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New perspectives on photophosphorylation have been offered from the standpoint of the torsional mechanism of energy transduction and ATP synthesis. New experimental data on the involvement of malate anions in ATP synthesis in an acid-base malate bath procedure has been reported on spinach chloroplast thylakoids as the model system. The data cannot be reconciled with the chemiosmotic theory but has been shown to be naturally explained by the torsional mechanism. The path of malic acid in the acid and base stages of the experiment has been traced, offering further strong support to the new paradigm. Classical observations in the field have been re-interpreted in the light of these findings. A new concept of ion translocation, energy transduction and coupling at the overall physiological level in photophosphorylation has been presented and a large number of novel experimentally testable predictions have been made and shown to arise as logical consequences of the new perspectives.

The extrinsic and intrinsic membrane sectors of F1F0-ATPases are linked by a slender stalk 40-50 A in length. The stalk transmits the energy produced by oxidative or photosynthetic phosphorylation from the intrinsic sector, F0, to the catalytic sites in the extrinsic F1 sector. How this is achieved is unknown, but long-range conformational changes linked to transmembrane proton transport may be involved. In bacterial and chloroplast F1F0-ATPases, the stalk is probably a composite of subunits delta and epsilon, part of the gamma-subunit, and the extrinsic membrane domains of 2 subunits (identical or non-identical according to the species) that are bound to the membrane by their N-terminal regions. The stalk in the bovine mitochondrial enzyme appears to be more complex, and the gamma, delta, epsilon, OSCP, F6, b and d subunits all contribute to it. A bovine stalk complex has been assembled in vitro from bacterially expressed OSCP, F6, b and d, both in the presence and in the absence of F1-ATPase. One molecule of each of these subunits is present in the assembled complex, as there is also in each native F1F0-ATPase assembly. Providing that suitable crystals can be obtained, the stalk complex and the F1.stalk complex may permit the high resolution structure of bovine F1-ATPase to be extended into the stalk domain.

By the early 1970s, the chemiosmotic hypothesis of Peter Mitchell was widely accepted by bioenergetics researchers as the best conceptual scheme to explain how ATP is formed in oxidative and photosynthetic phosphorylation. At about the same time, however, work from a few laboratories suggested that some aspects of that elegant, relatively simple hypothesis required revision — not abandonment, but refinement to accommodate more complex movements of protons in the ATP formation mechanism than originally envisioned by Peter Mitchell. In some situations it appeared that protons were constrained to localized domains rather than always delocalized within an enclosed vesicle as envisioned by chemiosmosis. This minireview tells that story from my perspective, as one of the researchers involved in the experimental approaches that revealed more complex energy coupling proton flux patterns. Ionic conditions during isolated thylakoid storage were found to reversibly switch the \( \Delta \tilde \mu _{H^ + } \) gradient driving ATP formation between delocalized and localized energy coupling modes. Thylakoid accessible Ca2+ ions proved to be the switching factor that was responding to the ionic conditions in the storage treatment. The mechanism of Ca2+ was at least partially demystified when it was shown that the reversible switching between \( \Delta \tilde \mu _{H^ + } \) energy coupling modes involved Ca2+ interactions with the 8 kDa CF0 (the H+ channel) subunit in a type of H+ flux gating action. Other experiments showed that the Ca2+ gating of H+ flux into the lumen may be a critical regulatory factor in controlling the lumen pH and thereby help regulate the activity of the violaxanthin de-epoxidase enzyme, a key part of the chloroplast photoprotective response to over-energization (excess light) stress.

Photosynthetic electron transport is coupled to ATP synthesis. This process – photosynthetic phosphorylation – proceeds by several alternative electron-transport pathways in isolated chloroplasts. The question: ‘Which of these works in real life?’ has long occupied students of photosynthesis. Recent results from structural biology and genomics suggest that the answer is ‘All of them’. The interplay between the pathways might explain the flexibility of photosynthesis in meeting different metabolic demands for ATP.

