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Uncoupling of photophosphorylation appears to have positive effect on lipid production of microalgae, however, underlying mechanisms involved in the effect are still unresolved. In this study, an uncoupler of photophosphorylation, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), was used for the promotion of microalgal lipid accumulation. The results indicated that addition of CCCP was able to enhance the lipid productions of all tested species of Chlorella, especially when cultivated under mixotrophic condition. Based on the transcriptome and functional gene expression analyses, the functional genes involving in photosynthetic electron transport (PET), fatty acids synthesis, tricarboxylic acid (TCA) cycle, glycolysis and pentose phosphate (PP) pathway were found to be up-regulated while the genes participating in photosystem II, ATP synthesis during photosynthesis and calvin cycle were to be down-regulated by the presence of CCCP. According to these observations, the mechanism of CCCP to the promotion of lipid synthesis probably through channeling the NAD(P)H metabolic fluxes from carbon fixation towards fatty acid biosynthesis and lipid production, which was accomplished by (1) down-regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase genes that involving in CO2 fixation via calvin cycle to reduce the NADPH consumption; (2) up-regulation of genes involved in TCA cycle and PP pathway to produce more NAD(P)H. The results of this study provide a new method to promote microalgal lipid production.

The gene cycA encodes a periplasmic protein, cytochrome c2 (cyt c2), which dominates electron transfer from the membrane-bound ubiquinol: cyt c2 oxidoreductase (cyt bc1) to the photosynthetic reaction center, contributing to the production of transmembrane proton potential and then the synthesis of ATP. For photosynthetic bacteria, the total energy supply for light anaerobic growth and hydrogen production comes from photophosphorylation. As a result, the key protein encoding gene plays an important role in hydrogen production. To figure out the specific effect of cycA expression level on H2 production ability of Rhodobacter sphaeroides HY01, cycA-expression plasmids derived from pRK415 and pBBR1MCS-2 were constructed and then crossed into the parent strain R. sphaeroides HY01 for H2 production test. And further verification by RT-PCR suggested that there was about 20% enhancement of cycA expression level by pBBR1MCS-2 where the H2 production performance of corresponding strain was improved by 6–8% compared with blank control. In contrast, cycA expression level was about 3.4 folds by vector pRK415 compared with control strain, but corresponding strains showed slightly depressed H2 production performance. Besides, the mutant XJ01 with cycA gene overexpressing by 70% in the genome of HY01(hupSL:cycA) also showed positive effect on hydrogen production performance. The results demonstrated that slightly overexpression of cycA could enhance the hydrogen production rate, but too much higher level of cycA-expression could show negative effect on H2 production performance of R. sphaeroides HY01.

Fo·F1H+-ATPase/synthase in coupled plasma membrane vesicles of Paracoccus denitrificans catalyzes ATP hydrolysis and/or ATP synthesis with comparable enzyme turnover. Significant difference in pH-profile of these alternative activities is seen: decreasing pH from 8.0 to 7.0 results in reversible inhibition of hydrolytic activity, whereas ATP synthesis activity is not changed. The inhibition of ATPase activity upon acidification results from neither change in ADP(Mg2+)-induced deactivation nor the energy-dependent enzyme activation. Vmax, not apparent KmATP is affected by lowering the pH. Venturicidin noncompetitively inhibits ATP synthesis and coupled ATP hydrolysis, showing significant difference in the affinity to its inhibitory site depending on the direction of the catalysis. This difference cannot be attributed to variations of the substrate-enzyme intermediates for steady-state forward and back reactions or to possible equilibrium between ATP hydrolase and ATP synthase Fo·F1 modes of the opposite directions of catalysis. The data are interpreted as to suggest that distinct non-equilibrated molecular isoforms of Fo·F1 ATP synthase and ATP hydrolase exist in coupled energy-transducing membranes.

Cyclic electron flow around photosystem I (CEF) is critical for balancing the photosynthetic energy budget of the chloroplast by generating ATP without net production of NADPH. We demonstrate that the chloroplast NADPH dehydrogenase complex, a homolog to respiratory Complex I, pumps approximately two protons from the chloroplast stroma to the lumen per electron transferred from ferredoxin to plastoquinone, effectively increasing the efficiency of ATP production via CEF by 2-fold compared with CEF pathways involving non-proton-pumping plastoquinone reductases. By virtue of this proton-pumping stoichiometry, we hypothesize that NADPH dehydrogenase not only efficiently contributes to ATP production but operates near thermodynamic reversibility, with potentially important consequences for remediating mismatches in the thylakoid energy budget.

Theories of biological energy coupling in oxidative phosphorylation (OX PHOS) and photophosphorylation (PHOTO PHOS) are reviewed and applied to ATP synthesis by an experimental system containing purified ATP synthase reconstituted into liposomes. The theories are critically evaluated from the standpoint of the principle of electrical neutrality. It is shown that the obligatory requirement to maintain overall electroneutrality of bulk aqueous phases imposes strong constraints on possible theories of energy coupling and molecular mechanisms of ATP synthesis. Mitchell's chemiosmotic theory is found to violate the electroneutrality of bulk aqueous phases and is shown to be untenable on these grounds. Purely electroneutral mechanisms or mechanisms where the anion/countercation gradient is dissipated or simply flows through the lipid bilayer are also shown to be inadequate. A dynamically electrogenic but overall electroneutral mode of ion transport postulated by Nath's torsional mechanism of energy transduction and ATP synthesis is shown to be consistent both with the experimental findings and the principle of electrical neutrality. It is concluded that the ATP synthase functions as a proton-dicarboxylic acid anion cotransporter in OX PHOS or PHOTO PHOS. A logical chemical explanation for the selection of dicarboxylic acids as intermediates in OX PHOS and PHOTO PHOS is suggested based on the pioneering classical thermodynamic work of Christensen, Izatt, and Hansen. The nonequilibrium thermodynamic consequences for theories in which the protons originate from water vis-a-vis weak organic acids are compared and contrasted, and several new mechanistic and thermodynamic insights into biological energy transduction by ATP synthase are offered. These considerations make the new theory of energy coupling more complete, and lead to a deeper understanding of the molecular mechanism of ATP synthesis.

As the chief energy source of eukaryotic cells, it is important to determine the thermodynamic efficiency of ATP synthesis in oxidative phosphorylation (OX PHOS). Previous estimates of the thermodynamic efficiency of this vital process have ranged from Lehninger's original back-of-the-envelope calculation of 38% to the often quoted value of 55-60% in current textbooks of biochemistry, to high values of 90% from recent information theoretic considerations, and reports of realizations of close to ideal 100% efficiencies by single molecule experiments. Hence this problem has been reinvestigated from first principles. The overall thermodynamic efficiency of ATP synthesis in the mitochondrial energy transduction OX PHOS process has been found to lie between 40 and 41% from four different approaches based on a) estimation using structural and biochemical data, b) fundamental nonequilibrium thermodynamic analysis, c) novel insights arising from Nath's torsional mechanism of energy transduction and ATP synthesis, and d) the overall balance of cellular energetics. The torsional mechanism also offers an explanation for the observation of a thermodynamic efficiency approaching 100% in some experiments. Applications of the unique, molecular machine mode of functioning of F1FO-ATP synthase involving direct inter-conversion of chemical and mechanical energies in the design and fabrication of novel, man-made mechanochemical devices have been envisaged, and some new ways to exorcise Maxwell's demon have been proposed. It is hoped that analysis of the fundamental problem of energy transduction in OX PHOS from a fresh perspective will catalyze new avenues of research in this interdisciplinary field.

