A second mechanism for sodium extrusion in Halobacterium halobium: a light-driven sodium pump.

Membrane vesicles from a red mutant of $\text{Halobacterium}\text{halobium}$ R₁ accumulate protons when illuminated causing the pH of the suspension to rise. Sodium is extruded from the vesicles and a membrane potential is formed. This potential and the proton uptake are abolished by valinomycin if K⁺ is present. In contrast, Na⁺-efflux is uninhibited by valinomycin even though no membrane potential is detectable and H⁺ influx does not occur. $\text{Bis}$ (hexafluoracetonyl)acetone (1799) stimulates proton uptake but does not abolish membrane potential. We propose that a light-dependent sodium pump is present. Passive proton uptake occurs in response to the electrical gradient created by this light-driven Na⁺ pump in contrast to the $\text{active}$ proton, and passive Na⁺ flux that occurs in response to the light-driven proton pump described in vesicles of the parent strain of $\text{H.}\text{halobium}$ R₁.     

Factors defining the functional coupling of bacteriorhodopsin and ATP synthase in liposomes

Optimal conditions for the co-reconstitution of bacteriorhodopsin and yeast mitochondria ATP synthase were determined. Reconstitution was achieved with a quick two-step procedure. Preparations obtained by this method displayed in optimal cases 2–3-times higher activities (up to 500 nmol ATP/min per mg protein) compared with maximal values reported in the literature, when light-driven ATP synthesis was measured under similar conditions. The final activities depended on the purification method used for the ATP synthase, and it is shown that the oligomycin-sensitive ATP hydrolysis activity was not a good measure for the ability of the ATP synthase preparations to perform ATP synthesis after co-reconstitution. Light-driven ATP synthesis activities depended also on the type of phospholipid used, soybean phospholipid giving the best results. A close relation to the bacteriorhodopsin proton pump activity was found. Using different phospholipids, different $\text{H}{}^{\mathrm{+}}\text{ATP}$ ratios were found, calculated from ATP synthesis activities and initial and steady-state light-driven proton pump activities. From this, together with the findings that the ATP synthase displayed the same ATP hydrolysis and ATP-³²P_i exchange activities with these different phospholipids used, it is concluded that the protein distribution for the two proteins among the liposomes is different relative to each other for the different phospholipids. The light-driven ATP synthesis activity did not correlate with the variation in leakiness of the membrane for protons when different phospholipids were used. An explanation is given by the finding that at high light intensities, the ATP synthesis became independent of the presence of protonophore.     

Photophosphorylation in cell envelope vesicles from Halobacteriumhalobium

We have prepared vesicles from cell envelope membranes of $\text{Halobacterium}\text{halobium}$ strains R₁ and ET-15 which are able to synthesize ATP in response to illumination. This photophosphorylation is inhibited by dicyclohexylcarbodiimide (DCCD) and by phloretin. ATP synthesis in L vesicles from the R₁ strain (which contain bacteriorhodopsin) is inhibited by the protonophore 1799 but not by valinomycin. In M vesicles from the R₁ strain and in ET-15 vesicles (both contain halorhodopsin) photophosphorylation is inhibited by both 1799 and valinomycin. These data are consistent with the idea that light-driven ATP synthesis can be coupled to the electrochemical H⁺ gradient generated by bacteriorhodopsin or by halorhodopsin through the membrane potential component of protonmotive force.     

ESR studies of light-dependent volume changes in cell envelope vesicles from Halobacterium halobium

Volume changes in illuminated cell envelope vesicles, prepared from various Halobacterium halobium strains, were measured with an ESR method. We demonstrated light-dependent swelling of vesicles which contained halorhodopsin (an inward-directed light-driven chloride pump), and shrinking of vesicles which contained bacteriorhodopsin (an outward-directed light-driven proton pump coupled to a proton/sodium antiporter). The swelling of the halorhodopsin vesicles was not inhibited by uncouplers or gramicidin, but the shrinking of the bacteriorhodopsin-vesicles was abolished by these ionophores. These findings confirm earlier models for ion circulation in these systems. Vesicles from strains which contained both pigments showed relatively small net volume changes upon illumination. A scheme of ionic transport in H. halobium cells is suggested, in which the inward movement of K⁺ exceeds the outward movement of Na⁺, and the difference equals the Cl⁻ uptake, so as to provide the net gain of KCl necessary for volume increases during cell growth.     