Osmotically disrupted chloroplasts catalyze a rapid, light and AMP and ATP dependent 32 P i incorporation into ATP. Light does not stimulate [ 14 C] AMP incorporation into ATP in this system. AMP in the presence of P i inhibits electron flow in a manner analogous to ADP inhibition in the absence of P i . The inhibition of AMP + P i is reversed on addition of ADP.

energy-that is, into ATP-within the so-called energy-coupling membranes of mitochondria and chloroplasts and within the plasma membranes of respiring or photosynthesizing bacteria (Figure 1). In the previous article, Prince describes events underlying redox-associated pumping of protons; I focus here mainly on the remarkable enzyme that actually catalyzes ATP synthesis. For comprehensive and more technical information, consult the reviews by Cross (1981), McCarty and Carmeli (1982), and Senior and Wise (1983). Several different names have been applied to the enzyme that couples ATP synthesis (and sometimes ATP hydrolysis) to proton fluxes. Because in most organisms it synthesizes ATP, the enzyme is often called ATP synthase (or ATP synthetase). The term protonATPase (or H+-ATPase) describes the reverse reaction from the one usually catalyzed but emphasizes the enzyme's ion-translocating nature. The enzyme is also frequently referred to as Fo-Fi or some minor variant. Here I use the term

The role of transmembrane electric potential difference (Δψ) in mitochondria, chloroplasts, and bacteria has been considered. Since the electric capacitance of membranes is much lower than the pH buffer capacitance of water phases, Δψ proves to be the primary form of energy produced by generators of electrochemical H+ potential difference [Formula: see text]. There are 11 distinct types of [Formula: see text]-generating systems in coupling membranes, involved in respiratory and light-dependent electron and proton transfer, as well as in ATP and PPi hydrolysis and synthesis. Bacteriorhodopsin is the simplest [Formula: see text] generator. However, even in this case, the molecular mechanism of Δψ production remains obscure. Many types of work can be supported by [Formula: see text] with no ATP involved so that [Formula: see text] proves to be not only a transient intermediate of oxidative and photosynthetic phosphorylation but also a convertible energy currency for the cell. Among the [Formula: see text]-supported activities, mechanical work was recently demonstrated. It can be exemplified by the motility systems of (i) flagellar bacteria and (ii) blue–green algae. As was found in multicellular cyanobacteria, [Formula: see text] can be used for a power transmission over distances as long as 1 mm. It seems to be probable that in large cells of eukaryotes (e.g., in muscle fibers) giant mitochondria may serve as power-transmitting structures. Na+–K+ gradients can be used to stabilize [Formula: see text] in bacteria. It is suggested that the primary function of unequal distribution of these cations between the microbial cell and the medium is [Formula: see text] buffering.

The concentrations of ADP and ATP were measured during illumination of a reconstituted chloroplast system to which NADP+, 3-phosphoglycerate and ribose 5-phosphate were successively added. Their effects on photosynthetic oxygen evolution and on the concentrations of ATP and ADP are discussed in terms of the regulation of photosynthetic carbon assimilation.

Summary Photophosphorylation has been achieved in isolated chloroplasts of Phaseolus, without inhibition, by the addition of bovine serum albumin. Cyclic and non-cyclic photophosphorylation and four partial reactions of photosynthetic phosphorylation were studied in the system with chloroplasts of different ages. There seems to be evidence for a correlation between a minimum structural complexity of the chloroplast and the ability to carry out the reactions studied.

Phenazine methosulfate (PMS) as well as pyocyanine appears to function as a catalytic cofactor for photosynthetic phosphorylation by spinach chloroplasts. CMU inhibition of pyocyanine, or of PMS in red light, is severe in air, intermediate under microaerobic conditions, and is relieved almost completely by complete absence of oxygen. The CMU inhibition of PMS under microaerobic conditions is also relieved when the PMS is reduced nonenzymically, either by ascorbate or by white light. CMU inhibition of FMN or vitamin K-catalyzed phosphorylations is not relieved by eliminating oxygen or by adding ascorbate. CMU inhibition of TPN reduction is relieved by adding a mixture of ascorbate and PMS, or of ascorbate and trichlorophenolindophenol. These results are consistent with a very general scheme proposed for electron transport in chloroplasts recognizing that repetitions of the initial step require the oxidation of one catalyst and the reduction of a second. From the nature of the response to CMU it is inferred that photooxidation of ascorbate plus trichlorophenolindophenol, and the accompanying ATP formation, represent a complex mixture of at least two reactions.

The influence of tobacco mosaic virus infection upon the Hill reaction and photosynthetic phosphorylation activities of isolated chloroplasts from directly inoculated leaves has been investigated. These enzymatic activities are not affected until approximately the seventh day after infection. The rates in chloroplasts from infected leaves then begin to decline, falling to about 60% of those from uninfected at about the ninth day after inoculation. Evidence is presented to show that this loss of enzymatic activity is a secondary effect of virus infection. Under certain conditions of plant growth and with supplemental fertilizer nitrogen, Hill reaction activity is not affected by virus infection while virus multiplication is significantly stimulated. These results have been interpreted as suggesting that virus multiplication can affect some intercellular processes by inducing a state of nitrogen stress at the cellular level.

