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Significant strides toward producing biochemical fuels have been achieved by mimicking natural oxidative and photosynthetic phosphorylation. Here, different from these strategies, we explore boric acid as a fuel for tuneable synthesis of energy-storing molecules in a cell-like supramolecular architecture. Specifically, a proton locked in boric acid is released in a modulated fashion by the choice of polyols. As a consequence, controlled proton gradients across the lipid membrane are established to drive ATP synthase embedded in the biomimetic architecture, which facilitates tuneable ATP production. This strategy paves a unique route to achieve highly efficient bioenergy conversion, holding broad applications in synthesis and devices that require biochemical fuels.

High light causes disturbances in photosynthetic phosphorylation or damage to the photosystem II (PSII) structure or even assimilation tissues. The value of the red/ far-red ratio (R/FR) provides the plant with information on the environmental light conditions, regulating, among others, photosynthetic activity and pigment composition of the plant. The response of the photosynthetic apparatus of the sporotrophophylls and nest leaves of Platycerium bifurcatum, grown for 6 months at the low or high R/FR ratio, were studied. Later, the plants were transferred to high light (1,200 µmol quantum • m−2 • s−1). Changes in PSII photochemical activity were determined based on non-destructive methods of chlorophyll a fluorescence kinetics analysis. The measurement of radiation reflectance from the leaves allowed to determine the content of selected pigments related to the photosynthesis process and to assess changes in the Photochemical Reflectance Index. The calculation of reflectance difference and sensitivity analysis was used to identify so-called “stress-sensitive wavelengths”. Plant growth at high R/FR ratio prepares photosynthetic apparatus of ferns to high light and enables more efficient conversion of absorbed photons. The increase in the amount of photoprotective compounds allows the protection against photoinhibition in the sporotrophophyll leaves that play key roles in plant nutrition and reproduction.

The effects of superoxide dismutase (SOD) inhibitors, diethyldithiocarbamate (DDC), triethylenetetramine (trien), and their combination with glucose on cells of the epidermis from pea leaves of different age (rapidly growing young leaves and slowly growing old leaves) was investigated. DDC and trien caused death of the guard cells as determined by destruction of their nuclei. Glucose did not affect destruction of the nuclei induced by SOD inhibitors in the cells from old leaves, but intensified it in the cells from young leaves. 2-Deoxyglucose, an inhibitor of glycolysis, and propyl gallate, SOD-mimic and antioxidant, suppressed destruction of the nuclei that was caused by SOD inhibitors and glucose in cells of the epidermis from the young, but not from the old leaves. Glucose and trien stimulated, and propyl gallate reduced generation of reactive oxygen species (ROS) in the pea epidermis as determined by the fluorescence of 2',7'-dichlorofluorescein (DCF). Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a protonophoric uncoupler of oxidative and photosynthetic phosphorylation, suppressed the DCF fluorescence in the guard cells. Treatment of the cells with CCCP followed by its removal with washing increased destruction of the nuclei caused by SOD inhibitors and glucose. In young leaves, CCCP was less effective than in old ones. The findings demonstrate the effects of SOD inhibitors and glucose on the cell death and generation of ROS and could indicate glycolysis-dependent ROS production.

Textbooks of biochemistry will explain that the otherwise endergonic reactions of ATP synthesis can be driven by the exergonic reactions of respiratory electron transport, and that these two half-reactions are catalyzed by protein complexes embedded in the same, closed membrane. These views are correct. The textbooks also state that, according to the chemiosmotic coupling hypothesis, a (or the) kinetically and thermodynamically competent intermediate linking the two half-reactions is the electrochemical difference of protons that is in equilibrium with that between the two bulk phases that the coupling membrane serves to separate. This gradient consists of a membrane potential term Δψ and a pH gradient term ΔpH, and is known colloquially as the protonmotive force or pmf. Artificial imposition of a pmf can drive phosphorylation, but only if the pmf exceeds some 150–170 mV; to achieve in vivo rates the imposed pmf must reach 200 mV. The key question then is ‘does the pmf generated by electron transport exceed 200 mV, or even 170 mV?’ The possibly surprising answer, from a great many kinds of experiment and sources of evidence, including direct measurements with microelectrodes, indicates it that it does not. Observable pH changes driven by electron transport are real, and they control various processes; however, compensating ion movements restrict the Δψ component to low values. A protet-based model, that I outline here, can account for all the necessary observations, including all of those inconsistent with chemiosmotic coupling, and provides for a variety of testable hypotheses by which it might be refined.

The vital coupled processes of oxidative phosphorylation and photosynthetic phosphorylation synthesize molecules of adenosine-5'-triphosphate (ATP), the universal biological energy currency, and sustain all life on our planet. The chemiosmotic theory of energy coupling in oxidative and photophosphorylation was proposed by Mitchell >50years ago. It has had a contentious history, with part of the accumulated body of experimental evidence supporting it, and part of it in conflict with the theory. Although the theory was strongly criticized by many prominent scientists, the controversy has never been resolved. Here, the mathematical steps of Mitchell's original derivation leading to the principal equation of the chemiosmotic theory are scrutinized, and a fundamental flaw in them has been identified. Surprisingly, this flaw had not been detected earlier. Discovery of such a defect negates, or at least considerably weakens, the theoretical foundations on which the chemiosmotic theory is based. Ad hoc or simplistic ways to remedy this defect are shown to be scientifically unproductive and sterile. A novel two-ion theory of biological energy coupling salvages the situation by rectifying the fundamental flaw in the chemiosmotic theory, and the governing equations of the new theory have been shown to accurately quantify and predict extensive recent experimental data on ATP synthesis by F1FO-ATP synthase without using adjustable parameters. Some major biological implications arising from the new thinking are discussed. The principles of energy transduction and coupling proposed in the new paradigm are shown to be of a very general and universal nature. It is concluded that the timely availability after a 25-year research struggle of Nath's torsional mechanism of energy transduction and ATP synthesis is a rational alternative that has the power to solve the problems arising from the past, and also meet present and future challenges in this important interdisciplinary field of research.

