Determination of H+/e- ratios in chloroplasts with flashing light.

Using a rapid pH electrode, measurements were made of the flash-induced proton transport in isolated spinach chloroplasts. To calibrate the system, we assumed that in the presence of ferricyanide and in steady-state flashing light, each flash liberates from water one proton per reaction chain. We concluded that with both ferricyanide and methylviologen as acceptors two protons per electron are translocated by the electron transport chain connecting Photosystem II and I. With methyl viologen but not with ferricyanide as an acceptor, two additional protons per electron are taken up due to Photosystem I activity. One of these latter protons is translocated to the inside of the thylakoid while the other is taken up in H2O2 formation. Assuming that the proton released during water splitting remains inside the thylakoid, we compute H+/e- ratios of 3 and 4 for ferricyanide and methylviologen, respectively. In continuous light of low intensity, we obtained the same H+/e- ratios. However, with higher intensities where electron transport becomes rate limited by the internal pH, the H+/e- ratio approached 2 as a limit for both acceptors. A working model is presented which includes two sites of proton translocation, one between the photoacts, the other connected to Photosystem I, each of which translocates two protons per electron. Each site presents a approximately 30 ms diffusion barrier to proton passage which can be lowered by uncouplers to 6-10 ms.     

Sulfide-dependent photosynthetic electron flow coupled to proton translocation in thylakoids of the cyanobacterium Oscillatoria limnetica

Light-induced proton translocation coupled to sulfide-dependent electron transport has been studied in isolated thylakoids of the cyanobacterium Oscillatoria limnetica. The thylakoids are obtained by osmotic shock of washed spheroplasts, prepared with glycine-betaine as the osmotic stabilizer. 13C NMR studies suggests that betaine is the major osmoregulator in O. limnetica. Thylakoid preparations obtained from both sulfide-induced anoxygenic cells and noninduced oxygenic cells are capable of proton pumping coupled to phenazinemethosulfate-mediated cyclic electron flow. However, only in the induced thylakoids can sulfide-dependent proton gradient (delta pH) formation be measured, using either NADP or methyl viologen as the terminal acceptor. Sulfide-dependent delta pH formation correlates with a high-affinity electron donation site (apparent Km 44 microM at pH 7.9). This site is not lost upon washing of the thylakoids. In addition, both sulfide-dependent electron transport and delta pH formation are sensitive to inhibitors of the cytochrome b6f complex such as 2-n-nonyl-4-hydroxyquinoline-N-oxide, 2,4-dinitrophenyl ether of 2-iodo-4-nitrothymol, or stigmatellin. Sulfide-dependent NADP photoreduction of low affinity (which does not saturate by as much as 7 mM sulfide) is detected in both induced and noninduced thylakoids, but this activity is insensitive to the inhibitors and is not coupled to proton transport. It is suggested that the adaptation of O. limnetica to anoxygenic photosynthesis involves the induction of a thylakoid factor(s) which creates a high-affinity site for sulfide, and the transfer of its electrons via the cytochrome b6f complex, coupled to proton translocation.     

Characteristics of energy-linked proton translocation in liposome reconstituted bovine cytochrome bc1 complex. Influence of the protonmotive force on the H+/e- stoichiometry.

A study is presented on the H+/e- stoichiometry for proton translocation by the isolated cytochrome bc1 complex under level-flow and steady-state conditions. An experimental procedure was used which allows the determination of pure vectorial proton translocation in both conditions in a single experiment. The results obtained indicate an H+/e- ratio of 1 at level-flow and 0.3 at steady-state. The ratios appear to be independent of the rate of electron transfer through the complex. Making use of pyranine-entrapped bc1 vesicles, a respiration-dependent steady-state delta pH value of 0.4 was determined in the presence of valinomycin. This value could be either decreased by subsaturating concentrations of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) or increased by introducing bovine serum albumin in the assay mixture. The steady-state H+/e- ratio appeared to be in linear inverse correlation with the delta pH. This indicates that delta pH exerts a control on the proton pump of the bc1 complex at the steady state. The effect of valinomycin-mediated potassium-diffusion potential on electron-transfer and proton-translocation activities is also shown. The experiments presented show that the H+/e- ratio is unaffected, both at level flow and steady state, by an imposed diffusion potential up to around 100 mV. At higher potential values the level-flow H+/e- ratio slightly decreased. Measurements as a function of imposed membrane potential of the rate of electron transfer at level flow and of the rate of the pre-steady-state reduction of b and c1 cytochromes in the complex indicate activation of electron transfer at potential values of 40-50 mV. This activation appears, however, to involve a rate-limiting step which remains normally coupled to proton translocation.     

