ADP and ATP binding to noncatalytic sites of thiol-modulated chloroplast ATP synthase

A modified ‘cold chase’ technique was used to study tight [¹⁴C]ADP and [¹⁴C]ATP binding to noncatalytic sites of chloroplast ATP synthase (CF₀F₁). The binding was very low in the dark and sharply increased with light intensity. Dissociation of labeled nucleotides incorporated into noncatalytic sites of CF₀F₁ or CF₁ reconstituted with EDTA-treated thylakoid membranes was also found to be light-dependent. Time dependence of nucleotide dissociation is described by the first order equation with a k _d of about 5 min⁻¹. The exposure of thylakoid membranes to 0.7–24.8 μM nucleotides leads to filling of up to two noncatalytic sites of CF₀F₁. The sites differ in their specificity: one preferentially binds ADP, whereas the other – ATP. A much higher ATP/ADP ratio of nucleotides bound at noncatalytic sites of isolated CF₁ dramatically decreases upon its reconstitution with EDTA-treated thylakoid membranes. It is suggested that the decrease is caused by conformational changes in one of the α subunits induced by its interaction with the δ subunit and/or subunit I–II when CF₁ becomes bound to a thylakoid membrane.     

Photoaffinity labeling of soluble chloroplast adenosine 5′-triphosphatase with 3′-O-(4-benzoyl)benzoyl ADP

3′-O-(4-benzoyl)benzoyl ADP (BzADP) acts as a reversible inhibitor of the chloroplast coupling factor 1 ATPase (CF₁) when incubated with the enzyme in the dark. The V_max of ATP hydrolysis is decreased and the kinetics of the reaction are altered from noncooperative to cooperative with respect to ATP. Photoactivation of the benzophenone group in BzADP by irradiation with ultraviolet light (366 nm) results in the covalent binding of BzADP to the enzyme and inactivation of its enzymic activity. Polyacrylamide gel electrophoresis of CF₁-ATPase in the presence of sodium dodecyl sulfate shows that the analog is bound primarily to the enzyme's β subunit. Complete inactivation of the activated CF₁-ATPase occurs upon covalent binding of 2.45 mol BzADP/mol CF₁. Binding of BzADP and inactivation of the ATPase are prevented if ADP, but not ATP, is present during the photoactivation step. The presence of Ca²⁺ during irradiation enhances the rate of BzADP covalent binding as well as the rate of inactivation of the enzyme.     

pH dependent changes in ADP and ATP affinity for the tight nucleotide-binding site of chloroplast coupling factor 1

The pH-dependence of ADP and ATP affinity for CF₁ tight nucleotide-binding sites was studied under conditions of equilibrium between bound and free labeled nucleotides. With the nucleotide/CF₁ ratio>1, the ATP content in tightly bound nucleotides depended only slightly on medium pH. With the nucleotide/CF₁ ratio approaching 1, tightly bound ATP content grew rapidly with decreasing pH. Calculations of ADP/ATP ratio in free and tightly bound nucleotides showed that decreasing the pH from 8.0 to 6.0 induced a 150 times greater affinity of the nucleotide-binding site for ATP than for ADP. The data indicates that ATP-ADP equilibrium at the CF₁ tight nucleotide-binding site depends on protonation of specific acid-base groups of the enzyme.Abbreviations CF₁, BF₁, and MF₁ coupling factors of chloroplasts, bacteria, and mitochondria, respectively - AdN adenine nucleotide     

Fragmentation of chloroplast coupling factor in dependence of bound nucleotides Preparation of a reconstitutionally active form of subunit δ

Previous studies on the ability of CF₁, fragments to reconstitute photophosphorylation in CF₁,-depleted thylakoids have shown that the degree of reconstitution was correlated with the presence of subunit δ in the fragment. This was taken as evidence that subunit δ was necessary for plugging the active proton channel CF₀ [(1986) Eur. J. Biochem. 160, 635–643]. We questioned whether or not δ alone had this ability. In order to obtain δ we investigated the role of bound nucleotides in the stability of CF₁. Starting from ammonium sulfate-precipitated CF₁, we found that a low content of bound ADP (1 mol ADP/mol CF₁) seemed to stabilize the β—δ interaction, while loosening the interaction between α,β and γ. By elution from an anion-exchange column in the presence of the nonionic surfactant Mega 9 we obtained β₃δ and CF₁(—δ) (both containing one ADP) or, after washing with alcohol/glycerol mixtures, β (nucleotide-free) and CF₁/CF₁(—ϵ). On the other hand, with a further 2 ADP and 2 ATP bound to CF₁, (after incubation with excess ATP) the α-β-γ interaction was stabilized in such a way that subunit δ alone could be isolated from the complex. Subunit δ, when isolated by this procedure and added back to CF₁-depleted thylakoids, reconstituted a high rate of photophosphorylation.     

