhibit neither respiration nor CO2 fixation. They do not respire because the H2O2 produced in the light destroys respiration. Thus, the rule that no CO2 fixation is possible without respiration, has been established. § 69 Quinone Catalysis and Phosphorylation In § 51 we discussed Anion's discovery that green grana suspended in phos- phate solution at pH 8 are able to synthesize ATP from ADP upon illumina- tion. Warburg (21) found that substances, such as 0.0001 N phenanthroline and 0.01 A^ HCN (at low O2 pressures), which inhibit quinone catalysis also inhibit light phosphorylation. However, at high O2 pressures 0.01 A^ HCN does not or only very slighdy inhibits both quinone catalysis and light phos- phorylation. This specific behavior with respect to HCN proves that a rela- tionship between both processes must exist. It seems therefore contradictory that nevertheless ATP production has no influence whatever upon quinone catalysis. The speed of O2 evolution elicited by the quinone remains the same whether ADP has been added or not, provided, of course, that phosphate is present in sufficient quantities. The explanation lies in the fact that in quinone reactions phosphate is bound under all circumstances. In the ab- sence of ADP this bound phosphate is freed again during the O2 evolution and the amount of free phosphate remains constant in the over-all reacdon. It acts as a catalyzer. In the presence of ADP, phosphate is trapped and the over-all reaction shows its decrease. Warburg found that, in the presence of ADP, one molecule phosphate is bound when one molecule HoOo is produced. In other words, one molecule phosphate is bound when one molecule quinone takes up two H atoms. This relationship of 1 ATP and 2 H is the same as that Warburg and Christian found in the oxidation reaction of fermentation (see §44), the only reaction in which the mechanism of phosphorylation has been chemically defined. Thus, the energetics of light phosphorylation can be identified with the ener- getics of dark phosphorylation (21). It is certainly permissible to speculate about the substance in the quinone reactions which may be responsible for the binding of phosphate. Warburg proposes a phosphorylated peroxide of COo according to the following reac- tion scheme: OPO3H2 / quinone + CO> + H,0 + H:iP04 > hydroquinone + C=0 O— OH OPOnHo C=0 + H..O > H,P04 + H.COOH + O, \ O— OH THE CHEMISTRY OF PHOTOSYNTHESIS 183 quinone + H COOH > hydroquinone + CO2 2 quinone + 2H'20 > 2 hydroquinone + O2 If we accept the hypothesis of water photolysis, a similar scheme can be out- lined with the intermediate production of a phosphorylated HoOo: 2 quinone + 2H.O + 2H:iP04 > 2 hydroquinone + 2H3PO5 2H,P05 > 2H3P04'+ O2 2 quinone + 2H2O > 2 hydroquinone + O2 The formation of H4P2O9 instead of H3PO5 may be envisaged : 2 quinone + H2O + 2H:iP04 > 2 hydroquinone + H4P2O9 It does not seem very probable that such phosphorylated hydrogen peroxides (peroxyphosphoric acid and peroxypyrophosphoric acid) play a biological role. The investigations discussed in this Section are being continued in War- burg's institute. This experimental work is difficult and painstaking but the results already obtained show the prime importance of CO2. The many interesting facts drawn from the experiments with fluoride and quinone have enriched our knowledge of the very complicated process of photosynthesis. § 70 Some Final Remarks In this chapter some basically important results of research in the chemis- try of photosynthesis have been discussed. It must be admitted that, with respect to the extreme complexity of the problem, these results are modest. Most of them are far from being certain and are not universally accepted. Generally, the work done consists of tentative approaches and there is a con- siderable amount of disagreement between the investigators working in the field. The main risk in all the schemes worked out in the last years to de- scribe chemical pathways in photosynthesis lies in the fact that the student may forget that most of it is pure "paper" chemistry. It is evident that such schemes may