1. 1. It is demonstrated that the major site of biosynthesis of saturated fatty acids from C10 to C18 and of oleic acid in leaves is the chloroplast. 2. 2. The co-factors required in the presence of light are ATP, CoA, Mg2+, CO2 and inorganic phosphate. 3. 3. In the dark greatly diminished synthesis occurs despite the presence of a full complement of cofactors. 4. 4. As in photosynthetic phosphorylation the synthesis of fatty acids is inhibited by NH3 and 3(p-chlorophenyl) 1,1-dimethyl urea but not by dinitrophenol. 5. 5. Under anaerobic conditions the biosynthesis of oleic acid drops markedly but that of the saturated acids is relatively unaffected. 6. 6. Isolated chloroplasts are less effective in utilising the C8, C10, C12 and C14 saturated fatty acids for the biosynthesis of oleic acid than is the intact leaf. Both malonic and acetic acids are, however, readily utilised.

Cyclic electron flow in photosynthetic phosphorylation was found to be inhibitable by concentrations of CMU higher than those needed to inhibit oxygen evolution. The inhibition studied here usually occurred only when using low concentrations of broken, aged chloroplasts and could be reversed by adding back fairly large amounts of the supernatant solution after breaking the chloroplasts. The factor responsible for reversing this inhibition was purified considerably and seemed to be associated with a diaphorase activity capable of oxidizing both TPNH and DPNH. The results were integrated in a speculative scheme in which ( a ) the higher concentration of CMU inhibited at a new site, on the reducing side of pyocyanine; and ( b ) the enzyme which can be depleted and added back functions as a CMU-resistant alternative pathway for electron flow. Relationship of this enzyme to previous ones with similar activities found in chloroplasts is discussed.

Inhibition of the Hill reaction, and of photosynthetic phosphorylation with various cofactors, has been studied using calcium, p-chloromercuribenzoate (PCMB), o-phenanthroline, and p-chlorophenyldimethylurea (CMU). Calcium and PCMB inhibit phosphorylation before the Hill reaction is affected. o-Phenanthroline and CMU appear to have the same site of action; they inhibit all reactions except phosphorylation with phenazine methosulfate. A scheme is postulated which can account for the inhibition pattern.

Illuminated spinach chloroplasts reduce quarternary dipyridyl salts with oxidation-reduction potentials ranging from −342 to −656 mV. The accumulation of reduced dipyridyl was dependent upon the oxidation-reduction potential of each dipyridyl, ranging from 100% with the 1,1′-ethylene-2,2′-dipyridylium dibromide (E0′ = −342 mV) to 3% with the 1,1′-trimethylene-2,2′-dipyridylium-4,4′-methyl dibromide (E0′ = −656 mV). The data suggest that the oxidation-reduction potential of a natural photosynthetic reductant(s) may be near −521 mV. Photosynthetic phosphorylation occurs concurrent with reduction under both argon and air. In the presence of oxygen the reduced dipyridyls are oxidized and H2O2 is produced. The dipyridyls could be substituted for spinach ferredoxin in a dark reduction of cytochrome c in the presence of NADPH and NADP+ reductase (EC 1.6.99.4). The polagraphic half-wave oxidation-reduction potential of the dipyridyls is reported.

1. 1. The extract from acetone-dried powder of washed chloroplasts shows a marked DPNH and TPNH-cytochrome c reductase activity. TPNH-cytochrome c reductase has been further purified to a specific activity nine times greater than in the extract. The enzyme requires FMN or FAD for full activity, and is severely inhibited by p -chloro-mercuribenzoic acid, while practically insensitive to KCN, NaN 3 , iodoacetate and o -iodosobenzoate. A marked TPNH oxidase activity seems associated with the reductase activity of the preparation. 2. 2. Isolated whole chloroplasts actively reduce cytochrome c in the dark in the presence of TPNH or DPNH. The rate of reduction of cytochrome is greatly enhanced by FMN, particularly with TPNH as electron donor. Participation of the TPNH-cytochrome c reductase system in the photosynthetic activity of the chloroplasts is indicated by the fact that FMN and TPN + significantly increase the rate of reduction of cytochrome c by illuminated chloroplast suspensions. 3. 3. The addition of cytochrome c to chloroplast suspensions almost doubles their photosynthetic phosphorylation activity. When TPN + is added as well, phosphorylation is further increased, but TPN + alone has little effect. The presence of FMN, a powerful activator of photosynthetic phosphorylation, augments, on an absolute basis, the effects of either cytochrome c or cytochrome c plus TPN + . 4. 4. These results, together with those of other authors, are interpreted as indicating that the role of TPN in photosynthesis is not only that of a reducing agent for phosphoglycerate, but also that of an intermediary electron carrier in photosynthetic phosphorylation.