Energy transduction by the reconstituted b-c1 complex from yeast mitochondria. Inhibitory effects of dicyclohexylcarbodiimide

A purified cytochrome b-c1 complex isolated from yeast mitochondria has been reconstituted into proteoliposomes. The reconstituted comp]lex catalyzed antimycin A-sensitive electron transfer from different analogues of coenzyme Q to cytochrome c. The reconstituted complex was also capable of energy conservation as indicated by uncoupler-stimulated rates of electron transfer, electrogenic proton ejection, and reversed electron flow from cytochrome b to coenzyme Q2 in the presence of antimycin A driven by a valinomycin-induced K+-diffusion potential (negative inside). Close to four protons were ejected per two electrons transported through the reconstituted b-c1 complex with ferricyanide as an artificial and impermeable electron acceptor.l The H+/2e- ratio decreased to two in the presence of the proton-conducting agent, carbonyl cyanide m-chlorophenylhydrazone. The same processes were studied in parallel in energy-conserving site 2 of rat liver mitochondria with similar results. In the reconstituted b-c1 complex, dicyclohexylcarbodiimide (DCCD) blocked the function of the electrogenic proton translocating device in the forward direction of proton ejection as well as in the backwards direction, measured as reversed electron flow from cytochrome b to coenzyme Q2 driven by a K+-diffusion potential. The primary effect of DCCD is localized on the proton ejection process, as the low proton conductance of the proteoliposome membrane was totally preserved after DCCD treatment.     

Characteristics of the redox-linked proton ejection in beef-heart cytochrome c oxidase reconstituted in liposomes

In this paper a study is presented of the characteristics of redox-linked proton ejection exhibited by isolated beef-heart cytochrome c oxidase incorporated in asolectin vesicles. The enzyme was 90% oriented 'right-side out' as in the mitochondrial membrane. The effects on the H+/e- stoichiometry of the modalities of activation of electron flow, the pH of the medium and its ionic composition were investigated. The results obtained show that, whilst ferrocytochrome c pulses of the aerobic oxidase vesicles at neutral pH and in the presence of saturating concentrations of valinomycin and K+ to ensure charge compensation produced H+/e- ratios around 1 (as has been shown previously), oxygen pulses of reduced anaerobic vesicles supplemented with cytochrome c, gave H+/e- ratios around 0.3. The H+/e- ratios exhibited, with both reductant and oxidant pulses, a marked pH dependence. Maximum values were observed at pH 7.0-7.7, which decreased to negligible values at acidic pH with apparent pKa of 6.7-6.3. Mg2+ and Ca2+ caused a marked depression of the H+/e- ratio, which in the presence of these cations and after a few ferrocytochrome pulses, became negligible. Analysis of cytochrome c oxidation showed that the modalities of activation of electron flow and divalent cations exerted profound effects on the kinetics of cytochrome c oxidation by oxidase vesicles. The observations presented seem to provide interesting clues for the nature and mechanism of redox-linked proton ejection in reconstituted cytochrome c oxidase.     

pH dependence of proton translocation by Escherichia coli.

Proton translocation in spheroplasts from Escherichia coli has been studied in two mutants, one of which expresses cytochrome o and the other cytochrome d as the terminal oxidase. Using the O2 pulse method, the H+/e- ratio of proton translocation associated with cytochrome o was confirmed to be near 2 at neutral pH, but was found to decrease considerably when the medium pH was raised above 8. At high pH there was an increase in H+/OH- permeability of the cell membrane, but this was not sufficient to explain the decline in proton ejection. The pH effect was confined to cytochrome o-linked activity. It was not present when cytochrome d generated the electrochemical proton gradient. This makes it improbable that the Na+/H+ antiporter is responsible. The most likely explanation for our finding is that there is a "slip" in the proton-pumping mechanism of cytochrome o at high pH.     