Energy-dependent exchange of adenine nucleotides on chloroplast coupling factor (CF1)

1. [¹⁴C]ADP is incorporated into washed broken chloroplasts in the light. The bound labelled nucleotides which cannot be removed by washing are almost exclusively related to coupling factor CF₁. [¹⁴C]ADP binding exhibits a monophasic concentration curve with a K_m of 2 μM.2. By illumination of the chloroplasts, previously incorporated labelled nucleotides are released. A fast release is obtained in the presence of unlabelled ADP and ATP, indicating an energy-dependent exchange. A slow and incomplete release is induced by light in the absence of unlabelled adenine nucleotides. Obviously, under those conditions, an adenine nucleotide depleted CF₁ conformation is established.3. Re-binding of [¹⁴C]ADP by depleted membranes is an energy-independent process. Even after solubilization of adenylate-depleted CF₁, [¹⁴C]ADP is incorporated into the protein. By re-binding of ADP in the dark, CF₁ is converted to a non-exchangeable form.4. Energy-dependent adenine nucleotide exchange on CF₁ is suggested to include three different conformational states of the enzyme: (1) a stable, non-exchangeable form which contains firmly bound nucleotides, is converted to (2), an unstable form containing loosely bound adenine nucleotides. This conformation allows adenylate exchange; it is in equilibrium with (3) a metastable, adenylate-depleted form. The transition from state (1) to state (2) is the energy-requiring step.     

Properties of noncatalytic sites of thioredoxin-activated chloroplast coupling factor 1

Nucleotide binding properties of two vacant noncatalytic sites of thioredoxin-activated chloroplast coupling factor 1 (CF₁) were studied. Kinetics of nucleotide binding to noncatalytic sites is described by the first-order equation that allows for two nucleotide binding sites that differ in kinetic features. Dependence of the nucleotide binding rate on nucleotide concentration suggests that tight nucleotide binding is preceded by rapid reversible binding of nucleotides. ADP binding is cooperative. The preincubation of CF₁ with Mg²⁺ produces only slight effect on the rate of ADP binding and decreases the ATP binding rate. The ATP and ADP dissociation from noncatalytic sites is described by the first-order equation for similar sites with dissociation rate constants k₋₂(ADP)=1.5×10⁻¹ min⁻¹ and k₋₂(ATP)≅10⁻³ min⁻¹, respectively. As follows from the study, the noncatalytic sites of CF₁ are not homogeneous. One of them retains the major part of endogenous ADP after CF₁ precipitation with ammonium sulfate. Its other two sites can bind both ADP and ATP but have different kinetic parameters and different affinity for nucleotides.     

Cooperativity Between the Enzymatic Sites of F1-ATPase Revisited by the Use of HPLC Methods

The fundamental question of the cooperativity between the enzymatic sites of F₁-ATPase is examined in the light of new measurements of the enzymatic rate of ATP hydrolysis by CF₁, the enzyme isolated from spinach chloroplasts. The experimental data, obtained with a chromatographic method, fit a model that involves two kinds of independent enzymatic sites working with metal-free ATP, with no need of cooperativity between the sites. Binding measurements between ADP or ATP and CF₁ by the chromatographic method of Hummel and Dreyer (1962) also support this conclusion. The present data and interpretation are in agreement with those reported recently (Reynafarje and Pedersen, 1996) which show that the first order rate constant of ATP hydrolysis by MF₁, the analogous enzyme from mitochondria, is virtually constant under experimental conditions involving either unisite or multisite hydrolysis of ATP. The present data and interpretation are discussed together with those reported previously, in particular with regard to the methods that were used to support the commonly accepted opposite viewpoint.     