27. SALTMAN, P., G. M. KUNITAKE, AND C. STIrr. 1956. The dark fixation of CO2 by succulent leaves. The first products. Plant Physiol. 31: 464-68. 28. SANTARIUS, K. A., V. HEBER, W. ULLRICH, AND W. URBACH. 1964. Intracellular translocation of ATP, ADP and inorganic phosphate in leaf cells of Elodea densa in relation to photosynthesis. Biochem. Biophys. Res. Commun. 15: 139-46. 29. SCOTT, G. C. AND H. R. HAYWARD. 1953. Metabolic factors influencing sodium and potassium distribution in UTlva lactuca. J. Gen. Physiol. 36: 659-71. 30. SMILIE, R. M. AND R. C. FULLER. 1959. Ribulose 1-5-diphosphate carboxylase activity in relation to photosynthesis by intact leaves and isolated chloroplasts. Plant Physiol. 34: 651-56. 31. WANG, C. H. Metabolism studies by radiorespirometry. In: Advances in Trace Methodology. S. Rothchild, ed. Plenum Press, New York, 1961. Vol 1, p 274-90. 32. WHATLEY, F. R., M. B. ALLEN, AND D. I. ARNON. 1959. Photosynthesis by isolated chloroplasts. VII. Vitamin K and riboflavin phosphate as cofactors of photosynthetic phosphorylation. Biochim. Biophys. Acta 32: 32-46. 33. WHATLEY, F. R., M. B. ALLEN, A. V. TREBST, AND D. I. ARNON. 1960. Photosynthesis by isolated chloroplasts. IX. Photosynthetic phosphorylation and CO2 assimilation in different species. Plant Physiol. 35: 188-93. 34. ZUCKER, M. AND H. T. STINSON. 1962. Chloroplasts as the major protein bearing structures in oenothera leaves. Arch. Biochem. Biophys. 96: 637-44.

1. Maize chloroplasts contain a trypsin-, dithiothreitol-, and Ca 2+ -activated ATPase. This enzyme, which can serve as a coupling factor for photosynthetic phosphorylation, differs slightly in a few properties but in general resembles a similar one in spinach plastids which was described earlier by others. 2. Maize etioplasts (immature plastids in dark-grown plants) also contain this ATPase, and it is shown that NaCl-EDTA extracts of etioplasts can restore photosynthetic phosphorylation activity to depleted green membranes of chloroplasts. 3. Electron microscopy of maize etioplast and chloroplast membranes demonstrates the presence of protruding knobs, approx. 90 A in diameter. Removal and reassociation of knobs with membranes can be correlated with the ability to carry on photosynthetic phosphorylation. 4. Most or possibly all of the coupling factor (measured as ATPase) activity of a chloroplast may be present in the etioplast from which it develops. The photosynthetic membrane of the chloroplast can be formed in stages. 5. The significance of these observations is discussed with regard to membrane formation in general and plastid membrane development in particular.

The isolation and purification of a new substance named phosphodoxin from photosynthetic organisms was described recently (7, 8). Phosphodoxin is a naturally occurring thermostable, water-soluble compound(s) which catalyzes photophosphorylation with both chloroplasts and chromatophores in the absence of exogenous electron acceptors. This report will present further properties of spinach phosphodoxin and the effects of spinach phosphodoxin on some photochemical reactions catalyzed by spinach chloroplast fragments.

Illumination of a suspension of Chlamydomonas reinhardi causes an increase in the pH of the medium which is reversed in the dark. This pH change is a manifestation of CO(2) uptake in the light and its evolution in the dark. Simultaneous measurements of pH changes and oxygen evolution reveal that the photosynthetic coefficient approaches one.Intact cells of F-60, a mutant strain of C. reinhardi that lacks an active phosphoribulokinase, do not exhibit the light-dependent pH increase or oxygen evolution. However, chloroplast fragments prepared from the cells of the mutant strain exhibit a normal "proton pump" activity.The light-dependent pH increase shown by intact cells can be inhibited by KCN, by uncouplers of photosynthetic phosphorylation, and by Dio-9. It is markedly increased upon the addition of potassium bicarbonate, and all inhibitors tested inhibit the pH increase in both the presence and absence of potassium bicarbonate.The results of the present work negate the conclusion of other workers that the light-dependent pH changes in intact cells of C. reinhardi (and probably in other algae as well) are due to the operation of the "proton pump."