Coupling Ratios H+/e=3 versus H+/e=2 in Chloroplasts and Quantum Requirements of Net Oxygen Exchange during the Reduction of Nitrite, Ferricyanide or Methylviologen

Using intact and osmotically ruptured chloroplasts, ratios ofcoupling between deposition of protons in the intrathylakoidspace and light-dependent transport of electrons from waterto an external acceptor were determined. The data indicate couplingbetween proton and electron transport at a ratio of H⁺/e=3 withmethylviologen as electron acceptor in thylakoids and with nitriteas electron acceptor in intact chloroplasts. With ferricyanideas electron acceptor in thylakoids, values close to H⁺/e=2 wereobserved. Evidence is discussed that H⁺/e=3 is a fixed valuein intact chloroplasts at levels of thylakoid energization sufficientfor supporting effective carbon assimilation. In the presence of methylviologen and ascorbate, the minimumquantum requirement of oxygen uptake by thylakoids was about2.7 quanta of 675 nm light per O₂ indicating an e/O₂ ratio of1.33. In the absence of ascorbate, and with KCN present in additionto methylviologen, e/O₂ ratios up to 4 were observed. The minimumquantum requirement of oxygen evolution by thylakoids in thepresence of ferricyanide and by intact chloroplasts in the presenceof nitrite was about 8 quanta/O₂. (Received May 1, 1995; Accepted October 2, 1995)     

The role of the membrane-bound hydrogenase in the energy-conserving oxidation of molecular hydrogen by Escherichia coli.

H2-dependent reduction of fumarate and nitrate by spheroplasts from Escherichia coli is coupled to the translocation of protons across the cytoplasmic membrane. The leads to H+/2e- stoicheiometry (g-ions of H+ translocated divided by mol of H2 added) is approx. 2 with fumarate and approx. 4 with nitrate as electron acceptor. This proton translocation is dependent on H2 and a terminal electron acceptor and is not observed in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone and the respiratory inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide. H2-dependent reduction of menadione and ubiquinone-1 is coupled to a protonophore-sensitive, but 2-n-heptyl-4-hydroxy-quinoline N-oxide-insensitive, proton translocation with leads to H+/2e- stoicheiometry of approx. 2. H2-dependent reduction of Benzyl Viologen (BV++) to its radical (BV+) liberates protons at the periplasmic aspect of the cytoplasmic membrane according to the reaction: H2 + 2BV++ leads to 2H+ + 2BV+. It is concluded that the effective proton translocation observed in the H2-oxidizing segment of the anaerobic respiratory chain of Escherichia coli arises as a direct and inevitable consequence of transmembranous electron transfer between protolytic reactions that are spatially separated by a membrane of low proton-permeability.     

Phospholipid vesicles containing bovine heart mitochondrial cytochrome c oxidase and subunit III-deficient enzyme: analysis of respiratory control and proton translocating activities

Phospholipid vesicles containing bovine heart mitochondrial cytochrome c oxidase (COV) or subunit III (Mr 29884)-deficient enzyme (COV-III) were characterized for electron transfer and proton translocating activities in order to investigate the relationship between the respiratory control ratio (RCR) and the apparent proton translocated to electron transferred stoichiometry (H+/e- ratio) in these preparations. We did not observe a quantitative correlation between the RCR value and the H+/e- ratio in the preparations. Significant deviation between these two parameters was observed in COV-III and also in COV. However, a new parameter, RCRval, did show a linear relationship with the H+/e- ratio of each preparation. Subunit III (SIII)-deficient cytochrome c oxidase isolated by either native gel electrophoresis or chymotrypsin treatment and incorporated into COV-III exhibited H+/e- ratios of 0.34 +/- 0.10, compared to 0.63 +/- 0.09 for COV, emphasizing that the 50% decrease of proton translocating activity is independent of the method of removal of SIII from the enzyme. COV and COV-III also showed similar rates of alkalinization of the extravesicular media after the initial proton translocation reaction (0.07-0.09 neq OH-/s), suggesting that these two preparations had similar endogenous proton permeabilities. In contrast, cytochrome c oxidase (COX) treated with Triton X-100 (3 mg/mg COX) and incorporated into phospholipid vesicles [COV (+TX)] exhibited slower rates of alkalinization (0.04 neq OH-/s), while having a H+/e- ratio similar to that of COV (0.66 +/- 0.10). The passive proton permeabilities of these preparations were tested by valinomycin-induced K+/H+ exchange activity. COV (+TX) and COV-III exhibited similar pseudo-first-order rate constants (10 peq OH-/s), while COV had a 20-fold higher rate constant. These results taken together suggest that the different preparations of COX-containing phospholipid vesicles have different biophysical properties. In addition, the decrease in proton-pumping activity observed in COV-III is due to removal of SIII from COX, suggesting that SIII may act either as a passive proton-conducting channel or as a regulator of COX conformation and/or functional activities.     