Gemmatimonas phototrophica is, so far, the only described phototrophic species of the bacterial phylum Gemmatimonadetes. Its cells contain a unique type of photosynthetic complex with the reaction center surrounded by a double ring antenna, however they can also grow in the dark using organic carbon substrates. Its photosynthesis genes were received via horizontal gene transfer from Proteobacteria. This raises two questions; how the horizontally transferred photosynthesis apparatus has integrated into the cellular machinery, and how much light-derived energy actually contributes to the cellular metabolism? To address these points, the photosynthetic reactions were studied on several levels, from photophysics of the reaction center to cellular growth. Flash photolysis measurements and bacteriochlorophyll fluorescence kinetic measurements documented the presence of fully functional type-2 reaction centers with a large light harvesting antenna. When illuminated, the bacterial cells reduced their respiration rate by 58 ± 5%, revealing that oxidative phosphorylation was replaced by photophosphorylation. Moreover, illumination also more than doubled the assimilation rates of glucose, a sugar that is mostly used for respiration. Finally, light increased the growth rates of Gemmatimonas phototrophica colonies on agar plates. All the presented data provide evidence that photosynthetic complexes are fully integrated into cellular metabolism of Gemmatimonas phototrophica, and are able to provide a substantial amount of energy for its metabolism and growth.

In a recent publication, Manoj raises criticisms against consensus views on the ATP synthase. The radical statements and assertions are shown to contradict a vast body of available knowledge that includes i) pioneering single-molecule biochemical and biophysical studies from the respected experimental groups of Kinosita, Yoshida, Noji, Borsch, Dunn, Graber, Frasch, and Dimroth etc., ii) state-of-the-art X-ray and EM/cryo-EM structural information garnered over the decades by the expert groups of Leslie-Walker, Kuhlbrandt, Mueller, Meier, Rubinstein, Sazanov, Duncan, and Pedersen on ATP synthase, iii) the pioneering energy-based computer simulations of Warshel, and iv) the novel theoretical and experimental works of Nath. Valid objections against Mitchell's chemiosmotic theory and Boyer's binding change mechanism put forth by Manoj have been addressed satisfactorily by Nath's torsional mechanism of ATP synthesis and two-ion theory of energy coupling and published 10 to 20 years ago, but these papers are not cited by him. This communication shows conclusively and in great detail that none of his objections apply to Nath's mechanism/theory. Nath's theory is further consolidated based on its previous predictive record, its consistency with biochemical evidence, its unified nature, its application to other related energy transductions and to disease, and finally its ability to guide the design of new experiments. Some constructive suggestions for high-resolution structural experiments that have the power to delve into the heart of the matter and throw unprecedented light on the nature of coupled ion translocation in the membrane-bound FO portion of F1FO-ATP synthase are made.

We attempted to elucidate possible reasons of a decline in fresh and dry mass accumulation by Chinese cabbage (Brassica chinensis L.) plants under combined narrowband red and blue light emitting diodes (LEDs) in comparison with illumination by a broad-spectrum high-pressure sodium lamps (HPSL) of equal photosynthetically active radiation intensity. Analysis of photosynthetic activity, sugar content and composition, antioxidant activity, and membrane peroxidation revealed neither inhibition of nor damage to the photosynthetic apparatus. There was also no detectable oxidative stress and related photoassimilate loss, the source-sink relations were also unimpaired in LED-grown plants. Our findings suggest that the light distribution in the plant canopy might be an essential factor limiting plant growth under the LED light. The results are discussed in view of the role of the radiation in the yellow–green range of the spectrum in driving photosynthesis.

Like the evolutionary related F1FO ATP synthases and V1VO ATPases, the A1AO ATP synthases from archaea are multisubunit, membrane-bound transport machines that couple ion flow to the synthesis of ATP. Although the subunit composition is known for at least two species, nothing is known so far with respect to the function of individual subunits or amino acid residues. To pave the road for a functional analysis of A1AO ATP synthases, we have cloned the entire operon from Methanosarcina mazei into an expression vector and produced the enzyme in Escherichia coli. Inverted membrane vesicles of the recombinants catalyzed ATP synthesis driven by NADH oxidation as well as artificial driving forces. Δ μ ˜ H + as well as ΔpH were used as driving forces which is consistent with the inhibition of NADH-driven ATP synthesis by protonophores. Exchange of the conserved glutamate in subunit c led to a complete loss of ATP synthesis, proving that this residue is essential for H+ translocation. Exchange of two conserved arginine residues in subunit a has different effects on ATP synthesis. The role of these residues in ion translocation is discussed.

Physiological, biochemical and electron microscopy analyses were used to investigate the photosynthetic performance of flag leaves in rice white stripe mutant 6001 during the senescence process. Results showed that the chlorophyll content at the heading and milk-ripe stages in rice mutant 6001 were about 34.78% and 3.00% less than those in wild type 6028, respectively. However, the chlorophyll content at the fully-ripe stage in rice mutant 6001 was higher than that in wild type 6028. At the heading stage, the net photosynthetic rate (Pn) in rice mutant 6001 was lower than that in wild type 6028. Rice mutant 6001 also exhibited a significantly slower decrease rate of Pn than wild type 6028 during the senescence progress, especially at the later stage. Furthermore, Ca2+-ATPase, Mg2+-ATPase and photophosphorylation activities exhibited the similar trends as the Pn. During the senescence process, the 68 kDa polypeptide concentrations in the thylakoid membrane proteins exhibited a significant change, which was one of the critical factors that contributed to the observed change in photosynthesis. We also observed that the chloroplasts of rice mutant 6001 exhibited higher integrity than those of wild type 6028, and the chloroplast membrane of rice mutant 6001 disintegrated more slow during the senescence process. In general, rice mutant 6001 had a relatively slower senescence rate than wild type 6028, and exhibited anti-senescence properties.

Currently light emitting diodes (LEDs) are considered to be most preferable source for space plant growth facilities. We performed a complex study of growth and photosynthesis in Chinese cabbage plants (Brassica chinensis L.) grown with continuous LED lighting based on red (650 nm) and blue (470 nm) LEDs with a red to blue photon ratio of 7:1. Plants grown with high-pressure sodium (HPS) lamps were used as a control. PPF levels used were about 100 μmol/(m2 s) (PPF 100) and nearly 400 μmol/(m2 s) (PPF 400). One group of plants was grown with PPF 100 and transferred to PPF 400 at the age of 12 days. Plants were studied at the age of 15 and 28 days (harvest age); some plants were left to naturally end their life cycle. We studied a number of parameters reflecting different stages of photosynthesis: photosynthetic pigment content; chlorophyll fluorescence parameters (photosystem II quantum yield, photochemical and non-photochemical chlorophyll fluorescence quenching); electron transport rate, proton gradient on thylakoid membranes (ΔpH), and photophosphorylation rate in isolated chloroplasts. We also tested parameters reflecting plant growth and productivity: shoot and root fresh and dry weight, sugar content and ascorbic acid content in shoots. Our results had shown that at PPF 100, plants grown with LEDs did not differ from control plants in shoot fresh weight, but showed substantial differences in photophosphorylation rate and sugar content. Differences observed in plants grown with PPF 100 become more pronounced in plants grown with PPF 400. Most parameters characterizing the plant photosynthetic performance, such as photosynthetic pigment content, electron transport rate, and ΔpH did not react strongly to light spectrum. Photophosphorylation rate differed strongly in plants grown with different spectrum and PPF level, but did not always reflect final plant yield. Results of the present work suggest that narrow-band LED lighting caused changes in Chinese cabbage plants on levels of the photosynthetic apparatus and the whole plant, concerning its development and adaptation to a varying PPF level.