The photochemical esterification of inorganic phosphate into adenosine triphosphate by chloroplasts was investigated under anaerobic conditions in the presence of either vitamin K3 or flavin mononucleotide. Evidence is presented in support of the conclusion that vitamin K3 and flavin mononucleotide are involved in separate pathways for photosynthetic phosphorylation. A tentative scheme for the generation of adenosine triphosphate in chloroplasts, consistent with this conclusion, is given.

The fatty acids of plastids from several types of mineral-deficient and senescent tissues were analyzed. Incorporation of acetate into long-chain fatty acids of leaf tissue and of plastids from nitrogen-deficient and normal plants was determined. In general, the senescent and nitrogen-deficient chloroplasts contained a higher ratio of saturates to unsaturates than did plastids from younger tissues and from tissues grown on a complete nutrient. Nitrogen-deficient leaf tissue and plastids were capable of rapidly incorporating acetate into some of the fatty acids, especially palmitic and oleic acids. However, the comparative rate of acetate incorporation into linolenic acid in nitrogen-deficient chlorophyllous tissue was less than in tissue grown on a complete nutrient. With the addition of UDP-glucose to a reaction mixture containing added cofactors for noncyclic photosynthetic phosphorylation the relative incorporation of acetate into linolenate as compared to palmitate was increased in both the nitrogen-deficient and normal leaf tissue. This would indicate that nitrogen-deficient tissues have the enzymic systems for forming long-chain fatty acids but that the reduced photosynthesis limits the amount of precursors for the formation of lipids, especially galactolipids. However, nothing is known about the rate of fatty acid degradation under these conditions.

The photochemical reduction of TPN by isolated chloroplasts was investigated. A comparison of the rate of TPN reduction with that of photosynthetic phosphorylation provided evidence that the generation of ATP in the presence of vitamin K 3 or FMN is not coupled with the reoxidation of TPNH by the oxidized product of the photolysis of water. Photosynthetic phosphorylation could proceed unimpaired under conditions in which the chloroplasts had lost their ability to reduce TPN. On the other hand, TPN reduction could be considerably stimulated by a chloroplast extract which did not affect photosynthetic phosphorylation. These results are discussed in relation to the recent finding that the reduction of TPN by chloroplasts is accompanied by ATP formation.

It was postulated and confirmed here that light scattering changes in chloroplast membranes are manifest during photosynthetic phosphorylation. The correlation of the light scattering with photophosphorylation was demonstrated by both processes having the same requirements (phosphate, ADP, and Mg ++ ) and by their inhibition in the presence of NH 4 + ions, under conditions where photosynthetic electron transport proceeds in the presence or absence of these factors. The precise interpretation of the nature of the structural changes in chloroplast fragment systems as indicated by scattering changes is still undefined, but it may be of considerable importance that the energy transfer systems both of mitochondria and chloroplasts are capable of bringing about structural changes in their membranes by the action of the phosphorylation reactions in a predictable manner.

— Measurements were made of the 3.7 msec delayed light emission of chloroplasts treated with a variety of agents which affect the rate of electron transport (Hill reaction) or photosynthetic phosphorylation. The presence of the electron acceptors ferricyanide or pyocyanine increased delayed light emission. Inhibitors of electron transport (3-(3,4-dichlorophenyl)-1, -1-dimethylurea or 1,10(ortho)-penanthroline) inhibited delayed light emission. The addition of a phosphate acceptor system inhibited delayed light emission. This inhibition was reversed by inhibitors of the phosphorylation reaction, e.g. Dio-9 or phlorizin. From these results it was concluded that the 3.7 msec delayed light emission probably occurs as a result of back reactions of intermediates in the coupled electron transport and photosynthetic phosphorylation systems.

Carbonylcyanide m -chlorophenylhydrazone is a powerful inhibitor of both photosynthetic phosphorylation and photoreduction of O 2 in isolated spinach chloroplasts. The inhibition of phosphorylation was observed in three cyclic systems (PMS, vitamin K 3 and FMN) and in three non-cyclic systems (ferricyanide-water, TPN + -water and TPN + -ascorbate). Inhibition of O 2 evolution was also obtained in the ferricyanide-water and TPN + -water systems but only at higher concentrations of CCCP. The only photochemical activity of chloroplasts which was found to be resistant to CCCP inhibition was the photoreduction of TPN + with ascorbate-dichlorophenol-indophenol as the electron donor (TPN + -ascorbate system). In the cyclic system (PMS) but not in the non-cyclic system (ferricyanide-water) CCCP inhibition of photophosphorylation varied with light intensity. Thus, in the PMS system, at 1000 lux, 0.4 μM CCCP gave 50% inhibition of photophosphorylation, whereas at 50 000 lux, 8 μM CCCP was required to give 50% inhibition. A model is proposed to account for the differences between cyclic and non-cyclic phosphorylation. The inhibition of photophosphorylation by CCCP was completely reversed by the addition of cysteine or by washing.