be useful as working hypotheses but more or less definite con- clusions should only be drawn when every intermediate reaction of a reaction scheme is found to accord with experimental facts. It must be emphasized again that at the present time the chemistry of photosynthesis is still in its infancy. Three main directions of research can be distinguished to-day. They are inspired by Arnon, Calvin and Warburg. Arnon's concept of light phos- phorylation and Calvin's photosynthetic carbon cycle certainly show new and important ways, though the general theories as advanced by these investi- gators are difficult to accept. All theories based upon the photolysis of water — a hypothesis for which not the slightest evidence can be advanced and which was put forward about 20 years ago to explain the reactions discovered by 184 PROBLEMS OF PHOTOSYNTHESIS Hill — must be regarded as more or less suspect. Phosphorylation due to the action of light still remains very problematical. Warburg's work in the field of photosynthesis chemistry deals mainly with the importance of COo. His studies with fluoride and with quinone have already resulted in many im- portant discoveries which were discussed in the last section of this chapter. They are all based upon reliable measurements by means of new manometric methods. Warburg found considerable evidence that respiration belongs to the chemical mechanism of photosynthesis. This follows from the one- quantum theory discovered in 1950. In 1958 he found that respiration and photosynthesis decrease in the same proportion at very low Oo pressures (§ 42). The experiments discussed in § 67 showing that no COo fixation with- out respiration can be detected provided further support. Recently War- burg and Kayser (9) measured the CO induced inhibitions of respiration and photosynthesis in cotyledons of barley of various ages (6 to 21 days) and found a significant relationship. When respiration in older leaves was less inhibited by CO, the same decrease in CO inhibition was noted in photo- synthesis. Six day old leaves showed inhibition values of 56% and 59% for the two processes. In 14 day old leaves values of 24% and 20% were found, whereas in 21 day old leaves there was no CO induced inhibition in respiration and in photosynthesis. Thus, these experiments clearly show that when respiration is inhibited to a certain degree, photosynthesis is equally inhibited to the same extent. We may also recall here the inhibitory action of O2 withdrawal upon photosynthesis attributed to the decomposition of glutamic acid (§ 61). In § 36 and § 57 we discussed the production of glycolic acid in photo- synthesis of Chlorella. There is evidence that this compound may be of particular importance. Zelitch (24) draws the attention to the central posi- tion of glycolic acid in the processes of respiration and photosynthesis in tobacco leaves. Warburg and Krippahl (19) show that Chlorella suspensions in appropriate carbonate buffer solutions provoking a low CO2 pressure (0.05 atm %) produce upon strong illumination (800 jul quanta/min) with pure Oo in the gas phase about one molecule glycolic acid for two molecules CO., con- sumed. In increasing the CO2 pressure, glycolic acid formation is inhibited. At a COo pressure of 1.70 atm % only traces of the acid are detectable. It was possible to separate CO2 fixation and glycolic acid formation. When the gas phase contains 2 atm % CO2 and Oo, it is shown that in the beginning of the experiment COo disappears without formation of glycolic acid. After a while the COo pressure is decreased to such an extent that glycolic acid can be produced in more or less pure Oo atmosphere. The chemical mechanism is not yet elucidated but there is evidence that COo reduction on the one hand (e.g., to glycolaldehyde) and oxidation of the resulting reduction products (e.g., to glycolic acid) on the other hand are competitive reactions with respect to an oxidizing enzyme. This view is in full agreement with the fact that in photosynthesis COo has to be oxidized (to the state of the photolyte) before it can be reduced (19). THE