The pathway of adenosine triphosphate (ATP) synthesis in living organisms consists of two autonomous stages; this must be taken into account during the design and analysis of chemical models of the abiogenesis of this key participant of metabolic processes. The first stage is construction of an adenine heterocycle linked to a ribose-5-phosphate molecule to yield AMP, while the following stage is the attachment of phosphoryl residues to the nucleotide molecule by macroergic phosphoanhydride bonds. Involvement of the same set of precursor molecules in both de novo biosynthesis of AMP and abiogenesis of this nucleotide is a very important issue for the analysis of metabolic pathways. Photochemical matrix systems that convert light energy into macroergic bond energy are functional prototypes of photosynthetic phosphorylation; they are of special interest for the construction of abiotic phosphorylation models. Interaction of substrates with a purely mineral matrix (montmorillonite particles) under UV irradiation resulted in the formation of ATP from ADP and orthophosphate. Micro-and nanostructures that formed upon the interaction of the mineral component (sodium polysilicate) with an abiogenic organic pigment (a flavin conjugate with a random amino acid polymer) exhibited phosphorylating activity as well when irradiated with visible light. The properties of AMP and ATP abiosynthesis models investigated are in good accordance with the current views on the environmental conditions of the ancient Earth; evident structural differences exist between these models and the biosynthetic systems in modern organisms.

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.

Cyanide is an apoptosis inducer in stoma guard cells from pea leaf epidermis. Unlike CN-, the uncoupler of oxidative and photosynthetic phosphorylation carbonyl cyanide m-chlorophenylhydrazone (CCCP), the combination of CCCP, 3-(3 ,4 -dichlorophenyl)-1,1-dimethylurea (DCMU), benzylhydroxamate (BH), myxothiazol, antimycin A, and a glycolysis inhibitor 2-deoxyglucose (DG) did not induce destruction of guard cell nuclei for 20 h of incubation of epidermal peels in the light. DCMU prevented the effect of CN- as a programmed cell death (PCD) inducer. CCCP, the combination of DCMU and CCCP, or the combination of DCMU, CCCP, BH, myxothiazol, antimycin A, and DG supplemented by CN- caused destruction of cell nuclei; the number of the cells lacking nuclei in this case was higher than with CN- alone. DG and CCCP caused cell destruction after longer incubation of the isolated epidermis - after 2 days and to a greater degree after 4 days. The effect of DG and CCCP was reduced by illumination. Cell destruction during long-term incubation was prevented by the combination of DG and CCCP. From data of electron microscopy, DCMU and dinitrophenyl ester of iodonitrothymol (DNP-INT) prevented apoptotic changes of the nuclear ultrastructure induced by CN-. The suppression of the destruction of the guard cell nuclei under combined action of DG and CCCP was apparently caused by switching of cell death from PCD to necrosis. Thus, the type of cell death - via apoptosis or necrosis - is controlled by the level of energy provision.

Cytochrome c oxidase is essential for aerobic life as a membrane-bound energy transducer. O(2) reduction at the haem a(3)-Cu(B) centre consumes electrons transferred via haem a from cytochrome c outside the membrane. Protons are taken up from the inside, both to form water and to be pumped across the membrane (M.K.F. Wikström, Nature 266 (1977) 271; M. Wikström, K. Krab, M. Saraste, Cytochrome Oxidase, A Synthesis, Academic Press, London, 1981 ). The resulting electrochemical proton gradient drives ATP synthesis (P. Mitchell, Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation, Glynn Research, Bodmin, UK, 1966 ). Here we present a molecular mechanism for proton pumping coupled to oxygen reduction that is based on the unique properties of water in hydrophobic cavities. An array of water molecules conducts protons from a conserved glutamic acid, either to the Delta-propionate of haem a(3) (pumping), or to haem a(3)-Cu(B) (water formation). Switching between these pathways is controlled by the redox-state-dependent electric field between haem a and haem a(3)-Cu(B), which determines the water-dipole orientation, and therefore the proton transfer direction. Proton transfer via the propionate provides a gate to O(2) reduction. This pumping mechanism explains the unique arrangement of the metal cofactors in the structure. It is consistent with the large body of biochemical data, and is shown to be plausible by molecular dynamics simulations.

The perspectives and enthusiasms recorded in this review describe the events I witnessed and, in small ways, contributed to. Two great rewards emerged from my experiences – the pleasure of doing experiments and the great wealth of friendships with students and colleagues. As a graduate student, phenomena appeared at the bench before me which clarified the coupling of electron transport to ATP synthesis. My first PhD graduate student measured concentrations of pyridine nucleotides in chloroplasts and his results have been often confirmed and well used. All of the many graduate students who followed contributed to our understanding of photosynthesis. I have taken much pleasure from documenting the details of photosynthetic phosphorylation and electron transport in cyanobacteria. Studies of the `c' type cytochromes in these organisms continue to fascinate me. My experiences in government in its efforts to promote research are unusual, perhaps unique. A rare event outside the laboratory – a natural bloom of cyanobacteria – stimulated new thoughts and special opportunities for laboratory science. Photosynthesis seems magisterial in its shaping of our planet and its biology and in the details of its cleverness that were revealed in the time of my witness.

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.