Nitric oxide (NO) is an important signalling molecule in plants under physiological and stress conditions. Here we review the influence of NO on chloroplasts which can be directly induced by interaction with the photosynthetic apparatus by influencing photophosphorylation, electron transport activity and oxido-reduction state of the Mn clusters of the oxygen-evolving complex or by changes in gene expression. The influence of NO-induced changes in the photosynthetic apparatus on its functions and sensitivity to stress factors are discussed.

Background Sulfate uptake was analyzed in photosynthetic Euglena gracilis grown in sulfate sufficient or sulfate deficient media, or under Cd2+ exposure or Cys overload, to determine its regulatory mechanisms and contribution to Cys homeostasis. Results In control and sulfate deficient or Cd2+-stressed cells, one high affinity and two low affinity sulfate transporters were revealed, which were partially inhibited by photophosphorylation and oxidative phosphorylation inhibitors and ionophores, as well as by chromate and molybdate; H+ efflux also diminished in presence of sulfate. In both sulfate deficient and Cd2+-exposed cells, the activity of the sulfate transporters was significantly increased. However, the content of thiol-metabolites was lower in sulfate-deficient cells, and higher in Cd2+-exposed cells, in comparison to control cells. In cells incubated with external Cys, sulfate uptake was strongly inhibited correlating with 5-times increased intracellular Cys. Re-supply of sulfate to sulfate deficient cells increased the Cys, γ-glutamylcysteine and GSH pools, and to Cys-overloaded cells resulted in the consumption of previously accumulated Cys. In contrast, in Cd2+ exposed cells none of the already elevated thiol-metabolites changed. Conclusions (i) Sulfate transport is an energy-dependent process; (ii) sulfate transporters are over-expressed under sulfate deficiency or Cd2+ stress and their activity can be inhibited by high internal Cys; and (iii) sulfate uptake exerts homeostatic control of the Cys pool.

Direct degradation of macromolecules by photosynthetic bacteria (PSB) is important for the industrial application of PSB wastewater treatment. Light and oxygen are the most important parameters in PSB growth. This paper studied the PSB macromolecule degradation process under three different light and oxygen conditions: light-anaerobic, natural light-microaerobic and dark-aerobic. The results showed that under three different light-oxygen conditions, PSB degradation of macromolecules was higher than 90%; the removal ratios of COD, TN, TP, total sugar and protein were also high; and the biomass yield reached nearly 0.5 mg-biomass/mg-COD-removal. Light and oxygen significantly influenced the efficiency. Macromolecules and pollutants removals were higher under oxygen condition than those under light-anaerobic condition. Theoretical analysis showed that under aerobic condition, PSB carried out oxidative phosphorylation, in which pollutants were sufficiently utilized with high mineralization degree. Under light-anaerobic condition, PSB carried out photophosphorylation and fermentation, which led to low pollutants removal efficiency.

Salvinia possess inherent capacity to accumulate high levels of various heavy metals. Accumulation of Cr, Fe, Ni, Cu, Pb and Cd ranged between 6 and 9 mg g(-1)dry wt., while accumulation of Co, Zn and Mn was ∼4 mg g(-1)dry wt. Heavy metal accumulation affected the physiological status of plants. Photosystem II activity noted to decline in Ni, Co, Cd, Pb, Zn and Cu exposed plants, while Photosystem I activity showed enhancement under heavy metal stress in comparison to control. The increase in PS I activity supported build up of transthylakoidal proton gradient (ΔpH), which subsequently helped in maintaining the photophosphorylation potential. Ribulose 1,5 dicarboxylase/oxygenase (Rubisco) activity noted a decline. Alterations in photosynthetic potential of Salvinia result primarily from changes in carbon assimilation efficiency with slight variations in primary photochemical activities and photophosphorylation potential. Studies suggest that Salvinia possess efficient photosynthetic machinery to withstand heavy metal stress.

The effects of three antibiotics (erythromycin, ciprofloxacin and sulfamethoxazole) on photosynthesis process of Selenastrum capricornutum were investigated by determining a battery of parameters including photosynthetic rate, chlorophyll fluorescence, Hill reaction, and ribulose-1.5-bisphosphate carboxylase activity, etc. The results indicated that three antibiotics could significantly inhibit the physiological progress including primary photochemistry, electron transport, photophosphorylation and carbon assimilation. Erythromycin could induce acute toxic effects at the concentration of 0.06 mg L(-1), while the same results were exhibited for ciprofloxacin and sulfamethoxazole at higher than 1.0 mg L(-1). Erythromycin was considerably more toxic than ciprofloxacin and sulfamethoxazole and may pose a higher potential risk to the aquatic ecosystem. Some indices like chlorophyll fluorescence, Mg(2+)-ATPase activity and RuBPCase activity showed a high specificity and sensitivity to the exposure of erythromycin, and may be potentially used as candidate biomarkers for the exposure of the macrolide antibiotics.

6α,7β-Dihydroxyvouacapan-17β-oic acid (1) and methyl 6α,7β-dihydroxyvouacapan-17β-oate (8) were isolated from Pterodon polygalaeflorus Benth. 1 was modified to obtain 6α-hydroxyvouacapan-7-β,17β lactone (2). Then, 6-oxovouacapan-7β,17β lactone (3) was obtained from 2. The furanoditerpene ester derivatives: propyl 7β-hydroxy-6-oxovouacapan-17β-oate (4), butyl 7β-hydroxy-6-oxovouacapan-17β-oate (5), 2-methoxyethyl 7β-hydroxy-6-oxovouacapan-17β-oate (6) and 3-methylbut-2-enyl 7β-hydroxy-6-oxovouacapan-17β-oate (7) were synthesized from (3) and methyl 6α,7β-thiocarbonyldioxyvouacapan-17β-oate (9) was obtained from (8). In this work, the lactone ester derivatives 4–7 and 9 were tested on photosynthetic activities in an attempt to search for new compounds as potential herbicide agents that affect photosynthesis. All compounds inhibited ATP synthesis and electron flow from water to MV, therefore, they act as Hill reaction inhibitors, being 4- to 9-fold more potent than 2 and 3 as inhibitors of ATP synthesis. Their interaction site was located at PSII in a similar way to diuron. Furthermore, furanoditerpene esters 6 and 7 act as uncouplers, and were corroborated by enhancement of the light-activated Mg2+-ATPase, while 5 act as an energy transfer inhibitor. Finally 5–7 behave as herbicides, since they inhibit the biomass production of weeds assay.

ATP provided by oxidative phosphorylation supports highly complex and energetically expensive cellular processes. Yet, in several pathological settings, mitochondria could revert to ATP consumption, aggravating an existing cellular pathology. Here we review (i) the pathological conditions leading to ATP hydrolysis by the reverse operation of the mitochondrial F(o)F(1)-ATPase, (ii) molecular and thermodynamic factors influencing the directionality of the F(o)F(1)-ATPase, (iii) the role of the adenine nucleotide translocase as the intermediary adenine nucleotide flux pathway between the cytosol and the mitochondrial matrix when mitochondria become ATP consumers, (iv) the role of the permeability transition pore in bypassing the ANT, thereby allowing the flux of ATP directly to the hydrolyzing F(o)F(1)-ATPase, (v) the impact of the permeability transition pore on glycolytic ATP production, and (vi) endogenous and exogenous interventions for limiting ATP hydrolysis by the mitochondrial F(o)F(1)-ATPase.