Our earlier results are confirmed by new experiments with ultrasonically disrupted isolated thylakoid systems and with “broken chloroplasts”: Chloroplast membrane systems are not agglutinated directly by antisera against ferredoxin-NADP reductase, although this protein is an integral part of the surface of the thylakoids. An indirect agglutination of the preparations, however, occurs, when the thylakoid systems are incubated with antisera against the reductase and then an antiserum against rabbit-γ-globulin or soluble reductase is added. Therefore the hypothetical deepenings discussed in the former publication must be located in the surface of the outer membranes of the thylakoid stacks. The thylakoid systems cannot be agglutinated directly by antibodies against reductase, because the antibody molecules are too short to connect the reductase located in the deepenings of the particles. The minimum depth of the cavities therefore must be about 60 A. The maximum depth is estimated experimentally to be about 200 A. By treatment with EDTA the molecular structure of the thylakoid surface is altered in such a manner, that after this treatment the thylakoid systems can be agglutinated by antibodies against reductase directly. This treatment with EDTA is said to detach protruding knobs of the coupling factor of the photosynthetic phosphorylation. Thus our results are in agreement with the hypothesis, that the coupling factor is a protruding knob. Ferredoxin-NADP reductase would be located in the surface of the outer membranes of the thylakoid system between the knobs. - Other possible hypotheses to explain our immunological results are excluded by several control experiments.

12. GIBBS, M. AND N. CALO. 1960. Studies on CO2 fixation by chloroplasts and the effect of arsenite and other factors. Biochim. Biophys. Acta 44: 341-47. 13. HEYTLER, P. G. 1963. Uncoupling of oxidative phosphorylation by carbonyl cyanide phenylhydrozones. I. Some characteristics of m-Cl-CCP action in mitochondria and chloroplasts. Biochemistry 2: 357-61. 14. KEISTER, D. L., A. SAN PIETRO, AND F. E. STOLZENBACH. 1960. Pyridine nucleotide transhydrogenase from spinach. I. Purification and properties. J. Biol. Chem. 235: 2989-96. 15. KEissE, D. L., A. SAN PIETRO, AND F. E. STOLZENBACH. 1961. Photosynthetic pyridine nucleotide reductase. III. Effect of phosphate acceptor system on triphosphopyridine nucleotide reduction. Arch. Biochem. Biophys. 94: 187-95. 16. KEISTER, D. L., A. SAN PIETRO, AND F. E. STOLZENBACH. 1962. Pyridine nucleotide transhydrogenase from spinach. II. Requirement of enzyme for photochemical accumulation of reduced pyridine nucleotides. Arch. Biochem. Biophys. 98: 235-44. 17. KROGMANN, D. W. 1960. Further studies on oxidative photosynthetic phosphorylation. J. Biol. Chem. 235: 3630-34. 18. KROGMANN, D. W. AND B. VENNESLAND. 1959. Oxidative photosynthetic phosphorylation by spinach chloroplasts. J. Biol. Chem. 234: 2205-10. 19. OHMURA, T. 1958. Photophosphorylation by chloroplasts. Biochem. J. 45: 319-31. 20. SAN PIETRo, A. 1961. Photochemical reduction of triphosphopyridine nucleotide by illuminated chloroplasts. In: Light and Life. W. D. McElroy and B. Glass, eds. Johns Hopkins Press, Baltimore. p 631-42. 21. TURNER, J. F., C. C. BLACK, AND M. GIBBS. 1962. Studies on photosynthetic processes. I. The effect of light intensity on triphosphopyridine nucleotide reduction, adenosine triphosphate formation, and CO2 assimilation in spinach chloroplasts. J. Biol. Chem. 237: 577-79.

Under improved experimental conditions, rates of photosynthetic phosphorylation up to 500 micromoles of orthophosphate esterified per hour per mg of chlorophyll were obtained with isolated chloroplast fragments. Expressed on a nitrogen basis these rates correspond to about 780 micromoles of orthophosphate esterified per hour per mg N. These rates are similar to maximum rates of photosynthesis in intact leaves of land plants with optimal light and CO 2 supply (about 180 micromoles of CO 2 fixed per hour per mg of chlorophyll) and are several times higher than rates of oxidative phosphorylation from mitochondria from various plant and animal sources. The significance of these rates in the evaluation of the role of photosynthetic phosphorylation as a source of ATP in photosynthesis is discussed.