My twenty-five year fascination with membrane ATPases grew out of my experiences in the laboratories of André Jagendorf and Efraim Racker. André introduced me to photosynthetic phosphorylation and Ef, to whose memory this article is dedicated, convinced me that ATPases had much to do with ATP synthesis. Astounding progress has been made in the H+-ATPase field in just two decades. By the early 1970s, it was generally recognized that oxidative and photosynthetic ATP synthesis were catalyzed by membrane enzymes that could act as H+-ATPases and that the common intermediate between electron transport and phosphorylation is the electrochemical proton gradient. At that time, it had been shown that a cation-stimulated ATPase activity was associated with plasma membrane preparations from plant roots. The endomembrane or vacuolar ATPases were unknown. The application of improved biochemical methods for membrane isolation and purification, as well as membrane protein reconstitutions, led rapidly to the conclusion that there are three major classes of membrane H+-ATPases, P, V and F. P-ATPases, which will not be considered further in this article, are phosphorylated during their catalytic cycle and have a much simpler polypeptide composition than V- or F-ATPases. The plasma membrane H+-ATPase of plant, yeasts and fungal cells is one example of this class of enzymes (see Pedersen and Carafoli, 1987, for a comparison of plasma membrane ATPases). Biochemical and gene sequencing analysis have revealed that V- and F-ATPases resemble each other structurally, but are distinct in function and origin. The 'V' stands for vacuolar and the 'F' for F1Fo. F1 was the first factor isolated from bovine heart mitochondria shown to be required for oxidative phosphorylation. Fo was so named because it is a factor that conferred oligomycin sensitivity to soluble F1. Other F-ATPases are often named to indicate their sources. For example, chloroplast F1 is denoted CF1 (see Racker, 1965, for early work on F1). Recent successes in reconstitution of vacuolar ATPase have led to a V1Vo nomenclature for this enzyme as well. The term 'ATP synthase' is now in general use to describe F-ATPases. This term emphasizes the facts that although F-ATPases function to synthesize ATP, they do not catalyze, normally, ATP hydrolysis linked to proton flux. In contrast, V-ATPases are very unlikely to operate as ATP synthases. Thus, F-ATPases are proton gradient consumers, whereas V-ATPases generate proton gradients at the expense of hydrolysis. In this brief review, I will compare the structures of F- and V-ATPases. Also, I give some insight into the mechanisms that help prevent wasteful ATP hydrolysis by the chloroplast ATP synthase (CF1Fo).

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.

This history traces the development of photosynthesis research in Canada from 1945 to 1975, starting with the work of Gleb(1) Krotkov and his students, Paul Vittorio, Tony(1) Bidwell, Don(1) Nelson, Jim(1) Craigie, Bruce Tregunna, Andreas Hauschild, Geoff Lister and others in the Department of Biology at Queen's University, Kingston, Ontario. They focused on the influence of taxonomy and light quality on the path of carbon into early products, photorespiration and photosynthesis in young trees. During the same period, Ken(1) Clendenning and one of the authors (PRG) at the National Research Council of Canada (NRCC) laboratory in Ottawa began studies of chloroplast photoreduction and leaf carboxylases. They were joined by Don(1) Mortimer, who showed that the path of carbon varies with species of plant and by Morris Kates, who studied phospholipid enzymology in chloroplasts and leaves. Stan(1) Holt researched the chemistry and distribution of chlorophylls in different taxa. In 1952, Ralph Lewin joined NRCC's new Atlantic Regional Laboratory in Halifax, Nova Scotia, followed by Jim Craigie, Jack McLachlan and Tony Bidwell who mainly investigated the products of photosynthesis in marine algae. Tony Bidwell continued these studies in 1959 at the Department of Botany, University of Toronto. Dave(1) Canvin joined the staff at Queen's in 1965 and became involved in solving the mystery of photorespiration. Tony Bidwell returned to Queen's in 1969 and studied photosynthesis of algal chloroplasts using an 'artificial leaf.' In 1965, Don Nelson established a group at Simon Fraser University in Burnaby, British Columbia that included his former student, Geoff Lister who produced the first photosynthetic action spectra for trees, and Bill(1) Vidaver, who showed the useful relation between chlorophyll fluorescence and photosynthetic activity. In 1970, Mário Fragata headed a group at the Université du Québec à Trois Rivières, Québec, that began with studies of Photosystem II in chloroplasts and particles.

Uncouplers of photosynthetic phosphorylation, CCCP, TTFB and PCP, inhibited light-induced O2 evolution in the Hill reaction with SiMo (I50 approximately 20, 3 and 45 microM, respectively), but only insignificantly diminished SiMo photoreduction by pea chloroplasts. The same properties were exhibited by the ADRY agent ANT2p. CCCP, TTFB and PCP are oxidizable compounds with redox potentials of +1.17, +1.18 and +1.09 V (pH 6.0), as determined by cyclic voltammetry. Similarly to NH2OH, the tested uncouplers can apparently serve as electron donors for photosystem II.

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.

This chapter presents a general introduction to functions and structures of membrane transport proteins, which catalyze the translocation of substrates through the hydrophobic bilayer membrane of cells. It defines some terms used in the characterization of transport processes, such as passive diffusion, facilitated diffusion, active transport, and group translocation. Thousands of transport processes, each catalyzed by its own protein, have been identified; however, there are relatively few strategies found in coupling metabolic energy to the translocation process. The chapter describes these strategies to provide a formal basis for a preliminary classification of all the processes. It describes the chemiosmotic theory of oxidative and photosynthetic phosphorylation and applies it to explain substrate transport mechanism. It tabulates the names and examples of several families of transport proteins. Facilitated diffusion transport systems usually contain a single protein. Secondary active transport systems usually contain one protein, if those that generate the driving ion gradient are not counted. Primary active transport systems may occasionally contain one protein, for example, bacteriorhodopsin; however, most appear to comprise a protein complex, involving from as few as 2–20 or more domains.

Nitrosubstituted phenols (NP), the compounds widely used for explosives production, dye, polymers, drugs and pesticide synthesis, and released into the environment with industrial wastes and with agricultural chemical preparations, are highly toxic pollutants. Toxicity of NP is based on their capacity to uncouple oxidative and photosynthetic phosphorylation and to inhibit envelope and membrane associated enzymes. According to the data of the EPA (USA) (cited in Ref. [1]) o-,p-nitro and 2,4-dinitrophenols are the most toxic [1–3].