The ADP/ATP and ATP-Mg/Pi carriers are widespread among eukaryotes and constitute two systems to transport adenine nucleotides in mitochondria. ADP/ATP carriers carry out an electrogenic exchange of ADP for ATP essential for oxidative phosphorylation, whereas ATP-Mg/Pi carriers perform an electroneutral exchange of ATP-Mg for phosphate and are able to modulate the net content of adenine nucleotides in mitochondria. The functional interplay between both carriers has been shown to modulate viability in Saccharomyces cerevisiae. The simultaneous absence of both carriers is lethal. In the light of the new evidence we suggest that, in addition to exchange of cytosolic ADP for mitochondrial ATP, the specific function of the ADP/ATP carriers required for respiration, both transporters have a second function, which is the import of cytosolic ATP in mitochondria. The participation of these carriers in the generation of mitochondrial membrane potential is discussed. Both are necessary for the function of the mitochondrial protein import and assembly systems, which are the only essential mitochondrial functions in S. cerevisiae.

Our previous study has demonstrated that both RuBP carboxylation limitation and RuBP regeneration limitation exist simultaneously in rice grown under free-air CO2 enrichment (FACE, about 200 mmol mol � 1 above the ambient air CO2 concentration) conditions [G.-Y. Chen, Z.-H. Yong, Y. Liao, D.-Y. Zhang, Y. Chen, H.-B. Zhang, J. Chen, J.-G. Zhu, D.-Q. Xu, Photosynthetic acclimation in rice leaves to free-air CO2 enrichment related to both ribulose-1,5-bisphosphate carboxylase limitation and ribulose1,5-bisphosphate regeneration limitation. Plant Cell Physiol. 46 (2005) 1036–1045]. To explore the mechanism for forming of RuBP regeneration limitation, we conducted the gas exchange measurements and some biochemical analyses in FACE-treated and ambient rice plants. Net CO2 assimilation rate (Anet) in FACE leaves was remarkably lower than that in ambient leaves when measured at the same CO2 concentration, indicating that photosynthetic acclimation to elevated CO2 occurred. In the meantime the maximum electron transport rate (ETR) (Jmax), maximum carboxylation rate (Vcmax) in vivo, and RuBP contents decreased significantly in FACE leaves. The whole chain electron transport rate and photophosphorylation rate reduced significantly while ETR of photosystem II (PSII) did not significantly decrease and ETR of photosystem I (PSI) was significantly increased in the chloroplasts from FACE leaves. Further, the amount of cytochrome (Cyt) f protein, a key component localized between PSII and PSI, was remarkably declined in FACE leaves. It appears that during photosynthetic acclimation the decline in the Cyt f amount is an important cause for the decreased RuBP regeneration capacity by decreasing the whole chain electron transport in FACE leaves.

The light-dependent regulation of chloroplast ATP synthase activity depends on an intricate but ill defined interplay between the proton electrochemical potential across the thylakoid membrane and thioredoxin-mediated redox modulation of a cysteine bridge located on the ATP synthase gamma-subunit. The abnormal light-dependent regulation of the chloroplast ATP synthase in the Arabidopsis thaliana cfq (coupling factor quick recovery) mutant was caused by a point mutation (G to A) in the atpC1 gene, which caused an amino acid substitution (E244K) in the vicinity of the redox modulation domain in the gamma-subunit of ATP synthase. Equilibrium redox titration revealed that this mutation made the regulatory sulfhydryl group energetically much more difficult to reduce relative to the wild type (i.e. raised the Em,7.9 by 39 mV). Enzymatic studies using isolated chloroplasts showed significantly lower light-induced ATPase and ATP synthase activity in the mutant compared with the wild type. The lower ATP synthesis capacity in turn restricted overall rates of leaf photosynthesis in the cfq mutant under low light. This work provides in situ validation of the concept that thioredoxin-dependent reduction of the gamma-subunit regulatory disulfide modulates the proton electrochemical potential energy requirement for activation of the chloroplast ATP synthase and that the activation state of the ATP synthase can limit leaf level photosynthesis.

The three major polyamines are normally found in chloroplasts of higher plants and are implicated in plant growth and stress response. We have recently shown that putrescine can increase light energy utilization through stimulation of photophosphorylation [Ioannidis et al., (2006) BBA-Bioenergetics, 1757, 821-828]. We are now to compare the role of the three major polyamines in terms of chloroplast bioenergetics. There is a different mode of action between the diamine putrescine and the higher polyamines (spermidine and spermine). Putrescine is an efficient stimulator of ATP synthesis, better than spermidine and spermine in terms of maximal % stimulation. On the other hand, spermidine and spermine are efficient stimulators of non-photochemical quenching. Spermidine and spermine at high concentrations are efficient uncouplers of photophosphorylation. In addition, the higher the polycationic character of the amine being used, the higher was the effectiveness in PSII efficiency restoration, as well as stacking of low salt thylakoids. Spermine with 50 microM increase F(V) as efficiently as 100 microM of spermidine or 1000 microM of putrescine or 1000 microM of Mg(2+). It is also demonstrated that the increase in F(V) derives mainly from the contribution of PSIIalpha centers. These results underline the importance of chloroplastic polyamines in the functionality of the photosynthetic membrane.

Fo·F1-ATP synthase in inside-out coupled vesicles derived from Paracoccus denitrificans catalyzes Pi-dependent proton-translocating ATPase reaction if exposed to prior energization that relieves ADP·Mg2+-induced inhibition (Zharova, T.V. and Vinogradov, A.D. (2004) J. Biol. Chem.,279, 12319–12324). Here we present evidence that the presence of medium ADP is required for the steady-state energetically self-sustained coupled ATP hydrolysis. The initial rapid ATPase activity is declined to a certain level if the reaction proceeds in the presence of the ADP-consuming, ATP-regenerating system (pyruvate kinase/phosphoenol pyruvate). The rate and extent of the enzyme de-activation are inversely proportional to the steady-state ADP concentration, which is altered by various amounts of pyruvate kinase at constant ATPase level. The half-maximal rate of stationary ATP hydrolysis is reached at an ADP concentration of 8 × 10−6 M. The kinetic scheme is proposed explaining the requirement of the reaction products (ADP and Pi), the substrates of ATP synthesis, in the medium for proton-translocating ATP hydrolysis by P. denitrificans Fo·F1-ATP synthase.

2,4-Dinitrophenol (2,4-DNP) is frequently used as a starting material in the production of pesticides, herbicides, and dyes. This compound is toxic to animals, plants, and microorganisms because it inhibits respiration in mitochondria and photophosphorylation in chloroplasts. Anabaena variabilis showed a high ability to remove 2,4-DNP in the concentration range of 5–150 μM under continuous light. However, 2-amino-4-nitrophenol (2-ANP), which is a degradation product of the 2,4-DNP removal by A. variabilis, accumulated in the culture. Although 2,4-DNP was completely removed when incubation was with a light and dark cycle, accumulation of 2-ANP was observed. By screening various microalgae and cyanobacteria for their ability to remove 2-ANP, we found that Chlamydomonas reinhardtii and Anabaena cylindrica showed the highest ability. However, a high concentration of 2-ANP remained when C. reinhardtii was cultivated with A. variabilis. On the other hand, a mixed culture of A. variabilis and A. cylindrica could remove 2,4-DNP completely, and only a low concentration of 2-ANP was detected compared to a culture of A. variabilis. Therefore, a cyanobacterial mixed culture of A. variabilis and A. cylindrica is expected to be useful for the removal of 2,4-DNP from industrial wastewater.