Inhibition of the transthylakoid gradient of electrochemical proton potential by the local anesthetic dibucaine

The effects of the local anesthetic dibucaine on coupling between electron transport and ATP synthesis-hydrolysis by the coupling-factor complex (CF₀CF₁ ATPase) were investigated in thylakoid membranes from Spinacia oleracea L. cv. Monatol. Evidence is presented that inhibition of ATP synthesis was produced by a specific uncoupling mechanism which was based on dibucaine-membrane surface interactions rather than on the interaction of dibucaine with the ATPase complex. Dibucaine reduced the osmotic space of thylakoid vesicles. At low pH of the medium it stimulated ATP hydrolysis beyond the rates obtained with optimum concentrations of ‘classical’ uncouplers. After addition of dibucaine, there was displacement of membrane-bound Mg²⁺ and strong thylakoid stacking in the presence of only low Mg²⁺ concentrations. Inhibition of ATP synthesis and transmembrane pH gradient increased with medium pH. Hydrolysis of ATP by isolated CF₁ and the CF₀CF₁ complex was only slightly affected by dibucaine. The data are discussed assuming the involvement of localized proton channels on the membrane surface in protonic coupling of electron transport and ATP synthesis. A hypothesis for the mechanisms of action of local anesthetics at the thylakoid membrane is presented.     

ATP binding to noncatalytic sites of chloroplast coupling factor CF1

A kinetic analysis of ATP binding to noncatalytic sites of chloroplast coupling factor CF₁ was made. The ATP binding proved to be unaffected by reduction of the disulfide bridge of the CF₁ -subunit. The first-order equation describing nucleotide binding to noncatalytic sites allowed for two vacant nucleotide binding sites different in their kinetics. As suggested by nucleotide concentration dependence of the rate of nucleotide binding, the tight binding was preceded by rapid reversible binding of nucleotides. Preincubation of CF₁ with Mg²⁺ resulted in a decreased rate of ATP binding. ATP dissociation from noncatalytic sites was described by the first order equation for similar sites with a dissociation rate constant k _d (ATP) 10^–3 min^–1. Noncatalytic sites of CF₁ were shown to be not homogeneous. One of them retained the major part of endogenous ADP after precipitation of CF₁ with ammonium sulfate. Its two other sites differed in kinetic parameters and affinity for ATP. Anions of phosphate, sulfite, and especially, pyrophosphate inhibited the interaction between ATP and the noncatalytic sites.     

Covalent modification of the non-catalytic sites of the H+-ATPase from chloroplasts with 2-azido-[α-32P]ATP and its effect on ATP synthesis and ATP hydrolysis

Incubation of the isolated H⁺-ATPase from chloroplasts, CF₀F₁, with 2-azido-[α-³²P]ATP leads to the binding of this nucleotide to different sites. These sites were identified after removal of free nucleotides, UV-irradiation and trypsin treatment by separation of the tryptic peptides by ion exchange chromatography. The nitreno-AMP, nitreno-ADP and nitreno-ATP peptides were further separated on a reversed phase column, the main fractions were subjected to amino acid sequence analysis and the derivatized tyrosines were used to distinguish between catalytic (β-Tyr362) and non-catalytic (β-Tyr385) sites. Several incubation procedures were developed which allow a selective occupation of each of the three non-catalytic sites. The non-catalytic site with the highest dissociation constant (site 6) becomes half maximally filled at 50 μM 2-azido-[α-³²P]ATP, that with the intermediate dissociation constant (site 5) at 2 μM. The ATP at the site with the lowest dissociation constant had to be hydrolyzed first to ADP before a replacement by 2-azido-[α-³²P]ATP was possible. CF₀F₁ with non-covalently bound 2-azido-[α-³²P]ATP and after covalent derivatization was reconstituted into liposomes and the rates of ATP synthesis as well as ATP hydrolysis were measured after energization of the proteoliposomes by ΔpH/Δϕ. Non-covalent binding of 2-azido-ATP to any of the three non-catalytic sites does not influence ATP synthesis and ATP hydrolysis, whereas covalent derivatization of any of the three sites inhibits both, the degree being proportional to the degree of derivatization. Extrapolation to complete inhibition indicates that derivatization of one site (either 4 or 5 or 6) is sufficient to block completely multi-site catalysis. The rates of ATP synthesis and ATP hydrolysis were measured as a function of the ADP and ATP concentration from uni-site to multi-site conditions with covalently derivatized and non-derivatized CF₀F₁. Uni-site ATP synthesis and ATP hydrolysis were not inhibited by covalent derivatization of any of the non-catalytic sites, whereas multi-site catalysis is inhibited. These results indicate that multi-site catalysis requires some flexibility between β- and α-subunits which is abolished by covalent derivatization of β-Tyr385 with a 2-nitreno-adenine nucleotide. Conformational changes connected with energy transduction between the F₀-part and the F₁-part are either not required for uni-site ATP synthesis or they are not impaired by the derivatization of any of the three β-Tyr385.     