Ascorbate is oxidized to the free radical monodehydroascorbate by O2.- and by H2O2 (through the action of ascorbate peroxidase) formed in the Mehler reaction by isolated spinach (Spinacia oleracea) thylakoids. Light-dependent electron transport from water to monodehydroascorbate is shown to be coupled to ATP formation with a ratio ATP:O2 of 2. In the presence of ascorbate the net O2 exchange balance of the Mehler reaction is close to zero, and the synthesis of ATP is increased 2 to 3 times due to the extra electron transport to the monodehydroascorbate free radical. A scheme of the electron transport in the presence of ascorbate is discussed.

Publisher Summary This chapter discusses the structure and assembly of ATP synthase. The ATP synthase or F 0 F 1 -ATPase catalyses the formation of ATP from ADP and P i in oxidative or photosynthetic phosphorylation. This membrane-spanning enzyme is located in mitochondria, chloroplasts or the cytoplasmic membrane of bacteria and is structurally conserved. The multiple functional aspects of ATP Synthases are shared amongst many subunits. It is possible to discriminate two major types of enzyme complex organization. The first is the more simple bacterial and chloroplast type, which has a clearly demarcated F 1 sector carrying nucleotide binding and catalytic properties, and a membrane-embedded F 0 sector which contains the proton channel and, in conjunction with F 1 , the energy-transducing or coupling functions of the complex. The biogenesis of mitochondrial ATP synthase requires contributions from both the mitochondrial and nuclear genomes. At least two subunits are encoded in mitochondrial DNA of all organisms so far examined.

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.

Two electrophoretically distinguishable species of the 13S coupling factor of oxidative phosphorylation from Alcaligenes faecalis are detectable by standard polyacrylamide gel electrophoresis in the absence of urea, detergents, or any other protein-denaturing reagents. The slower species (type IA) can be converted into the faster species (type IB) by treatment with ATP, and the fast form converts into the slow form when aged at 4 degrees. The enzyme undergoes these conversions both when it is free in solution and when it is membrane bound. The ATP analog adenylyl imidodiphosphate (AMP-PNP) gives the conversion without being hydrolyzed and without causing any apparent change in the mass of the protein, which suggests that the conversion may be a ligand-induced conformational change. Types IA and IB can convert into three other electrophoretically distinguishable species (types IIA, IIB, and III) if the purification procedure involves chromatography on a DEAE-Sephadex column equilibrated in phosphate buffer. These conversions can be prevented if the column is eluted in morpholinoethanesulfonic acid (Mes) buffer and KCl. Type IIA is convertible into type IIB by ATP treatment. Types IA and IB will also convert into types IIA and IIB and finally into type III when aged for extended periods of time at 4 degrees, without a detectable change in mass. Coupling factor activity is lost when type I enzyme converts into type II enzyme, as is the ability of the enzyme to bind to the membrane. However, ATPase activity does not change significantly. The mitochondrial 13S coupling factor shows up to three electrophoretically distinguishable species. The use of phosphate buffer during DEAE-Sephadex chromatography gives conversion of slower species into faster species. ATP treatment does not give interconversions, and aging at 4 degrees gives only a slow dissociation of the enzyme into subunits. The chloroplast 13S coupling factor also shows up to three electrophoretic species. Incubation with ATP does not give interconversions, but a temperature-dependent conversion of the major species into a faster species occurs upon aging. The subunit composition of the three 13S enzymes is very similar by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the major difference being in the number of classes of small polypeptides.

Robinson, J. M. 1988. Does O2 photoreduction occur within chloroplasts in vivo? -Physiol. Plant. 72: 666–680. This discussion reviews evidence supporting the hypothesis that within intact chloroplasts in vivo, molecular O2 may serve as an alternative Hill oxidant (electron acceptor) on the reducing side of Photosystem I. Depending upon the availability of Hill oxidants such as NADP+ and NO−2, there is the potential within intact plastids in vivo, for photolytically derived reducing equivalents to reduce O2 to O−2 and H2O2 (the Mehler reaction). In chloroplasts of healthy tissues, the products of photosyn-thetic O2 reduction O−2 and H2O2) are rapidly removed by superoxide dismutase (EC 1.15.1.1) and L-ascorbate peroxidase (EC 1.11.1.11) to prevent toxicity. The presence of these two enzymes within chloroplasts in vivo reflects the potential for linear (non-cyclic) photosynthetic electron transport systems to draw upon molecular O2 as a terminal oxidant. In the intact plastid, O2 may act as an electron acceptor in the place of any other physiological Hill oxidant, e.g., NADP+, NO−2, and, presumably, oxidized thioredoxin. Under aerobic, physiological conditions, photo reduced ferre-doxin (Fdred), and/or reduced flavoprotein enzymes, e.g., ferredoxin:NADP+ oxidoreductase (EC 1.18.1.2), can donate electrons to O2; this reductive reaction appears to be non-enzymatic, but it is rapid. Stated from another viewpoint, O2 may serve as a Hill oxidant to support some linear electron flow when reductant supplies are in excess of reductant demands. For example, there are nitrogen assimilatory sites in the chloroplast, i.e., ferredoxin-nitrite reductase (NiR; EC 1.7.7.1) and glutamate synthase (ferredoxin) (GOGAT; EC 1.4.7.1), to which Fdred is allocated as reductant. Because NADH:nitrate reductase (NR; EC 1.6.6.1) is the rate limiting step of nitrogen assimilation, and, because NiR and GOGAT activities are in excess of NR activities by a factor of 2 or more, then an excess of unreacted Fdred could accumulate. Alternatively, the allocated Fdred would reduce the excess NiR and GOGAT sites, but the excess of reduced enzymes would not have substrates (e.g., NO−2, glutamine, and α-ketoglutarate) with which to react. Therefore, if ‘excess’ NiR and GOGAT binding sites were not employed, the available excess Fdred, and/or the reduced NiR and GOGAT proteins, would be susceptible to oxidation by O2. The resulting O2 photoreduction could account for nearly all of the observed in vivo Mehler type reactions. In vivo, apparent foliar O2 photoreduction occurs simultaneously with maximal CO2 photoassimilation, and, in high light, average rates have been determined by direct measurement to range from 10 to 40 μmol O2 consumed (mg Chl)−1 h−1. Therefore O2 reduction would support a low rate of linear (non-cyclic) electron flow which, in turn, could maintain a low, but significant rate of ATP production. However, there is not total agreement among researchers that the physiological role of O2 is that of serving as an alternative Hill oxidant in order to recycle unutilized Fdred or other photoreduced proteins. Also, there continues to be considerable controversy on whether or not O2 reduction supports significant photosynthetic phosphorylation. The total process of O2 photoreduction, and its physiological role(s), requires much more study before absolute functions can be assigned to O2 terminated, linear electron transport. Summary Molecular O2 possesses the physico-chemical properties that permit this molecule to serve as an alternative Hill oxidant within chloroplasts in vivo. Additionally, the physical and physiological properties within the chloroplast in vivo favor the potential for O2 to serve as an electron acceptor on the reducing side of Photosystem I. This may reflect an important ‘fail-safe mechanism’ which prevents over-reduction of linear photosynthetic electron transport chain proteins. This review has focused on the possibility that unutilized Fdred and/or other non-utilized, reduced plastid enzymes (e.g., NiR) may be electron donors to O2. It is hypothesized that this oxidation ultimately would be reflected as an in vivo Mehler reaction. However, it remains for future studies to establish without doubt, that in vivo, photoreduced chloroplast enzyme proteins can utilize O2 as a terminal electron acceptor. Further, that O2 photoreduction supports a significant level of photophosphorylation in vivo remains to be firmly established. Certainly, considerable evidence, gained with experiments utilizing isolates of intact chlo-roplasts and reconstituted chloroplast systems, supports the hypothesis that O2-terminated linear electron transport has the potential to support high rates of ATP production. However, in vivo studies e.g., with intact leaf tissues, which actually quantitate the relationship between O2 photoreduction and associated ATP production have not been conducted. These will be difficult experiments to perform, because, in vivo, it will be difficult to separate photosynthetic ATP production mediated by O2 from ATP production mediated by those other, more predominant Hill oxidants (e.g., NADP+, NO−2). Also, it continues to be a possibility that it is cyclic, and not pseudocyclic photophosphorylation that provides additional ATP to support photosynthetic cell metabolism. To establish beyond doubt that an in vivo role of the Mehler reaction is that of supplying ‘additional ATP’, remains for considerable future study.