New and old data pertinent to the electrochemical potentials across the inner mitochondrial membrane are reviewed with the intent of reconciling the various findings in the light of new perspectives provided by more recent knowledge. A careful scrutiny of old data permits ruling out the presence of a significant metabolically dependent electrical membrane potential. Recent technological advances make it possible to test the proposed alternatives. These proposals recast the original idea, and the possible mechanisms that are emerging also invoke a protonmotive force. Our conclusions that Δ Ψ is not involved in oxidative-phosphorylation finds parallel observations in Halobacterium halobium [H. Michel, D. Oesterhelt, Electrochemical proton gradient across the cell membrane of Halobacterium halobium : comparison of the light-induced increase with the increase of intracellular adenosine triphosphate under steady-state illumination, Biochemistry 19 (1980) 4615–4619] and thylakoid vesicles [D.R. Ort, R.A. Dilley, N.E. Good, Photophosphorylation as a function of illumination time II. Effects of permeant buffers, Biochim. Biophys. Acta 449 (1976) 108–129] in which light-induced ATP synthesis occurs in the absence of an apparent Δ Ψ or ΔpH, suggesting the presence of mechanisms similar to the one proposed for mitochondria.

Mass coral bleaching events are a worldwide phenomenon, which generally occur during periods of elevated sea surface temperature and intense sunlight. These conditions result in a decline in photochemical efficiency of symbiotic microalgae (zooxanthellae) which ultimately leads to the expulsion of these symbionts. The physiological mechanism which triggers the release of the zooxanthellae has yet to be adequately determined. Under bleaching conditions, non-photochemical quenching (NPQ) is used to dissipate excess energy from photosystem II (PSII). NPQ was partitioned into three components, (energy dependent quenching [qE], state transition quenching [qT] and photoinhibitory quenching [qI]), based on relaxation kinetics upon addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and darkening. This investigation revealed that for corals not exposed to bleaching stress, qE was the principle means of energy dissipation (∼60% of the total NPQ). In corals exposed to either high-light (475 μmol photons m−2 s−1 and 25 °C) or elevated temperature (225 μmol photons m−2 s−1 and 32 °C) treatments, the dominant component of NPQ was qE and the relative proportions did not change during the exposure period (1–8 h). When exposed to bleaching conditions (475 μmol photons m−2 s−1 and 32 °C) the contribution of the different components changed after 4 h and the total NPQ increased. At this time, the contribution of qT to the total NPQ significantly increased to equal that of qE (40%), suggesting state transitions become more important under such conditions. Throughout the exposure period in all treatments, no change in the proportion of qI was observed.

MRP1 transports glutathione-S-conjugated solutes in an ATP-dependent manner by utilizing its two NBDs to bind and hydrolyze ATP. We have found that ATP binding to NBD1 plays a regulatory role whereas ATP hydrolysis at NBD2 plays a dominant role in ATP-dependent LTC4 transport. However, whether ATP hydrolysis at NBD1 is required for the transport was not clear. We now report that ATP hydrolysis at NBD1 may not be essential for transport, but that the dissociation of the NBD1-bound nucleotide facilitates ATP-dependent LTC4 transport. These conclusions are supported by the following results. The substitution of the putative catalytic E1455 with a non-acidic residue in NBD2 greatly decreases the ATPase activity of NBD2 and the ATP-dependent LTC4 transport, indicating that E1455 participates in ATP hydrolysis. The mutation of the corresponding D793 residue in NBD1 to a different acidic residue has little effect on ATP-dependent LTC4 transport. The replacement of D793 with a non-acidic residue, such as D793L or D793N, increases the rate of ATP-dependent LTC4 transport. Along with their higher transport activities, their Michaelis constant Kms (ATP) are also higher than that of wild-type. Coincident with their higher Kms (ATP), their Kds derived from ATP binding are also higher than that of wild-type, implying that the rate of dissociation of the bound nucleotide from the mutated NBD1 is faster than that of wild-type. Therefore, regardless of whether the bound ATP at NBD1 is hydrolyzed or not, the release of the bound nucleotide from NBD1 may bring the molecule back to its original conformation and facilitate the protein to start a new cycle of ATP-dependent solute transport.

A treatment of leaves of Spinacia oleracea L. with light or with the thiol reagent dithiothreitol in the dark led to partly uncoupled thylakoids. After induction in intact leaves, the partial uncoupling was irreversible at the level of isolated thylakoids. We distinguish between uncoupling by proton slip, which means a decrease of the H+/e(-) -ratio due to less efficient proton pumping, and proton leak as defined by enhanced kinetics of proton efflux. Proton slip and proton leak made about equal contributions to the total uncoupling. The enhanced proton efflux kinetics corresponded to reduction of subunit CF1-gamma of the ATP synthase as shown by fluorescence labeling of thylakoid proteins with the sulfhydryl probe 5-iodoacetamido fluorescein. The maximum value of the fraction of reduced CF1-gamma was only 36%, which indicates that in vivo the reduction of CF1-gamma could be limited by fast reoxidation and/or restricted accessibility of CF1-gamma to thioredoxin. Measurements of the ratio ATP/2e indicated that only the uncoupling related to less efficient proton pumping led to a decrease in the ATP yield.

Novel insights into mechanistic, kinetic and thermodynamic details of ATP synthesis by F1F0-ATP synthase in oxidative phosphorylation and photophosphorylation have been offered from the perspective of the torsional mechanism of energy transduction and ATP synthesis [Nath, S., Adv. Biochem. Eng. Biotechnol. 85 (2003) 125–180]; [Nath, S., Adv. Biochem. Eng. Biotechnol. 74 (2002) 65–98]. A fundamental energetic analysis of membrane phosphorylation has been performed that highlights the merits of the new paradigm. Biological implications for energy transduction have been discussed in detail. The action of uncouplers and inhibitors of oxidative phosphorylation has been explained by a fresh and completely different rationale. New experimental data that further supports the torsional mechanism has been presented, and the consistency of proposed mechanisms with the laws of thermodynamics has been assessed. A general kinetic analysis of oxygen exchange has been shown to reveal the absence of site-site cooperativity in F1-ATPase. Details of the nanomechanics of coupling between F1 and F0 in ATP hydrolysis-driven proton pumping have been postulated. The original thinking and power of the theoretical approaches embodied in the torsional mechanism have been shown to prove invaluable to unravel the functioning of other biological machines through a new Molecular Systems Biology. The generality and universality of the principles in the theory have been illustrated by taking specific examples of other molecular motors such as myosin and kinesin, revealing the deep underlying unity in diverse energy transduction processes in biology. Changes required in our scientific thinking and industrial technology and particularly in the education of the next generation of biological scientists have been identified as challenging issues that urgently need to be addressed in the near future.