CHEMISTRY OF PHOTOSYNTHESIS 185 Recently, the half-value pressures of COo in the quinone reactions have been determined by Warburg (23) with the aid of a special manometric arrangement allowing the measurement of very low COo pressures. The values obtained are 7 to 8 mm HoO for Chlorella and only about 0.5 mm HgO lor chloroplasts and grana. These results provide definite proof that CO2 is indispensable for the Hill reactions. The hypothesis of water photolysis, therefore, can no longer be accepted. The following original scheme of the quinone reaction in Chlorella as well as in grana 2 quinone + 2H2O > 2 hydroquinone + O2 2 hydroquinone -|- CO 2 > 2 quinone + 2H2O + C CO2 ) C + O2 has to be replaced by 2 quinone + H4CO4 > 2 hydroquinone + 2O2 + C 2 hydroquinone + Oo * 2 quinone + 2H2O CO2 ) C + O2 According to Hill, Calvin and others, quinone oxidizes water and hydroquin- one reduces COo in the first scheme. The second scheme shows how, accord- ing to Warburg (23), quinone oxidizes CO2 (orthocarbonic acid: H4CO4) and hydroquinone reduces Oo. With respect to COo, the difTerence between photosynthesis and the Hill reactions lies in the fact that in photosynthesis the reduction product of COo is only partially (about two-thirds) reoxidized to COo. However, in the Hill reactions the reduction product of COo is completely reoxidized to CO2, so that CO2 does not appear in the over-all reaction. In the early part of this expose a clear-cut distinction was made between the energetics and the chemistry of photosynthesis. We considered them, for the sake of simplicity, to be two distinct problems. However, the reader will have noted that, in reality, there is no such line of demarcation. The more recent findings in chemistry have been successively developed from the earlier work on energetics. Warburg's discovery of the quantum requirement 3 led to the one-quantum theory advanced by Burk and Warburg. From this fruitful concept started the work done on the oxygen capacity, the labile car- bon dioxide and their relationships to chlorophyll which, in turn, led to War- burg's equations of photosynthesis and the notion of the photolyte. This fur- ther enabled Warburg to give a new explanation for the mechanism of the Hill reactions. Warburg's important fundamental discov^eries are the result of clear-sighted studies which are based not on simple theorizing but on painstaking measure- ments — measurements of a high standard with irreproachable techniques carried out along the lines of scientific thought and carefulness of the classical old style and which can stand up to objective criticism. The many unjusti- fied attacks of a polemical nature mostly hide the incompetence or lack of experimental ability of those making the attacks and will not stop the ad- 186 PROBLEMS OF PHOTOSYNTHESIS vance of scientific research. However, they will undermine the authority of Science over the uninitiated and, still worse, will bring confusion to the minds of the young generation of scientists from whom Humanity expects, as in the past, the search for truth. We may cite here Louis Pasteur's exclamation at a session of the Academy of Medicine in Paris when he had to counter the at- tacks made by the ignoramus Colin on his experiments dealing with the etiology of anthrax: "Jeunes gens qui siegez au haut de ces gradins et qui etes peut-etre I'espoir de I'avenir medical de notre pays, ne venez pas cher- cher ici les excitations de la polemique; venez vous instruire des methodes." REFERENCES 1. Kaufmann, T. and Kodding, R. : Zschr. Natiirf., I2b-J3S, 1957. 2. VisHNiAC, W. and Fuller, R. C. : Fed. Proc, 77:328, 1958. 3. Warburg, O. : Heavy Metal Prosthetic Groups. Clarendon Press, Oxford, 1949. 4. Warburg, O. : Chemischer Mechanismus der C02-Assimilation und die Theorie von Willstatter und Stoll. in Festschrift Prof. Arthur Stotl, Birkhauser, Basle, 1957. 5. Warburg, O.: Personal communication, 1957. 6. Warburg, C: Science, 725:68, 1958. 7. Warburg, O. and Christian, W. : Biochem. Zschr., 25/5:81, 1936. 8. Warburg, O., Gewitz, H.-S. and Volker, W. : Zschr. Naturf., 12b:122, 1957. 