Exposure of thylakoids free of vacuolar proteases to white light causes the loss of several thylakoid bound polypeptides. At a light intensity of 1,500 μ E m -2 s -1 , such loss is apparent within 5 min although this light intensity does not saturate the reaction. This degradation of thylakoid polypeptides proceeds most rapidly at a pH of 9.0. The rate of polypeptide degradation can be increased by incubation of thylakoids with low concentrations of the detergents Triton X-100 or SDS. Inclusion of an electron transport inhibitor or an uncoupler Of photosynthetic phosphorylation in the assay had no effect on the loss of thylakoid polypeptides in the light. Pre-digestion of thylakoids with trypsin or denaturing thylakoid proteins in a buffered solution of 2 % SDS, 6 M urea at 100 °C for five min prior to the assay did not prevent the loss of thylakoid polypeptides. These data strongly suggest that the light-stimulated loss of polypeptides is not mediated by a protease. The loss of thylakoid polypeptides could be prevented by a variety of reducing agents or by maintaining thylakoids in an anaerobic environment. These data suggest that a species of activated oxygen, probably singlet oxygen, is responsible for the loss of thylakoid polypeptides in the light.

Summary 2-Deoxyglucose is taken up by Chlorella vulgaris via the inducible non-phosphorylating hexose transport system. Since this hexose analogue is phosphorylated to a minor extent within the cells by hexokinase, the rate of uptake can be compared with the rate of an ATP dependent phosphorylation. The effect of the transphosphorylation inhibitors azide, DCCD, and oligomycin on these two processes has been investigated. The data especially with azide and DCCD clearly show that the ATP-dependent phosphorylation is much more severely inhibited than the rate of uptake. This was observed under aerobic conditions in the dark, as well as under anaerobic conditions in the light. Under anaerobiosis in the dark however there was no difference in the degree of inhibition. The same was true under all conditions with FCCP. These results are interpreted as evidence that a high energy intermediate of oxidative or photosynthetic phosphorylation drives the active sugar uptake of Chlorella. Under anaerobic conditions in the dark this high energy intermediate may be produced via ATP.

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.

Ammonia, methylamine and other uncouplers of photosynthetic phosphorylation promote in vivo the reversible conversion of the oxidized active form of Chlorella fusca nitrate reductase into its reduced inactive form. Concomitantly with the in vivo inactivation of the enzyme by the uncoupler, there occurs an increase in the cellular levels of the reduced forms of nicotinamide nucleotides and of the uncharged forms of adenine nucleotides. A general view of the regulation of photosynthesis by reducing power and energy charge is discyssed.

The role of photosynthesis in flower induction in the short-day plants Kalanchoe blossfeldiana and Xanthium pensylvanicum was investigated by chemical suppression of photosynthesis and prevention of chlorophyll formation in the induced leaf. 'Bleaching' leaves with streptomycin completely prevented flowering in X. pensylvanicum at concentrations shown to reduce the chlorophyll and carotenoid content of the leaf significantly. Such leaves were unable to induce flowering even when supplied with sugars and other photosynthetic products. Photosystem II inhibitors, DCMU and cadmium ion, inhibited induction in both species as well as suppressing photosynthesis (as tested by 02 evolution and starch production) whereas the photosystem I inhibitor, metronidazole, had no effect. Antimycin A inhibited flowering in K. blossfeldiana and may have a similar site of action to DCMU. Neither ammonium ion nor DBMIB. which acts upon plastoquinone (i.e. between PS I and PS II in the 'Z scheme'), had any effect on floral induction and it is argued that the inductive process is independent of photosynthetic phosphorylation but a step in the electron transport pathway between the sites of action of DCMU and DBMIB may be crucial. DSPD and its hydrolysis product, salicylaldehyde, suppressed flowering in K. blossfeldiana but the uncertainty regarding their chemistry precludes any firm conclusions regarding the nature of their action.