When bean plants are cultivated under certain conditions, liberation of cell contents by grinding with mortar and pestle yields chloroplasts of low Hill and photosynthetic phosphorylation activities, while grinding in a high speed blendor yields active chloroplasts. The inactivity of plastids obtained with mortar and pestle is due to the presence of an inactivator in the homogenate. The culture conditions that determine whether bean leaves ground with mortar and pestle contain inactivator were not determined.

The role of ascorbic acid (AA) in photosynthesis and respiration has been discussed in detail by various workers (Aberg, 1958; Mapson, 1958; Beevers, 1961; Chinoy, 1962; San Pietro et al., 1967; Rabinowitch and Govindjee, 1970; Krogmann, 1973). Conclusive evidence has been presented to show that chlorophyll bearing tissues (including leaves, green tomatoes etc.,) are responsible for the greater accumulation of AA in light grown plants compared with that in the dark grown ones (Reid, 1937; Prokoshev and Dancheva, 1946; Popovskaya, 1950; Haggen, 1953; Asselbergs, 1957; Hoffmann and Albrecht, 1966). Significant correlations between seasonal variations in photosynthetic phosphorylation, photochemical transphosphorylation and the Hill reaction of spinach leaves harvested in different seasons and the ability of these leaf extracts to synthesize AA from its precursor was reported (Mitsui and Oi, 1961). These authors Mitsui and Oi, 1961a) also found that all the photoproduct or the major portion of isolated chloroplast was AA, whereas, the heat labile precursor of AA was found in chloroplast fraction. Thus the chloroplast appears to be an important seat of AA in green tissues of plants.

In cellular energy transformations, the ATP-ADP cycle plays a central role by coupling both with energy-yielding reactions, e.g., oxidative phosphorylation in mitochondria and photosynthetic phosphorylation in chloroplasts, as well as with energy-requiring reactions including active transport, muscular contraction, and biosynthesis.

Photosynthetic phosphorylation, the light-driven synthesis of ATP from ADP and Pi, in chloroplasts was discovered over twenty years ago by Arnon et al. (1954). Within a few years, it was firmly established that photosynthetic phosphorylation (photophosphorylation in short) is coupled to light-dependent electron flow through an electron transport chain rather similar to that in mitochondria (Jagendorf, 1958; Arnon, 1958). However, the realization that ion fluxes might have something to do with photophosphorylation is only a decade old.

Ionising radiation influences major biochemical processes occurring in cell plastids. Changes were observed by several authors following β and γ irradiation. FUCHTBAUER (1961) reported inhibition of photosynthetic phosphorylation to 50% at the dose of 130 krads and of Hill’s reaction at 530 krads. There are no available data however, concerning the effects of neutron irradiation and of small doses of ionising radiation on chloroplasts.

Publisher Summary This chapter discusses role of cytochromes and other metalloproteins in the photosynthetic electron transport. Current understanding of the mechanism of photosynthesis leans heavily on a concept of a photosynthetic, i.e., light-induced, electron transport. Photosynthetic phosphorylation (photophosphorylation) in chloroplasts is subdivided into two types, cyclic and noncyclic. The cyclic and noncyclic photophosphorylation jointly account for the basic feature of photosynthesis, i.e., conversion of radiant energy into chemical energy. The wavelength dependence of the phosphorylation associated with electron flow from an artificial electron donor (DPIPH2) to NADP resembles the cyclic system. It is well established that treating chloroplasts with ferricyanide in the dark chemically oxidizes several chloroplast constituents including cytochromes. The spectrum of the photoinduced decrease in absorbance had a maximum at 550 nm that was suggestive of an α-peak of a new cytochrome of a c-type.

Chloroplasts from bean leaves stored in the dark at 0–4 °C. have low Hill activity but good photosynthetic phosphorylation activity. Hill activity can be restored by incubating leaves in the light at 20 °C. Comparison of photosynthetic activities of chloroplasts from cold-stored and fresh leaves suggests that cold-stored leaves have lost the ability to remove photosynthetically produced photooxidant by the process of oxygen evolution.

The light-dependent synthesis of ATP by chloroplasts has been studied without the addition of any redox cofactor. Under these conditions, photophosphorylation is oxygen dependent and 3-(p-chlorophenyl)-I,I-dimethylurea-sensitive. Addition of photosynthetic pyridine nucleotide reductase stimulates this “endogenous” photophosphorylation, a as does the addition of ascorbate. Ascorbate is acting as a catalyst in the system, and its effect is dependent on the presence of O2. Ascorbate-supported photophosphorylation is inhibited by cyanide, while the “endogenous” is not. The significance of these results is discussed, and a speculative interpretation is suggested.