Conformation and activity of chloroplast coupling factor exposed to low chemical potential of water in cells

(1) Photophosphorylation, fCa²⁺-ATPase and Mg²⁺-ATPase activities of isolated chloroplasts were inhibited 55–65% when the chemical potential of water was decreased by dehydrating leaves to water potentials (ψ_w) of ?25 bars before isolation of the plastids. The inhibition could be reversed in vivo by rehydrating the leaves.(2) These losses in activity were reflected in coupling factor (CF₁) isolated from the leaves, since CF₁ from leaves with low ?_w had less Ca²⁺-ATPase activity than control CF₁ and did not recouple phosphorylation in CF₁-deficient chloroplasts. In contrast, CF₁ from leaves having high ?_w only partially recoupled phosphorylation by CF₁-deficient chloroplasts from leaves having low ?_w. This indicated that low ?_w affected chloroplast membranes as well as CF₁ itself.(3) Coupling factor from leaves having low ψ_w had the same number of subunits, and the same electrophoretic mobility, and could be obtained with the same yields as CF₁ from control leaves. However, direct measurements of fluorescence polarization, ultraviolet absorption, and circular dichroism showed that CF₁ from leaves having low ?_w differed from control CF₁. The CF₁ from leaves having low ?_w also had decreased ability to bind fluorescent nucleotides (?-ATP and ?-ADP).(4) Exposure of isolated CF₁ to low ?_w in vitro by preincubation in sucrose-containing media inhibited the Ca²⁺-ATPase activity of the protein in subsequent assays without sucrose. Inclusion of 5 or 10 mM Mg²⁺ in the preincubation medium markedly inhibited Ca²⁺-ATPase activity.(5) These results show that CF₁ undergoes changes in cells which alter its phosphorylating ability. Since low cell ?_w changed the spectroscopic properties but not other protein properties of CF₁, the changes were most likely caused by altered conformation of the protein. This decreased the binding of nucleotides and, in turn, photophosphorylation. The inhibition of ATPase activity in CF₁ in vitro at low ?_w and high ion concentration mimicked the change in activity seen in vivo.     

Effective cryoprotection of thylakoid membranes by ATP

Freezing of isolated spinach thylakoids in the presence of NaCl uncoupled photophosphorylation from electron flow and increased the permeability of the membranes to protons. Addition of ATP prior to freezing diminished membrane inactivation. On a molar basis, ATP was at least 100 times more effective in protecting thylakoids from freezing damage than low-molecularweight carbohydrates such as sucrose and glucose. The cryoprotective effectiveness of ATP was increased by Mg²⁺. In the absence of carbohydrates, preservation of thylakoids during freezing in 100 mM NaCl was saturated at about 1–2 mM ATP, but under these conditions membranes were not fully protected. However, in the presence of small amounts of sugars which did not significantly prevent thylakoid inactivation during freezing, ATP concentrations considerably lower than 0.5 mM caused nearly complete membrane protection. Neither ADP nor AMP could substitute for ATP. These findings indicate that cryoprotection by ATP cannot be explained by a colligative mechanism. It is suggested that ATP acts on the chloroplast coupling factor, either by modifying its conformation or by preventing its release from the membranes. The results are discussed in regard to freezing injury and resistance in vivo.Abbreviations CF₁ chloroplast coupling factor - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - PMS phenazine methosulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propandiol     

Influence of the redox state and the activation of the chloroplast ATP synthase on proton-transport-coupled ATP synthesis/hydrolysis