The anaerobic photosynthetic bacterium Rhodopseudomonas sphaeroides was used to produce hydrogen gas from glucose. Light served as an energy source for the bacterium to synthesize ATP through photosynthetic phosphorylation. A new strain was developed R. sphaeroides VM81 through successive culturing and adaptation to glucose growth, which gave high yields and rates of hydrogen production. This paper reports optimization experiments on the growth and hydrogen production of R. sphaeroides VM81 at different initial glucose concentrations and pH. The optimum conditions for cell growth and hydrogen gas production were found to be at 7 g glu/l and pH = 7.2.

Spheroplasts of a Synechococcus sp. thermophilic cyanobacterium showed cyclic photophosphorylation activity of 1200μmolATP/mg Chlh at 45 C with ascorbate-phenazine methosulfate, and non-cyclic activity of 11μmol ATP/mg Chl h with H2O-methyl viologen. Thylakoid membranes exhibited less than one-tenth of the spheroplasts activities in both types of photophosphorylation. The spheroplasts gave optimal cyclic phosphorylation at 55°C and lost half of the activity at 67°C for 5min. It was suggested that the stability of the photosynthetic membranes to retain proton gradient across the membranes might be closely related to the thermostability of the photophosphorylation.

To clarify the debate on the localized character of proton gradients in energy-transducing membrane systems, the way in which the osmolarity and ionicity of the medium, which should affect the thylakoid lumen properties, may modulate the relative efficiency of Δ\gmH+ generated by PS I or PS II (restricted here to ΔpH: valinomycin present) was examined. Although the results depended on the preparations and the conditions, a trend towards proton delocalization, especially in 50 mM KCl vs. the usual 5–10 mM, was observed when thylakoids were suspended in a sorbitol-free buffer for only a short time before the experiment. It was also verified that the better efficiency of PS I vs. PS II protons was not due to the 9-aminoacridine method used to quantify ΔpH. One main argument is that similar results were found when the proton gradient was estimated by total H+ translocation, measured with a glass electrode, and by probe partitioning, followed in parallel. Lastly, it was observed that, even when protons are emitted by water-splitting enzymes, i.e., far from coupling factors, the rate of ATP synthesis is less sensitive to nigericin than expected from the ΔpH decrease. This suggests that protons are flowing, from redox to phosphorylating pumps, in an anisotropic medium. The role of vesicular configuration and topological organization of energy-transducing membranes in the microchemiosmotic behaviour of organelles is stressed. It is suggested that besides water, polypeptide chains, rather than lipid heads (owing to the limited effectiveness of lipophilic nigericin), may ensure the lateral H+ transport between their points of influx and efflux.

Summary When subjected to hypotonic shock, Dunaliella tertiolecta cells undergo a decrease in intracellular glycerol. If grown in an artificial seawater medium containing 1.5 M NaCl, and transferred to a medium containing 0.1 M NaCl, about 75% of the intracellular glycerol is lost: two-thirds of the glycerol lost from the cells is found in the external medium («leakage») whilst the remaining one-third appears to be metabolised to a non-osmotically active storage compound. Loss of intracellular glycerol following hypotonic shocks is essentially light-independent, but both leakage and metabolism are inhibited by the uncoupler carbonyl cyanide m -chlorophenyl hydrazone (CCCP), suggesting a dependence on ATP and that the permeability of the cell membrane to glycerol is low. Low concentrations of CCCP only inhibit loss of intracellular glycerol when cells are incubated in the dark after hypotonic stress: this inhibition is relieved by light when photosynthetic phosphorylation can occur, but loss of intracellular glycerol is then suppressed by the ATPase inhibitor tri- n -butyl tin chloride (TNBT). These data may help resolve the apparent discrepancy between those reports of glycerol leakage from Dunaliella , and those suggesting that the cell membrane of the alga has a very low permeability coefficient for glycerol.

Cyclic, pseudocyclic, and noncyclic photosynthetic phosphorylation are mutually interdependent. Evidence for this is reviewed, in particular where it has to do with regulation of net ATP synthesis during the onset and maintenance of photosynthesis in vivo. It is concluded that both cyclic and noncyclic phosphorylation contribute to photosynthesis and that their relative rates may be controlled by redox poising of their respective electron carriers. The effect of phosphorylation of light‐harvesting‐chlorophyll‐protein on electron transport is to maintain the redox poise of intersystem electron carriers, notably plastoquinone; it will thereby regulate both cyclic and noncyclic electron transport. The cyclic pathway of ATP synthesis is energetically advantageous only where there exists a mechanism for redistribution of excitation between the photosystems. Protein phosphorylation is therefore implicated in regulation of coupling and stoicheiometry of ATP production and in the maintenance of efficient energy c...

The discovery of photosynthetic phosphorylation (photophosphorylation) revealed the capacity of the photosynthetic apparatus to convert the electromagnetic energy of sunlight into the biochemical energy of ATP. Two types were found: cyclic photophosphorylation in which ATP is the sole product of energy conversion and noncyclic photophosphorylation in which ATP formation is accompanied by oxygen evolution and the generation of reducing power whose carrier is ferredoxin.