1. 1. Procedures are described for the preparation of chloroplasts capable of carrying out three photochemical reactions, each representing an increasingly complex phase of photosynthesis: photolysis of water (Hill reaction), esterification of inorganic phosphate into adenosine triphosphate (photosynthetic phosphorylation) and the reduction of carbon dioxide to the level of carbohydrates with a simultaneous evolution of oxygen. 2. 2. The three photochemical reactions were separable by variations in the technique for preparation of chloroplasts and by differential inhibition by several reagents. Inhibition of a more complex phase of photosynthesis does not affect the similar one which precedes it and, conversely, the inhibition of a simpler phase of photosynthesis is paralled by an inhibition of the more complex phase which follows. 3. 3. Reversible inhibition of CO 2 fixation and photosynthetic phosphorylation, but not of photolysis, by sulfhydryl group inhibitors suggests that sulfhydryl compounds (enzymes, cofactors, or both) are involved in phosphorylation and CO 2 fixation, but not in the primary conversion of light into chemical energy as measured by the Hill reaction. 4. 4. Evidence is presented in support of the conclusion that the synthesis of ATP by green cells occurs at two distinct sites: anaerobically in chloroplasts, by photosynthetic phosphorylation, and aerobically in smaller cytoplasmic particles, presumably mitochondria, by oxidative phosphorylation independent of light. 5. 5. A general scheme of photosynthesis by chloroplasts, consistent with these findings, is presented.

Chloroplast preparations from spinach leaves containing a high percentage of intact chloroplasts were capable of light dependent nitrite reduction at rates around 9 μmol/mg chlorophyll/h for at least 50 min. This reduction was inhibited by DCMU but unaffected by uncouplers of photosynthetic phosphorylation. Nitrite reduction was not accompanied by a stoichiometric evolution of oxygen evolution. The disappearance of nitrite was accompanied by an approximately stoichiometric formation of reduced nitrogen.

The phenomenon of light-induced changes of chloroplast volume is well known at present. It is related to the functional state of the photosynthetic apparatus, and first of all with the chain of reactions of photosynthetic phosphorylation coupled to electron transport (PACKER 1962, ITOH et al. 1963, IZAWA et al. 1963, PACKER 1963, KUSHIDA et al. 1964). The changes of chloroplast volume are to a large extent connected with the movement of ions and osmoregulating processes, which control water supply of chloroplasts (PACKER & SIEGENTHALER 1 9 6 6, PACKER et al. 1966, PACKER et al. 1967, NOBEL 1968, MURAKAMI & PACKER 1970, ALIEVA et al. 1971). The present report deals with some results of our studies on the ultrasotructural organization of chloroplasts, the content of pigment, the intensity of photosynthesis in vivo, phososynthetic phosphorylation, and photoinduced changes in the volume of isolated chloroplasts from leaves of plants grown under various water supply (ALIEVA et al. 1971).

It is well known that chromatophores of Rhodospirillum rubrum form pyrophosphate in the light in the absence of exogenous ADP (BALTSCHEFFSKY 1966) and that several metabolic processes may be driven by pyrophosphate in this organism: the reduction of cytochrome b (BALTSCHEFFSKY 1967), absorption changes of carotenoids (BALTSCHEFFSKY 1969), the energy-dependent transhydrogenase (KEISTER & YIKE 1967), C02-fixation (PFLUGER & BACHOFEN 1971) and ATP-formation itself in the dark (KEISTER & MINTON 1971). It seems therefore, that pyrophosphate is in close contact with the cytoplasmic membrane during its formation and hydrolysis.Since electron transport is bound to the membrane, it seems reasonable that the phosphate molecule is at least for a certain time fixed to compounds of the membrane, where the formation of ATP and PP can occur. The idea that a non-phosphorylated and a phosphorylated energy-rich intermediate participate in the reaction sequence forming ATP or PP is well accepted. So far no soluble energy-rich phosphorylated compound has been detected in oxidative or photosynthetic phosphorylation, while in mitochondria (CROSS et al. 1970) and chloroplasts (HINKSON & BOYER 1965) a rapid incorporation of P into insoluble proteins has been observed. Therefore it seems reasonable that the phosphorylated intermediate remains bound to the membrane particles. This communication is a preliminary report of an attempt to isolate membrane fragments with bound energy-rich phosphate.