Phospholipid vesicles containing bovine heart mitochondrial cytochrome c oxidase exhibit proton translocating activity in the presence of gramicidin.

Phospholipid vesicles containing bovine heart mitochondrial cytochrome c oxidase (COV) were characterized for electron transfer and proton translocating activities in the presence of the mobile potassium ionophore, valinomycin, and the channel-forming ionophore, gramicidin, in order to determine if the ionophores modify the functional properties of the enzyme. In agreement with previous work, incubation of COV with valinomycin resulted in a perturbation of the absorbance spectrum of oxidized heme aa3 in the Soret region (430 nm); gramicidin had no effect on the heme aa3 absorbance spectrum. Different concentrations of the two ionophores were required for maximum respiratory control ratios in COV; 40- to 70-fold higher concentrations of valinomycin were required to completely uncouple electron transfer activity when compared to gramidicin. The proton translocating activity of COV incubated with each inophore gave a similar apparent proton translocated to electron transferred stoichiometry (H+/e- ratio) of 0.66 +/- 0.10. However, COV treated with low concentrations of gramicidin (0.14 mg/g phospholipid) exhibited 1.5- to 2.5-fold higher rates of alkalinization of the extravesicular media after the initial proton translocation reaction than did COV treated with valinomycin, suggesting that gramicidin allows more rapid equilibration of protons across the phospholipid bilayer during the proton translocation assay. Moreover, at higher concentrations of gramicidin (1.4 mg/g phospholipid), the observed H+/e- ratio decreased to 0.280 +/- 0.020, while the rate of alkalinization increased an additional 2-fold, suggesting that at higher concentrations, gramicidin acts as a proton ionophore. These results support the hypothesis that cytochrome c oxidase is a redox-linked proton pump that operates at similar efficiencies in the presence of either ionophore. Low concentrations of gramicidin dissipate the membrane potential in COV most likely by a channel mechanism that is different from the carrier mechanism of valinomycin, yet does not make the phospholipid bilayer freely permeable to protons.     

Regulation of the proton/electron stoichiometry of mitochondrial ubiquinol:cytochrome c reductase by the membrane potential.

The electron transfer reaction catalysed by mitochondrial ubiquinol:cytochrome c reductase is linked to the outwards translocation of protons with an H+ e- stoichiometry of 1 under non-membrane potential condition. The effect of the electrical membrane potential on the H+/e- stoichiometry was investigated. The enzyme was isolated from Neurospora crassa, reconstituted into phospholipid vesicles and electrical membrane potentials of various values were generated across the membranes by means of the valinomycin-induced potassium-diffusion method. Using lithium ions as counterions for the intravesicular potassium, the induced membrane potential was stable for minutes and was not significantly changed by the protons ejected by the working enzyme. This allowed the assay of steady-state reaction rates at pre-given values of electrical membrane potential. The rate ratio between electron transfer and proton translocation declined from 1 to 0.6 with increase of the membrane potential from 0 to 100 mV. The activity of the quinol/cytochrome c redox reaction followed a parabolic dependence, being activated by low (less than 50 mV) potential and inhibited by high (greater than 100 mV) potential. This apparent non-linear dependence was interpreted in terms of a linear flow/force relationship plus a membrane-potential-dependent slip. Evaluation of the parabolic course by means of a modified linear flow/force relation also indicated a decline of the H+/e- stoichiometry from 1 to 0.5 with increase of the membrane potential from 0 to 120 mV. These observations suggest that the membrane potential controls a change of ubiquinol:cytochrome c reductase between two states that have different reaction routes.     

Voltammetric measurement of the plastoquinone redox state in isolated thylakoids

The oxidation of plastoquinol by the cytochrome bf complex is commonly believed to be the rate limiting step in photosynthetic electron transport. When input of electrons from PS II exceeds electron flow through the cytochrome bf complex the plastoquinone pool becomes reduced. A voltammetric technique previously used to measure the redox state of the ubiquinone pool in plant mitochondria, was modified to measure the redox state of the plastoquinone pool in thylakoids. The presence or absence of a proton gradient strongly influenced the relationship between the redox state of the plastoquinone pool and other photosynthetic parameters. A linear relationship between the rate of electron transport and the reduction of plastoquinone was found. The slope of this relationship was greater in coupled than in uncoupled thylakoids, indicating that under coupled conditions the plastoquinone pool is more reduced at any given rate of electron flow. A complex relationship was found between QA reduction, calculated as 1 – q_p, and the redox state of the plastoquinone pool. The extent of Q_A reduction was similar in coupled and uncoupled thylakoids, but at any given level of Q_A reduction, PQ was always more reduced in coupled thylakoids. These results suggest that the presence of a proton gradient changes the equilibrium constant between Q_A and PQ.     