2-anilino-1,3,4-thiadiazoles carrying various substituents in the 5-position as well as in the benzene-ring were synthesized. The compounds were tested with rat-liver-mitochondria and with spinach-chloroplasts and revealed to be potent uncouplers of both, oxidative and photosynthetic phosphorylation, with p I50-values rangeing from 6.79 to 4.05. At higher concentration all compounds are inhibitors of the Hillreaction. In mitochondria a fair correlation exists between pKa of the acidic NH-group and the uncoupling activity; a maximum is obtained around pKa= 6 .8 , whereas in chloroplasts activity is shifted to more acid pKa-values. The compounds meet the requirements for uncouplers according to the chemi-osmotic theory, being lipophilic weak acids. N-methylation causes total loss of activity in mitochondrial oxidative phosphorylation. The inhibitory action on photosynthetic electron transport is located within photosystem II. This latter activity is almost independent of substituent effects in contrast to uncoupling of either respiratory- or photo-phosphorylation

Stoicheometries and rates of proton translocation associated with respiratory reduction of NO3- have been measured for spheroplasts of Escherichia coli grown anaerobically in the presence of NO3-. Observed stoicheiometries [leads to H+/NO3- ratio; P. Mitchell (1966) Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation, Glynn Research, Bodmin] were approx. 4 for L-malate oxidation and approx. 2 for succinate, D-lactate and glycerol oxidation. Measurements of the leads to H+/2e- ratio with formate as the reductant and oxygen or NO3- as the oxidant were complicated by pH changes associated with formate uptake and CO2 formation. Nevertheless, it was possible to conclude that the site of formate oxidation is on the inner aspect of the cytoplasmic membrane, that the leads to H+/O ratio for formate oxidation is approx. 4, and that the leads to H+/NO3- ratio is greater than 2. Measurements of the rate of NO3- penetration into osmotically sensitive spheroplasts demonstrated an electrogenic entry of NO3- anion. The permeability coefficient for nitrate entry at 30 degrees C was between 10(-9) and 10(-10) cm- s(-1). The calculated rate of nitrate entry at the concentration typically used for the assay of nitrate reductase (EC 1.7.99.4) activity was about 0.1% of that required to support the observed rate of nitrate reduction by reduced Benzyl Viologen. Measurements of the distribution of nitrate between the intracellular and extracellular spaces of a haem-less mutant, de-repressed for nitrate reductase but unable to reduce nitrate by the respiratory chain, showed that, irrespective of the presence or the absence of added glucose, nitrate was not concentrated intracellularly. Osmotic-swelling experiments showed that the rate of diffusion of azid anion across the cytoplasmic membrane is relatively low in comparison with the fast diffusion of hydrazoic acid. The inhibitory effect of azide on nitrate reductase was not altered by treatments that modify pH gradients across the cytoplasmic membrane. It is concluded that the nitrate-reducing azide-sensitive site of nitrate reductase is located on the outer aspect of the cytoplasmic membrane. The consequences of this location for mechanisms of proton translocation driven by nitrate reduction are discussed, and lead to the proposal that the nitrate reductase of the cytoplasmic membrane is vectorial, reducing nitrate on the outer aspect of the membrane with 2H+ and 2e- that have crossed from the inner aspect of the membrane.

A new method for the isolation of photosynthetic membranes from the cyanobacterium Spirulina maxima has been developed. When illuminated, these membranes evolve oxygen in the presence of ferricyanide (Hill reaction) and consume oxygen in the presence of methyl viologen (Mehler reaction). When the membranes are left to stand at 4°C for 30 min, they develop the ability to consume oxygen in the light without an added, artificial electron acceptor. The Hill and Mehler reactions are not affected by the presence of ADP or uncouplers, but are inhibited by triphenyltin chloride. We have detected a cryptic ATPase activity stimulated by trypsin in the 2000×g supernatant fraction of the membrane preparation. In addition, the membrane vesicles contain an ATPase activity which is enhanced by treatment with dithiothreitol in the presence of light. These observations of ATPase led us to try a careful titration of the membrane vesicles with both triphenyltin chloride and N,N′-dicyclohexylcarbodiimide. When the vesicles were sealed with these reagents, we could observe both cyclic and stoichiometric photosynthetic phosphorylation.

Mutant strains of the unicellular green alga Chlamydomonas reinhardi which -have impaired photosynthesis are characterized by their inability to fix carbon dioxide in the light at the wild-type rate.l The incapacity of these mutant strains to fix carbon dioxide by photosynthesis can be attributed to one of three possibilities: (1) the loss of a step in the photosynthetic electron transport chain; (2) the loss of the capacity to carry out photosynthetic phosphorylation; or (3) the loss of one of the steps in photosynthetic carbon dioxide fixation associated with the reductive pentose phosphate cycle. The mutant strains of C. reinhardi described to date2-4 fall into the first category mentioned above. In this paper we shall describe ac-20, a mutant strain which lacks the capacity for photosynthetic carbon dioxide fixation as a consequence of the loss of RuDP carboxylase activity. Organisms and the Methods.-The organisms used in the experiments described below were the wild-type strain of C. reinhardi, 137c, and the mutant strain ac-20, derived from wild type by ultraviolet irradiation followed by a screening test'; for carbon dioxide fixation. Cells, in the logarithmic phase of growth, were harvested from shake cultures grown at 25?C in high salt minimal medium' supplemented with 0.2% sodium acetate. Light (2500 lux) was provided by daylight fluorescent lamps. Carbon dioxide fixation by whole cells was measured as previously described.7 The light intensity was 60,000 lux. The water-soluble products of carbon dioxide fixation by whole cells in the light were also examined. A cell suspension in minimal medium (30 ml, 10 mg/ml wet weight) was placed in a "lollipop" and illuminated with 20,000 lux. Temperature was maintained at 25?C. The suspension was aerated, and after 10 min C14-labeled sodium bicarbonate (8 ,moles, 50 /c/imole) was introduced into the "lollipop." Sixty sec later the reaction was terminated by emptying the contents of the "lollipop" into a hot methanol-chloroform mixture (12:5 v/v). The water-soluble products were then extracted according to the method of Bieleski and Young.' The products were separated on a Dowex-l chloride column (1 X 90 cm) using two stages of linear HC1 gradient elution. The gradient was as follows: tubes 1-35, water; tubes 36-155, a linear gradient from 0.028 N to 0.041 N HC1; tubes 156-265, a linear gradient from 0.070 N to 0.130 N HC1. Aliquots of 100 lambda from each tube were plated on planchets and counted. Twelve peaks were observed. Chloroplast fragments were prepared according to the method of Levine and Volkmann.K Crude extracts for enzyme assays were obtained from cells which had been washed once in 0.02 M Tris buffer, pH 7.5, and then resuspended in 5 ml of the same buffer. The cells were disrupted by sonic oscillation at 0?C for 21/2 min using a Mullard 20-kc ultrasonic disintegrator. The disrupted cell preparations were then centrifuged at 20,000 X g for 20 minl at 0?C. The green supernatant was used as the crude extract. The activity of the photosynthetic electron transport chain was measured by assaying the rate of TPN photoreduction by chloroplast fragments using the method described by Levine and Smillie.2