In chloroplasts, synthesis of ATP is energetically coupled with the utilization of a proton gradient formed by photosynthetic electron transport. The involved enzyme, the chloroplast ATP synthase, can potentially hydrolyze ATP when the magnitude of the transmembrane electrochemical potential difference of protons (DAH + ) is small, e.g. at low light intensity or in the dark. To prevent this wasteful consumption of ATP, the activity of chloroplast ATP synthase is regulated as the occasion may demand. As regulation systems DAH + activation, thiol modulation, tight binding of ADP and the role of the intrinsic inhibitory subunit q is well documented. In this article, we discuss recent progress in understanding of the regulation system of the chloroplast ATP synthase at the molecular level. D 2002 Elsevier Science B.V. All rights reserved.

ATP synthesis by oxidative phosphorylation and photophosphorylation, catalyzed by F1F0-ATP synthase, is the fundamental means of cell energy production. Earlier mutagenesis studies had gone some way to describing the mechanism. More recently, several X-ray structures at atomic resolution have pictured the catalytic sites, and real-time video recordings of subunit rotation have left no doubt of the nature of energy coupling between the transmembrane proton gradient and the catalytic sites in this extraordinary molecular motor. Nonetheless, the molecular events that are required to accomplish the chemical synthesis of ATP remain undefined. In this review we summarize current state of knowledge and present a hypothesis for the molecular mechanism of ATP synthesis.

The biflavonoids tepicanol 1 and crassifolin 2 and the flavonoids elongatin 3, tephrobotin 4, tephroleocarpin 5, glabranin 6, and methylglabranin 7 were isolated from Tephrosia tepicana, T. crassifolia, T. viridiflora, T. abbottiae, T. leiocarpa, and T. madrensis, respectively, Leguminoseae-Papilionidae, a large tropical and subtropical genus, and their in vitro effects on different photosynthetic activities were investigated. All the compounds isolated, except 1 and 3, induced a concentration-dependent inhibition of photophosphorylation. Compounds 2 and 4-7 interfered with the energetic metabolism of plants at the level of photosynthesis with different mechanisms of action. Compounds 2, 4, and 6 behave as Hill reaction inhibitors, and compounds 5 and 7 act as energy transfer inhibitors (H+-ATPase inhibitor).

Thylakoid membranes isolated from either spinach or chickpea leaves were used as a model system for evaluating the capacity of cyclitols to act as cryoprotectants. The effect of freezing for 3 h at -18 degrees C on cyclic photophosphorylation and electron transport was measured. The cyclitols, ononitol, O-methyl-muco-inositol, pinitol, quebrachitol and quercitol at 50-150 mol m(-3) decreased membrane damage by freezing and thawing to a similar degree as the well known cryoprotectants sucrose and trehalose. On addition of the cryotoxic solute NaCl (100 mol m(-3)) to the test system these methylated cyclohexanhexols again provided a protection comparable to that of the two disaccharides. Quercitol (cyclohexanpentol) was not effective when added in lower concentrations (50-100 mol m(-3)) and in case of this cyclitol a ratio of membrane toxic to membrane compatible solute of 0.66 was apparently needed to prevent a loss of cyclic photophosphorylation. Little difference was observed in the results from spinach or chickpea thylakoids although these plants naturally accumulate different cyto-solutes (spinach: glycinebetaine; chickpea: pinitol).

The ATP synthase from Escherichia coli was reconstituted into liposomes from phosphatidylcholine/phosphatidic acid. The proteoliposomes were energized by an acid-base transition and a K(+)/valinomycin diffusion potential, and one second after energization, the electrochemical proton gradient was dissipated by uncouplers, and the ATP hydrolysis measurement was started. In the presence of ADP and P(i), the initial rate of ATP hydrolysis was up to 9-fold higher with pre-energized proteoliposomes than with proteoliposomes that had not seen an electrochemical proton gradient. After dissipating the electrochemical proton gradient, the high rate of ATP hydrolysis decayed to the rate without pre-energization within about 15 s. During this decay the enzyme carried out approximately 100 turnovers. In the absence of ADP and P(i), the rate of ATP hydrolysis was already high and could not be significantly increased by pre-energization. It is concluded that ATP hydrolysis is inhibited when ADP and P(i) are bound to the enzyme and that a high Delta mu(H(+)) is required to release ADP and P(i) and to convert the enzyme into a high activity state. This high activity state is metastable and decays slowly when Delta mu(H(+)) is abolished. Thus, the proton motive force does not only supply energy for ATP synthesis but also regulates the fraction of active enzymes.

The mechanism of thiol modulation of the chloroplast ATP synthase by Escherichia coli thioredoxin was investigated in the isolated ATPase subcomplex and in the ATP synthase complex reconstituted in bacteriorhodopsin proteoliposomes. Thiol modulation was resolved kinetically by continuously monitoring ATP hydrolysis by the isolated subcomplex and ATP synthesis by proteoliposomes. The binding rate constant of reduced thioredoxin to the oxidized ATPase subcomplex devoid of its e subunit could be determined. It did not depend on the catalytic turnover. Reciprocically, the catalytic turnover did not seem to depend on thioredoxin binding. Thiol modulation by Trx of the e-bearing ATPase subcomplex was slow and favored the release of e. The rate constant of thioredoxin binding to the membrane-bound ATP synthase increased with the protonmotive force. It was lower in the presence of ADP than in its absence, revealing a specific effect of the ATP synthase turnover on thioredoxin-γ subunit interaction. These findings, and more especially the comparisons between the isolated ATPase subcomplex and the ATP synthase complex, can be interpreted in the frame of the rotational catalysis hypothesis. Finally, thiol modulation changed the catalytic properties of the ATP synthase, the kinetics of which became non-Michaelian. This questions the common view about the nature of changes induced by ATP synthase thiol modulation.

Coupling with electrochemical proton gradient, ATP synthase (F(0)F(1)) synthesizes ATP from ADP and phosphate. Mutational studies on high-resolution structure have been useful in understanding this complicated membrane enzyme. We discuss mainly the mechanism of catalysis in the beta subunit of F(1) sector and roles of the gamma subunit in energy coupling. The gamma-subunit rotation during catalysis is also discussed.

In ATP synthase, X-ray structures, demonstration of ATP-driven gamma-subunit rotation, and tryptophan fluorescence techniques to determine catalytic site occupancy and nucleotide binding affinities have resulted in pronounced progress in understanding ATP hydrolysis, for which a mechanism is presented here. In contrast, ATP synthesis remains enigmatic. The molecular mechanism by which ADP is bound in presence of a high ATP/ADP concentration ratio is a fundamental unknown; similarly P(i) binding is not understood. Techniques to measure catalytic site occupancy and ligand binding affinity changes during net ATP synthesis are much needed. Relation of these parameters to gamma-rotation is a further goal. A speculative model for ATP synthesis is offered.

ATP, the universal carrier of cell energy is manufactured from ADP and phosphate by the enzyme ATP synthase using the energy stored in a transmembrane ion gradient. The two components of the ion gradient (DeltapH or DeltapNa(+)) and the electrical potential difference Deltapsi are thermodynamically but not kinetically equivalent. In contrast to accepted wisdom, the electrical component is kinetically indispensable not only for bacterial ATP synthases but also for that from chloroplasts. Recent biochemical studies with the Na(+)-translocating ATP synthase of Propionigenium modestum have given a good idea of the ion translocation pathway in the F(0) motor. Taken together with biophysical data, the operating principles of the motor have been delineated.