H+/e- stoichiometry of mitochondrial cytochrome complexes reconstituted in liposomes. Rate-dependent changes of the stoichiometry in the cytochrome c oxidase vesicles.

The H+/e- stoichiometry of protonmotive cytochrome c oxidase, isolated from bovine heart mitochondria and reconstituted in liposomes, has been determined by making use of direct spectrophotometric measurements of the initial rates of e- flow and H+ translocation. It is shown that the ----H+/e- ratio for redox-linked proton ejection by the oxidase varies from around 0 to a maximum of 1 as a function of the rate of overall electron flow in the complex.     

Quinone interactions with the chloroplast cytochrome b6-f complex

The requirements for reconstitution of electron transfer activity with a plastoquinone (PQ)-depleted cytochrome b6-f complex from spinach have been considered. Full restoration of activity measured as plastocyanin reduction with either duroquinol in the dark or Photosystem II (PSII) in the light requires both PQ-9 and phospholipid. However, a substantial dark activity can be observed with duroquinol and phospholipid in the absence of any added PQ-9. PSII, with its associated PQ molecules, can also donate electrons in the light to the cytochrome complex which has been depleted of plastoquinone. Electron donation by duroquinol in the dark to the PQ-depleted cytochrome complex is stimulated by PSII, and this stimulation is dependent on the presence of the two PQ molecules in the PSII preparation. Measurements of proton translocation with the PQ-depleted complex indicate this quinone is not required for the observed H+/e- ratio of 2. Studies of cytochrome b6 kinetics with the free and liposome-incorporated PQ-depleted complex show this cytochrome undergoes redox reactions similar to those of a control complex which contains PQ. These results indicate the PQ that copurifies with the cytochrome complex is not essential for any of the measured activities. These findings are considered in relation to a quinone binding site(s) in the cytochrome complex which is not specific to PQ but can bind other quinones, such as duroquinol, in a lipid-dependent process.     

Redox-linked proton translocation in the b-c1 complex from beef-heart mitochondria reconstituted into phospholipid vesicles. General characteristics and control of electron flow by delta micro H+

A study is presented of the characteristics of redox-linked proton translocation in the b-c1 complex isolated from beef-heart mitochondria and reconstituted into phospholipid vesicles. Measurements of the H+/e- stoichiometry, with three different methods, show that four protons are released from the vesicles per 2e- flowing from quinols to cytochrome c, two of these protons formally deriving from scalar oxidation of quinols by cytochrome c. This H+/e- stoicheiometry is independent of the initial redox state of the b-c1 complex (fully reduced or oxidized) and the rate of electron flow through the complex. It does not change in the pH range 6.0 - 7.2, but declines to 1.5 going with pH from 7.2 - 8.3. This decrease is accompanied by enhancement of the rate of electron flow in the coupled state. Collapse of delta psi effected by valinomycin addition to turning-over b-c1 vesicles resulted in substantial oxidation of cytochrome b-566 and comparable reduction of cytochrome c1, with little oxidation of cytochrome b-562. Nigericin alone had no effect on the steady-state redox levels of b and c cytochromes. Its addition in the presence of valinomycin caused oxidation of b cytochromes but no change in the redox state of cytochrome c1. Valinomycin alone caused a marked enhancement of the rate of electron flow through the complex. Nigericin alone was ineffective, but caused further stimulation of electron flow when added in the presence of valinomycin. The data presented are discussed in terms of two mechanisms: the Q cycle and a model based on combination of protonmotive catalysis by special bound quinone and proton conduction along pathways in the apoproteins.     