Runeberg (20) found that desaspidin acts as a powerful uncoupler of oxidative phosphorylation. Baltscheffsky and de Kiewiet (13) introduced desaspidin to the study of photosynthetic phosphorylation. They subdivided 5 systems of light-induced phosphorylation into 2 groups with respect to their sensitivity to inhibition by desaspidin. In 1 group, the rate of ATP formation was reduced by half at a very low (l 0-7 M ) concentrationl of desaspidin, whereas in the second group a 100 times greater concentration of desaspidin was required to give the same degree of inhibition. In the present investigationi, the inhibitory action of desaspidin was used to test further the validity of the subdivision of photosynthetic phosphorylation by chloroplasts into 2 distinct types: a type discovered in 1954 (3, 6) anid later designated as cyclic photophosphorylationi (8). in wvhich ATP formation by illuminated chloroplasts proceeds with no consumption of either an electron donor or an electron acceptor, and a type discovered in 1957 (8) and later renamed noncyclic photophosphorylation (2), in which ATP formation is linked stoichiometrically with the consumption of an electron donor and an electron acceptor. The electron donor was water, and its oxidation liberated 02; the electron acceptor was either TIPN or ferricyanide (8. 9). The terms cyclic and noncyclic were introduced to denote the coupling of phosphorylation to a light-induced closed or open electron flow that liberates the requisite free energy for the synthesis of ATP (2). Sensitivity to desaspidin inhibition was also used in the present investigation to test the validity of characterizing pseudocyclic phosphorylation as a special case of noncyclic photophosphorylation in which an open electron flow is generated between water as the electron donor and 02 as the electron acceptor. This noncyclic electron flow from water to external 02 gives the appearance of a cyclic electron flow since manometric measuring techniques give no indication that an electron donor and acceptor are being consumed concomitantly with ATP formation. In pseudocyclic photophosphorylation the consumption of ?2 at the terminal end of the electronic pathway is balanlced by the production of 2., at the site of electron donationi by water (10). In addition, sensitivity to desaspidin inhibition proved to be useful in clarifying the type of photophosphorvlation bv chloroplaFts (19) that accompanies reduiction of TPN by illuminate(d chloroplasts in a noiuphysiological noncyclic systemii in which reduced dichlorophenol indophenol replaces water as the electroni (lonor (24). This type of photophosphorylationi w-as previously conisidered (19) to occcur at the same site as the photophosphorylation wvhiclh accompanies TPN reduction wheni water is the electroii donor (8, 9). A different interpretationi will now be proposed on the basis of the new results with desaspidin.

Chloroplast were prepared from leaves of Vicia faba L. Electron micrographs showed that crude chloroplast suspensions prepared by the classical differential centrifugation procedure were contaminated by mitochondria: the mitochondria were completely removed by a further centrifugation of the crude chloroplast preparations through a glycerol-sucrose density gradient. Most of the chloroplast in the crude preparations and all the chloroplast after centrifugation through the density gradient were devoid of external membranes and stroma. The mitochondria-free chloroplast suspensions were able to mediate photo-phosphorylation with 3·10−5 M FMN and 10−4 M vitamin K3 as co-factors. The rate of photophosphorylation, the product (ATP) and the nonadditive effect of the co-factors compare closely with the results of other workers who used spinach chloroplasts prepared by differential centrifugation alone. Thus, photphosphorylation would appear to be a real aspect of the metabolism of isolated chloroplast; mitochondria are not involved in the process.

A mutant strain of the unicellular green alga, Chlamydomonas reinhardi, is unable to fix carbon dioxide by photosynthesis because it is deficient in phosphoribulokinase activity. The absence of light-dependent carbon dioxide fixation in cells of the mutant strain supports the operation of the Calvin-Benson scheme of photosynthetic carbon dioxide fixation in this organism. No deficiency other than low phosphoribulokinase activity was found which would account for the inability of cells of the mutant strain to fix carbon dioxide by photosynthesis. Activities comparable to those in the wild-type strain were found for eight other enzymes of the Calvin cycle and two enzymes associated with the C(4) dicarboxylic acid pathway. The normal rates of nicotinamide adenine dinucleotide phosphate photoreduction and of photosynthetic phosphorylation observed in chloroplast fragments prepared from cells of the mutant strain indicated that the photosynthetic electron transport chain in the mutant is intact.

In spinach chloroplasts which have been shaken with n-heptane, photophosphorylation coupled to the reduction of nicotinamide adenine dinucleotide phosphate is inhibited about 95% while the photoreduction of NADP+ (and ferricyanide) is stimulated as much as 50%. Photophosphorylation catalyzed by phenazine methosulfate, however, is inhibited only 50%. These data are interpreted to indicate that there are two sites of photophosphorylation in spinach chloroplasts. Both sites are utilized in cyclic photophosphorylation, whereas one of the sites is common to both cyclic and noncyclic photophosphorylation. In heptane-treated chloroplasts the only site functioning is that site associated exclusively with cyclic electron flow. Heptane treatment also has a differential effect upon the adenosine triphosphatase activities of chloroplasts. Trypsin-activated Ca++-dependent ATPase is not affected by heptane treatment, whereas Mg++-dependent ATPase activated by light in the presence of dithiothreitol is inhibited 80 to 90%. After heptane treatment, the ability of chloroplasts to accumulate protons upon illumination is inhibited 40 to 60% and their ability to synthesize ATP when transferred from acidic to alkaline environment is inhibited 50%.