The membrane-bound ATP synthase from chloroplasts can occur in different redox and activation states. In the absence of reductants the enzyme usually is oxidized and inactive, E^ox_i. Illumination in the presence of dithiothreitol leads to an active, reduced enzyme, E^red_a. If this form is stored in the dark in the presence of dithiothreitol an inactive, reduced enzyme E^red_i is formed. The rates of ATP synthesis and ATP hydrolysis catalyzed by the different enzyme species are measured as a function of ΔpH (Δψ = 0 mV). The ΔpH was generated with an acid-base transition using a rapid-mixing quenched flow apparatus. The following results were obtained. (1) The oxidized ATP synthase catalyzes high rates of ATP synthesis, v^ox_max = 400 ATP per CF₀F₁ per s. The half-maximal rate is obtained at ΔpH = 3.4. (2) The active, reduced ATP synthase catalyzes high rates of ATP synthesis, v^red_max = 400 ATP per CF₀F₁ per s. The half-maximal rate is obtained at ΔpH = 2.7. It catalyzes also high rates of ATP hydrolysis v^red_max = −90 ATP per CF₀F per s at ΔpH = 0. (3) The inactive species (both oxidized and reduced) catalyze neither ATP synthesis nor ATP hydrolysis. The activation/inactivation of the reduced enzyme is completely reversible. (4) The activation of the reduced, inactive enzyme is measured as a function of ΔpH by measuring the rate of ATP hydrolysis catalyzed by the active species. Half-maximal activation is observed at ΔpH = 2.2. (5) On the basis of these results a reaction scheme is proposed relating the redox reaction, the activation and the catalytic reaction of the chloroplast ATP synthase.     

Properties of the solvent-stimulated ATPase activity of chloroplast coupling factor 1 from Chlamydomonas reinhardii

The effects of solvents on the ATPase activity of chloroplast coupling factor 1 (CF₁) isolated from wild-type Chlamydomonas reinhardii have been studied. Of the solvents examined, the following order summarizes their maximal ability to stimulate the ATPase activity of CF₁: ethanol > methanol>allyl alcohol >n-propanol > acetone≈dioxane > ethylene glycol. Glycerol inhibits the CF₁ activity at all concentrations. In the absence of organic solvents, 50% of the activity of the enzyme is irreversibly lost after a 10 min incubation at 65–70°C. Ethanol (23%) causes a 30°C drop in the temperature required for 50% inactivation. ATP partially stabilizes the CF₁ in the presence, but not in the absence, of ethanol. In the absence of organic solvents, both free Mg²⁺ and ADP inhibit the CF₁-ATPase. Mg²⁺ is a noncompetitive inhibitor with respect to MgATP, and the kinetic constants are: V, 6.3 μmol ATP hydrolyzed/mg protein per min; K_m(MgATP), 0.23 mM; K_ii(Mg²⁺), 27 μM; and K_is(Mg²⁺), 50 μM. In the presence of ethanol, double-reciprocal plots are no longer linear and have a Hill coefficient of about 1.8±0.1. V increases about 10–12-fold. The pattern of inhibition by Mg²⁺ appears to change from noncompetitive to competitive with respect to MgATP. In addition, ADP no longer inhibits the MgATPase activity of CF₁.     

Effect of pyridine homologues on proton flux through the CF0 · CF1 complex and photophosphorylation in chloroplasts

At concentrations below 1 mM, hydrophobic pyridine homologues decrease the rate of photophosphorylation and light-stimulated hydrolysis of ATP and light-activated exchange of the tightly bound nucleotides in chloroplasts, but increase the rate of the Hill reaction. Unlike uncoupling agents, the presence of the organic base at such low concentrations decreases the rate of light-dependent leakage and has no effect on the efficiency of two-stage photophosphorylation in broken chloroplasts. By assuming that the organic base is bound to independent equivalent sites in the thylakoid membrane, a simple expression can be derived which relates the observed rates of photophosphorylation and light-stimulated hydrolysis of ATP quantitatively to the concentration of the organic base in solution and gives dissociation equilibrium constants which are on the order of the relative hydrophobicities of the pyridine homologues. A possible mechanistic model for the CF₀ · CF₁ complex is proposed which could serve as the basis for a unified interpretation of the kinetics of proton translocation in illuminated chloroplasts, the steady-state rate of photophosphorylation, the light-stimulated ATPase activity, and the light-activated exchange of tightly bound adenine nucleotides.Abbreviations AMPPNP adenylylimidodiphosphate - Chl chlorophyll - CF₀ · CF₁ the coupling factor complex of chloroplasts - DCCD N,N-dicyclohexylcarbodiimide - DTT dithiothreitol - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - TCA trichloroacetic acid - Tricine N-tris-(hydroxymethyl)methylglycine     