The interactions between Mn accumulation and production and control of oxy radicals were investigated in rice (Oryza sativa L. cv. Safari) chloroplasts. Rice plants were grown for 21 days in nutrient solutions containing 2.3–582.5 μM Mn. The plant shoot and the photosynthetic membranes showed 18-fold increases over this range of treatments, but displayed different accumulation kinetics. Production of superoxide and hydroxyl radicals in the thylakoids decreased significantly up to the 36.4 μM Mn treatment, but increased at higher concentrations. In the thylakoid lamellae the total activity of superoxide dismutase (SOD) showed a similar pattern. The enzyme activities of the peroxide system coupled to the photophosphorylation varied significantly with increasing Mn concentrations. Between the 2.3 and the 36.4 μM Mn treatments, the Vmax of ascorbate peroxidase decreased, but thereafter, until the 582.5 μM an opposite trend was found. The activity of dehydroascorbate reductase did not vary significantly with Mn concentration. Between the 2.3 and the 9.1 μM Mn treatments the Vmax of glutathione reductase decreased significantly. In cellular extracts, the Vmax of catalase decreased significantly until the 145.6 μM Mn treatment but in the highest Mn treatment this enzyme activity increased. It was concluded that rice shows a biphasic response to Mn concentrations in the chloroplast lamellae. Until the 36.4 μM, the increasing photosynthetic rates along with the decrease of free radicals production and of the maximum activities of ascorbate peroxidase and SOD indicates that an efficient adaptation of chloroplasts to increasing Mn concentrations occurs. Moreover, from the 36.4 μM onwards, as this process is reversed, the main conclusion is that Mn accumulation starts to be toxic, leading to oxidative stress.

Summary The elevated content of endogenous cytokinin (CK), as the result of the introduction of the chimeric P ssu-ipt gene in tobacco, induced changes in growth, water regime and photosynthesis of transgenic grafts and rooted plants grown in a greenhouse. Despite closed stomata remarkable water stress was detected in P ssu-ipt plants. The ABA content was lower than in wild type tobacco, indicative of disturbances in ABA transport or metabolism. The rates of net photosynthesis (measured as CO 2 uptake or O 2 evolution) decreased by more than 50% in P ssu-ipt grafts and by 20% in P ssu-ipt rooted plants, respectively. The partial reactions of the electron transport chain in transgenic P ssu-ipt tobacco were differently influenced: the activity of the reaction centre of PSII was hardly affected; the PS I activity and the intersystem electron transport chain was inhibited up to 70%, particularly in the P ssu-ipt grafts. Although the slight decrease in the potential photochemical efficiency of PSII, expressed as F v /F m , was found in transgenic plants, the actual quantum yields, photochemical efficiencies, and qP under steady-state conditions indicated that transgenic plants were not seriously limited by the redox state of QA. The reaction centre of PSII was well preserved. In transgenic grafts, photophosphorylation capacity was strongly reduced, which could correspond with lower non-photochemical quenching. The above mentioned changes are the results of elevated CK content per se rather than the effect of altered water relations in the plants, caused by the disproportion of shoots and root system in both P ssu-ipt grafts and plants.

Site-directed mutations were made to the phosphate-binding loop threonine in the beta-subunit of the chloroplast F1-ATPase in Chlamydomonas (betaT168). Rates of photophosphorylation and ATPase-driven proton translocation measured in coupled thylakoids purified from betaT168D, betaT168C, and betaT168L mutants had10% of the wild type rates, as did rates of Mg2+-ATPase activity of purified chloroplast F1-ATPase (CF1). The EPR spectra of VO2+-ATP bound to Site 3 of CF1 from wild type and mutants showed that EPR species C, formed exclusively upon activation, was altered in CF1 from each mutant in both signal intensity and in 51V hyperfine parameters that depend on the equatorial VO2+ ligands. These data provide the first direct evidence that Site 3 is a catalytic site. No significant differences between wild type and mutants were observed in EPR species B, the predominant form of the latent enzyme. Thus, the phosphate-binding loop threonine is an equatorial metal ligand in the activated conformation but not in the latent conformation of Site 3. The metal-nucleotide conformation that gives rise to species B is consistent with the Mg2+-ADP complex that becomes entrapped in a catalytic site in a manner that regulates enzymatic activity. The lack of catalytic function of CF1 with entrapped Mg2+-ADP may be explained in part by the absence of the phosphate-binding loop threonine as a metal ligand.

7-Oxo-7-deacetoxygedunin, a limonoid isolated fromGuarea grandiflora(Meliaceae) has been found to act as an inhibitor of photophosphorylation in spinach thylakoids. ATP synthesis and phosphorylating electron flows were inhibited by 88 and 83%, respectively, at 300 μM of 7-oxo-7-deacetoxygedunin without affecting proton uptake, basal and uncoupled electron transports. The ketone group of 7-oxo-7-deacetoxygedunin is an important structural requirement for the displayed inhibitory activity on chloroplast H+-ATPase, since gedunin possessing a 7α-acetoxy group was a less potent energy transfer inhibitor. The light-activated membrane bound Mg2+-ATPase and the heat-activated Ca2+-ATPase of the isolated coupling factor were insensitive to 7-oxo-7-deacetoxygedunin. 7-oxo-7-deacetoxygedunin shows a competitive kinetic behavior with 5-O-β- D -galactopyranosyl-7-methoxy-3′,4′-dihydroxy-4-phenylcoumarin when they inhibit H+-ATPase activity, but a noncompetitive kinetic with DCCD. These results suggest that 7-oxo-7-deacetoxygedunin and the 4-phenylcoumarin have the same surrounding site of inhibition in C0. Ki for 7-oxo-7-deacetoxygedunin, 4-phenylcoumarin, and DCCD were 146, 220, and 10 μM, respectively.

Mass spectrometric analysis of the light-induced hydrogen gas exchange in the cyanobacteria Oscillatoria chalybea, Synechocystis PCC 6803, Synechococcus PCC 6301 (Synechococcus leopoliensis; Anacystis nidulans) and Synechococcus elongatus has been carried out by direct detection of molecular hydrogen at m/e = 2 in the H/D collector of a ‘delta’ mass spectrometer. The time curves of the signals reveal an initial outburst of hydrogen at the onset of light, with a subsequent superimposition of a hydrogen uptake in the case of Oscillatoria chalybea. With in vivo cultures of Synechocystis PCC 6803, which have not been shown to photoevolve molecular hydrogen so far, we are able to measure very small but clearly detectable evolution and uptake signals. The principal qualitative features of the transition from hydrogen evolution to uptake during the illumination period in the cases of Oscillatoria chalybea and Synechocystis PCC 6803 are about identical. Upon addition of increasing concentrations of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), an increased stable hydrogen photoevolution shows up with the hydrogen uptake being completely suppressed. In the case of Oscillatoria chalybea this effect is obtained with 5 μM CCCP, whereas with Synechocystis PCC 6803 generally higher CCCP concentrations are required to stabilize the hydrogen evolution over time. However, in the presence of CCCP the hydrogen evolution from Synechocystis PCC 6803 (which is nearly negligible in the controls without additions) reaches values very similar to those observed with Oscillatoria chalybea. The stimulatory effect of CCCP is clearly distinct from an uncoupling of electron transport and photophosphorylation, as the addition of ammonium chloride inhibits hydrogen evolution but exerts the expected effect of stimulation of photosynthetic oxygen evolution. However, modification of the pH value of the reaction buffer results in a clear dependency of the hydrogen gas exchange on the external proton concentration. Basic pH values at about > 9 diminish the gas exchange signal as a whole. Acidic pH at about 4–5 substantially increases the evolution and decreases the uptake part of the gas exchange signals. Thus, at a pH of 4.4, the hydrogen gas exchange signal largely corresponds to the one observed with an assay at pH 7.5 but in the presence of CCCP. When the cyanobacterial reaction assays are flushed with pure nitrogen or with N2/CO2 (1%), the hydrogen evolution rates are by no means decreased; in some cases the hydrogen photoevolution appears rather to be stimulated by the presence of CO2 in the flushing gas. Concomitant recording of the light-induced carbon dioxide reveals a strong initial CO2, uptake which is substantially diminished within the first minute of illumination. Thus, mass spectrometric analysis of the light-induced carbon dioxide gas exchange favours the CO2 concentrating mechanism discussed for cyanobacteria rather than the light activation of a dark inactive Calvin cycle.