9. Warburg, O., Kayser, D.: Zschr. Naturf., 74^:563, 1959. 10. Warburg, O., Klotzsch, H. and Krippahl, G. ; Naturw., 44:235, 1957. 11. Warburg, O., Klotzsch, H. and Krippahl, G.: Zschr. Naturf., J 2b -.266, 1957. 12. Warburg, O., Klotzsch, H. and Krippahl, G.: Zschr. Naturf., 12b:AS\, 1957. 13. Warburg, O., Klotzsch, H. and Krippahl, G. : Zschr. Naturf., J2b :622 1957. 14. Warburg, O. and Krippahl, G.: Zschr. Naturf., lib -.52, 1956. 15. Warburg, O. and Krippahl, G.: Zscht. Naturf., lib -.US, 1956. 16. Warburg, O. and Krippahl, G.: Svensk Kern. Tidskr., 69:143, 1957. 17. Warburg, O. and Krippahl, G.: Zschr. Naturf., J3b:63, 1958. 18. Warburg, O. and Krippahl, G.: Zschr. Naturf., 13b:509, 1958. 19. Warburg, O. and Krippahl, G.: Zschr. Naturf., 15b:\91, 1960. 20. Warburg, O., Krippahl, G., Gewitz, H.-S. and Volker, W.: Zschr. Naturf., 7,3/^:437, 1958. 21. Warburg, O., Krippahl, G., Gewitz, H.-S. and Volker, W.: Zschr. Naturf., 14h:l\2, 1959. 22. Warburg, O., Krippahl, G. and Schroder, W.: Naturw., 43:237, 1956. 23. Warburg, O., et al.: Zschr. Naturf., in press. 24. Zelitch, I.: J. Biol. Chem., 234:3077, 1959. Addendum A New Method for the Determination of the Quantum Requirement Recently Warburg and Krippahl* worked out an improved compensation method for determining the quantum requirement of Chlorella. In using the compensation method, dense cell suspensions are used so that the resulting great respiration has to be compensated with diffuse bluish light (20% blue and blue-green, 80% green, yellow and red). The compensation light is provided by a 100 watt metal filament lamp with a reflector fixed above the thermostat. Light absorption is nearly complete. As it is not possible to measure the absorbed intensity of such a diffuse compensation light with a bolometer, the manometer vessels are replaced by actinometer vessels con- taining a solution of pheophorbide and thiourea in pyridine (see § 24). The uptake of O2 in the actinometer divided by the O2 evolution in the cell suspen- sion equals the quantum requirement for the compensation light. The de- termination of the quantum requirement for the measured light is carried out in the usual way with the two-vessel method, the light beams entering the vessels from below. Respiration is compensated ; the intensities are measured with the bolometer and the degree of absorption which varies between 93 and 95% is found with the aid of the Ulbricht sphere. It has been found that the quantum requirement decreases with decreasing light intensity. At i = 0, a limit of \/^p is obtained which has the value 3.06 for the compensation light and 2.85 for the measured light (Fig. 71). The low value of 2.85 is very near to the theoretical quantum requirement 2.7 at 100% efficiency (see § 26). It is of course irrelevant that the value for the quantum requirement is not a whole number. According to the one- quantum reaction, one quantum causes the splitting of one molecule photolyte producing one molecule O2. As discussed in § 35, at the quantum require- ment 3 of the over-all reaction, 2/3 of the O2 evolved are used to produce the energy necessary for renewal of the photolyte. If the Oo, reacting back were somewhat less than 2/3, the quantum requirement of the over-all reaction would of course be somewhat less than 3. It may seem at first glance that quantum yield determinations in photo- synthesis should be as easy as those in simple photochemical reactions. However, this is not the case and it must be repeated once again that the cells must be forced to utilize the energy of light at its maximum. As a matter of fact they do not do so when they are not cultivated under fluctuating illumi- nation, when they are illuminated without blue-green light, when the neces- Warburg, O. and Krippahl, G. Zsclir. Naturf., 15b: 190, 1960. 187 !88 PROBLEMS OF PHOTOSYNTHESIS %,. 6^ 4- "1 — \ — I — I 1 I I r 6 8 10 12 14 16 18 20 Fig. 71. Determination of the quantum requirement. Dotted line: compensation of respiration. Continuous line: measured green light (5400-5680 A). Abscissa: i in /xl quanta/min. When i = 0, the values of \/