Modulation of proton efflux from chloroplasts in the light by external pH

The effects of external pH on the efflux of protons from illuminated spinach chloroplasts have been studied by monitoring the rates of proton-pumping electron transport under a variety of steady-state conditions. Phosphorylation-coupled proton efflux through the ATP synthase (CF₀-CF₁), determined from the rates of ATP formation and that portion of the total electron transport attributable to phosphorylation, is strongly dependent upon pH over the range 6–9, with little activity below pH 7 and half-maximal activity at pH ≈ 7.6. Noncoupled proton efflux through the ATP synthase, determined in the absence of ADP and phosphate, was also strongly pH sensitive, with little activity below pH 7.5 and half-maximal activity at pH ～- 7.9. When proton efflux via CF₀ was prevented by triphenyltin, the rate of passive proton leakage across the membrane was very low and practically insensitive to external pH indicating that the major pH-sensitive pathway(s) for proton efflux in the light involves CF₀ · CF₁. Modification of CF₁ sulfhydryls by Ag⁺ resulted in an apparent increase in proton efflux via the normally coupled CF₀ · CF₁ pathway (half-maximal activity = pH 7.6), whereas modification by Hg²⁺ resulted in an apparent increase in proton efflux via the noncoupled CF₀ · CF₁ pathway (half-maximal activity = pH 7.9).     

Purification and Characterization of a Glycerol-Resistant CF(0)-CF(1) and CF(1)-ATPase from the Halotolerant Alga Dunaliella bardawil

The isolation of the chloroplast ATP synthase complex (CF₀-CF₁) and of CF₁ from Dunaliella bardawil is described. The subunit structure of the D. bardawil ATPase differs from that of the spinach in that the D. bardawil α subunit migrates ahead of the β subunit and ε-migrates ahead of subunit II of CF₀ when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The CF₁ isolated from D. bardawil resembles the CF₁ isolated from Chladmydomonas reinhardi in that a reversible, Mg²⁺-dependent ATPase is induced by selected organic solvents. Glycerol stimulates cyclic photophosphorylation catalyzed by D. bardawil thylakoid membranes but inhibits photophosphorylation catalyzed by spinach thylakoid membranes. Glycerol (20%) also stimulates the rate of ATP-P_i exchange catalyzed by D. bardawil CF₀-CF₁ proteoliposomes but inhibits the activity with the spinach enzyme. The ethanol-activated, Mg²⁺-ATPase of the D. bardawil CF₁ is more resistant to glycerol inhibition than the octylglucoside-activated, Mg²⁺-ATPase of spinach CF₁ or the ethanol-activated, Mg²⁺-dependent ATPase of the C. reinhardi CF₁. Both cyclic photophosphorylation and ATP-P_i exchange catalyzed by D. bardawil CF₀-CF₁ are more sensitive to high concentrations of NaCl than is the spinach complex.     

Chloroplastic ATP synthase builds up a proton motive force preventing production of reactive oxygen species in photosystem I

Over‐reduction of the photosynthetic electron transport (PET) chain should be avoided, because the accumulation of reducing electron carriers produces reactive oxygen species (ROS) within photosystem I (PSI) in thylakoid membranes and causes oxidative damage to chloroplasts. To prevent production of ROS in thylakoid membranes the H⁺ gradient (ΔpH) needs to be built up across the thylakoid membranes to suppress the over‐reduction state of the PET chain. In this study, we aimed to identify the critical component that stimulates ΔpH formation under illumination in higher plants. To do this, we screened ethyl methane sulfonate (EMS)‐treated Arabidopsis thaliana, in which the formation of ΔpH is impaired and the PET chain caused over‐reduction under illumination. Subsequently, we isolated an allelic mutant that carries a missense mutation in the γ‐subunit of chloroplastic CF₀CF₁‐ATP synthase, named hope2. We found that hope2 suppressed the formation of ΔpH during photosynthesis because of the high H⁺ efflux activity from the lumenal to stromal side of the thylakoid membranes via CF₀CF₁‐ATP synthase. Furthermore, PSI was in a more reduced state in hope2 than in wild‐type (WT) plants, and hope2 was more vulnerable to PSI photoinhibition than WT under illumination. These results suggested that chloroplastic CF₀CF₁‐ATP synthase adjusts the redox state of the PET chain, especially for PSI, by modulating H⁺ efflux activity across the thylakoid membranes. Our findings suggest the importance of the buildup of ΔpH depending on CF₀CF₁‐ATP synthase to adjust the redox state of the reaction center chlorophyll P700 in PSI and to suppress the production of ROS in PSI during photosynthesis.