The integrally membrane-bound, proton-pumping inorganic pyrophosphate (PPi) synthase in phototrophic bacteria is hitherto the only described alternative to the ATP synthase in biological electron transport phosphorylation. We have identified and sequenced the first gene coding for a pyrophosphate synthase. The deduced protein contains 660 amino acid residues and 15 putative membrane-spanning segments. It is homologous to the vacuolar pyrophosphatases from plants.

We introduced mutations at the fully conserved residue Glu-195 in subunit beta of Rhodospirillum rubrum F1-ATPase. The activities of the expressed wild type (WT) and mutant beta subunits were assayed by following their capacity to assemble into the earlier prepared beta-depleted, membrane-bound R. rubrum enzyme (Philosoph, S., Binder, A., and Gromet-Elhanan, Z. (1977) J. Biol. Chem. 252, 8742-8747) and to restore ATP synthesis and/or hydrolysis activity. All three mutations, beta-E195K, beta-E195Q, and beta-E195G, were found to bind as the WTbeta into the beta-depleted enzyme. They restored between 30 and 60% of the WT restored photophosphorylation activity and 16, 45, and 105%, respectively of the CaATPase activity. The mutants required, however, much higher concentrations of divalent cations and could not restore any significant MgATPase or MnATPase activities. Only beta-E195G could restore some of these activities when assayed in the presence of 100 mM sulfite and high MgCl2 or MnCl2 concentrations. These results suggest that the observed difference in restoration of ATP synthesis and CaATPase, as compared with MgATPase and MnATPase, can be due to the tight regulation of the last two activities, resulting in their inhibition at cation/ATP ratios above 0.5. The R. rubrum F1beta-E195 is equivalent to the mitochondrial F1beta-E199, which points into the tunnel leading to the F1 catalytic nucleotide binding sites (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628). Our findings indicate that this residue, although not an integral part of the F1 catalytic sites, affects divalent cation binding and release of inhibitory MgADP, suggesting its participation in the interconversion of the F1 catalytic sites between different conformational states.

Soon after its formation the fruiting spathe of Zantedeschia aethiopica undergoes senescence which is characterized by the loss of its photosynthetic activity. During fruiting, spathe senescence is inhibited and regreening takes place. The amyloplasts present in the white spathe turn to functional chloroplasts and the spathe acquires photosynthetic capacity. In this work, thylakoid membranes were isolated from three distinct stages of spathe development (flora bud, white and regreened spathes) and used for the study of pigment-protein complexes, as well as for the determination of PSI and PSII activities and photophosphorylation rates. Our results indicate that, besides their ultrastructural similarity to leaf chloroplasts, regreened spathe chloroplasts showed values of photochemical and photophosphorylation rates lower than those found in leaf chloroplasts. These results are discussed in relation to the absence of the fatty acid Δ 3 - trans -hexadecenoic acid from regreened spathe thylakoid membranes, and we suggest that this fatty acid could be an important factor for optimal photochemical and photophosforylation activities.

In this investigation, we found that the main source of ATP necessary for envelope protein phosphorylation is generated by photophosphorylation. Indeed, seven envelope polypeptides exhibiting Mr of about 60, 51, 35, 33, 26, 14 and 13 kD were found to be fully labelled in organello in the presence of [32P]Pi and light. The majority of these phosphopolypeptides were also detected when envelopes were phosphorylated in vitro in the presence of [γ-32P]ATP. This supports the idea that the chloroplast stroma significantly supplies envelope protein-kinases with ATP in the light. Preincubation of intact chloroplasts in darkness or with the phosphate translocator inhibitor 4,4′-diisothiocianato-stilbene-2,2′-disulfonate (DIDS) strongly prevented chloroplast protein phosphorylation in the presence of [32P]Pi, thereby attesting the involvement of chloroplast ATP in the envelope protein phosphorylation process. In the dark, two chloroplast soluble polypeptides of about 94 and 67 kD were also labelled, when intact chloroplasts were fed with [32P]Pi, thus indicating that these two polypeptides are able to use dark-phosphorylation pathways, independent of the photophosphorylation process, to become phosphorylated. Our results show also that the 67 kD dark-phosphorylated protein is very likely to be a phosphoglucomutase, since its labelling was markedly reduced by the addition of exogenous glucose 1- or 6-phosphate.

The proton translocation stoichiometry (H+/ATP ratio) and other bioenergetic features were investigated in membrane vesicles from the moderately thermophile Synechococcus 6716 grown at 38°C and 50°C with saturating light intensity, and at 38°C with limiting light intensity. At 50°C growth is slower but proceeds to a higher cell density than at 38°C. Increasing the growth temperature from 38°C to 50°C resulted in an altered membrane fatty acid composition, with increased length and saturation of the acyl chains. At 38°C and lower light intensity chain length was somewhat decreased and saturation increased to a small extent. Membrane vesicles from cells grown at 50°C performed cyclic photophosphorylation at lower light intensities and lower threshold Δ μ H + than vesicles from cells grown at 38°C. The 50°C vesicles also displayed a diminished light-induced proton uptake, but ATP synthesis activity and the attained ΔGp remained constant. Moreover, ATP synthesis became more resistant to uncoupling. From acid–base transition induced ATP synthesis experiments the H+/ATP ratios were determined to be 3.9, 3.1 and 3.3 for membrane vesicles from cells grown at 50°C, 38°C and light-limited 38°C, respectively. In vesicles from cells grown at 50°C, ATP hydrolysis is inhibited by a lower valinomycin-induced K+-diffusion potential than in vesicles from cells grown at 38°C. A molecular mechanism to explain changes in H+/ATP as well as the physiological implications are discussed.

The effect of sulfite on ATP synthesis and hydrolysis activities is investigated in spinach chloroplasts and in membrane vesicles from the cyanobacterium Synechococcus 6716. Sulfite inhibits phenazine methosulfate-mediated cyclic photophosphorylation both in thiol-modulated chloroplasts and in cyanobacterial membranes with HSO3- (bisulfite) as the active ionic species. The observed inhibition is not due to inhibition of electron transfer or to uncoupling by sulfite. ATP synthesis in cyanobacterial membranes is more sensitive to sulfite when the inorganic phosphate concentration is decreased. This indicates competition between sulfite and phosphate for the same binding site on the ATP synthase. In cyanobacterial membranes sulfite can replace a proton gradient as activator of ATP hydrolysis in the same way as in reduced chloroplasts. By modeling, competition between sulfite and phosphate can fully explain the findings concerning both inhibition and activation.