Proton pump coupled to cytochrome c oxidase in Paracoccus denitrificans

The proton translocating properties of cytochrome c oxidase in whole cells of Paracoccus denitrificans have been studied with the oxidant pulse method. leads to H+/2e- quotients have been measured with endogenous substrates, added methanol and added ascorbate (+TMPD) as reductants, and oxygen and ferricyanide as oxidants. It was found that both the observed leads to H+/O with ascorbate (+TMPD) as reductant, and the differences in proton ejection between oxygen-and ferricyanide pulses, with endogenous substrates or added methanol as a substrate, indicate that the P. denitrificans cytochrome c oxidase translocates protons with a stoichiometry of 2H+/2e-. The results presented in this and previous papers are in good agreement with recent findings concerning the mitochondrial cytochrome c oxidase, and suggest unequal charge separation by different coupling segments of the respiratory chain of P. denitrificans.     

p-nitroacetophenoxime N-methylcarbamate,a new electron acceptor in isolated thylakoids

p-Nitroacetophenoxime N-methylcarbamate (MCPNA) is a rather potent inhibitor of the electron transfer in spinach class A chloroplasts. In isolated thylakoids, MCPNA is an electron acceptor at the level of photosystem I (PS I). It inhibits O₂ evolution in the presence of NADP and ferredoxin but not the reduction of ferricyanide. MCPNA is active as an acceptor between 3 μM and 100 μM. At concentrations higher than 300 μM, inhibition of photosystem II (PS II) occurs. MCPNA has no uncoupling effect on photophosphorylation. Reduction of MCPNA by thylakoids in the presence of light is in accordance with the E′_o of this compound (??0.57 V) and is followed by an electron transfer to O₂. This reaction probably explains the inhibitory effect of MCPNA on class A chloroplasts.     

pH dependence of proton translocation in the oxidative and reductive phases of the catalytic cycle of cytochrome c oxidase. The role of H2O produced at the oxygen-reduction site

A study is presented on the pH dependence of proton translocation in the oxidative and reductive phases of the catalytic cycle of purified cytochrome c oxidase (COX) from beef heart reconstituted in phospholipid vesicles (COV). Protons were shown to be released from COV both in the oxidative and reductive phases. In the oxidation by O2 of the fully reduced oxidase, the H+/COX ratio for proton release from COV (R --> O transition) decreased from approximately 2.4 at pH 6.5 to approximately 1.8 at pH 8.5. In the direct reduction of the fully oxidized enzyme (O --> R transition), the H+/COX ratio for proton release from COV increased from approximately 0.3 at pH 6.5 to approximately 1.6 at pH 8.5. Anaerobic oxidation by ferricyanide of the fully reduced oxidase, reconstituted in COV or in the soluble case, resulted in H+ release which exhibited, in both cases, an H+/COX ratio of 1.7-1.9 in the pH range 6.5-8.5. This H+ release associated with ferricyanide oxidation of the oxidase, in the absence of oxygen, originates evidently from deprotonation of acidic groups in the enzyme cooperatively linked to the redox state of the metal centers (redox Bohr protons). The additional H+ release (O2 versus ferricyanide oxidation) approaching 1 H+/COX at pH < or = 6.5 is associated with the reduction of O2 by the reduced metal centers. At pH > or = 8.5, this additional proton release takes place in the reductive phase of the catalytic cycle of the oxidase. The H+/COX ratio for proton release from COV in the overall catalytic cycle, oxidation by O2 of the fully reduced oxidase directly followed by re-reduction (R --> O --> R transition), exhibited a bell-shaped pH dependence approaching 4 at pH 7.2. A mechanism for the involvement in the proton pump of the oxidase of H+/e- cooperative coupling at the metal centers (redox Bohr effects) and protonmotive steps of reduction of O2 to H2O is presented.     

Porton uptake by isolated chloroplasts during cyclic and non-cyclic electron transport catalyzed by photosystem I in the presence of ferredoxin]

Proton uptake by isolated chloroplasts during cyclic electron transport in the presence of ferredoxin and under NADP+ reduction from the ascorbate--TMPD donor pair under anaerobic conditions was studied. It was found that during cyclic transport the proton uptake is less intensive than under NADP+ reduction. In the presence of ATP the proton uptake is increased in the first case and is decreased in the second one. During cyclic transport in the presence of gramicidin D the proton uptake is completely suppressed and under NADP+ reduction is decreased down to 0,08--0,09 mk equiv H+ per mg of chlorophyll, irrespective of ferredoxin or NADP+ concentrations. The role of ferredoxin NADP+ reductase in a proton uptake by thylakoids is discussed.