Characterization of a light-dependent glutamate synthase activity in Chlamydomonas reinhardtii

Photosynthetically active vesicles prepared from Chlamydomonas reinhardtii retained a light-dependent glutamate synthase activity which was highly specific for 2-oxoglutarate (K_m=2.1 mM) and L-glutamine (K_m=0.9 mM) as amido group acceptor and donor respectively. This activity was inhibited by azaserine, p-hydroxymercuribenzoate and 3-(p-chlorophenyl)-1,1-dimethyl urea.Light-dependent synthesis of glutamate was also obtained by coupling Chlamydomonas photosynthetic particles to purified ferredoxin-glutamate synthase, using ascorbate and 2,6-dichlorophenol-indophenol as electron donor. This system was also specific for 2-oxoglutarate (K_m=1 mM) and L-glutamine (K_m=0.8 mM) as substrates, and was stimulated by dithioerythritol. Azaserine and p-hydroxymercuribenzoate, but not 3-(p-chlorophenyl)-1,1-dimethyl urea, inhibited the reconstituted activity; high concentrations of 2-oxoglutarate were inhibitory.Abbreviations A Absorbance - CCP p-Trichlorometoxi-carbonylcyanide-phenylhydrazone - Chl Chlorophyll - CMU 3-(p-Chlorophenyl)-1,1-dimethyl urea - DPIP 2,6-Dichlorophenol-indophenol - DTE Dithioerythritol - MSX L-Methionine, D-L, sulfoximine - MV Methyl viologen     

pH-dependent Ca2+ binding to the F0 c-subunit affects proton translocation of the ATP synthase from Synechocystis 6803

It was shown before (Wooten, D. C., and Dilley, R. A. (1993) J. Bioenerg. Biomembr. 25, 557–567; Zakharov, S. D., Li, X., Red'ko, T. P., and Dilley, R. A. (1996) J. Bioenerg. Biomembr. 28, 483–493) that pH dependent reversible Ca²⁺ binding near the N- and C-terminal end of the 8 kDa subunit c modulates ATP synthesis driven by an applied pH jump in chloroplast and E. coli ATP synthase due to closing a proton gate proposed to exist in the F₀ H⁺ channel of the F₀F₁ ATP synthase. This mechanism has further been investigated with the use of membrane vesicles from mutants of the cyanobacterium Synechocystis 6803. Vesicles from a mutant with serine at position 37 in the hydrophilic loop of the c-subunit replaced by the charged glutamic acid (strain plc 37) has a higher H⁺/ATP ratio than the wild type and therefore shows ATP synthesis at low values of _H ⁺. The presence of 1 mM CaCl₂ during the preparation and storage of these vesicles blocked acid–base jump ATP formation when the pH of the acid side (inside) was between pH 5.6 and 7.1, even though the pH of the acid–base jump was thermodynamically in excess of the necessary energy to drive ATP formation at an external pH above 8.28. That is, in the absence of added CaCl₂, ATP formation did occur under those conditions. However, when the base stage pH was 7.16 and the acid stage below pH 5.2, ATP was formed when Ca²⁺ was present. This is consistent with Ca²⁺ being displaced by H⁺ ions from the F₀ on the inside of the thylakoid membrane at pH values below about 5.5. Vesicles from a mutant with the serine of position 3 replaced by a cysteine apparently already contain some bound Ca²⁺ to F₀. Addition of 1 mM EGTA during preparation and storage of those vesicles shifted the otherwise already low internal pH needed for onset of ATP synthesis to higher values when the external pH was above 8. With both strains it was shown that the Ca²⁺ binding effect on acid–base induced ATP synthesis occurs above an internal pH of about 5.5. These results were corroborated by ⁴⁵Ca²⁺- ligand blot assays on organic solvent soluble preparations containing the 8 kDa F₀ subunit c from the S-3-C mutant ATP synthase, which showed ⁴⁵Ca²⁺ binding as occurs with the pea chloroplast subunit III. The phosphorylation efficiency (P/2e), at strong light intensity, of Ca²⁺ and EGTA treated vesicles from both strains were almost equal showing that Ca²⁺ or EGTA have no other effect on the ATP synthase such as a change in the proton to ATP ratio. The results indicate that the Ca²⁺ binding to the F₀ H⁺ channel can block H⁺ flux through the channel at pH values above about 5.5, but below that pH protons apparently displace the bound Ca²⁺, opening the CF₀ H⁺ channel between the thylakoid lumen and H⁺ conductive channel.     

F0 cysteine,bCys21, in the Escherichia coli ATP synthase is involved in regulation of potassium uptake and molecular hydrogen production in anaerobic conditions

The single cysteine in the b subunit of the membranous F₀ sector and the 19 cysteines in extramembranous F₁ sector of the Escherichia coli ATP synthase were replaced by alanine. When cells were grown under anaerobic conditions on glucose, the k _cat for ATP hydrolysis of membrane vesicles containing the bCys21Ala mutant enzyme, but not enzymes with other cysteine replacements, was lower, while ATP-driven H⁺ pumping was unchanged. However, the ATP-dependent increase in the number of accessible thiol groups in membrane vesicles was negated. Furthermore, K⁺ uptake and molecular hydrogen production by whole cells and protoplasts was greatly decreased. These results indicate a role for the F₀ subunit bCys21 in the functionality of F₀F₁ and coupling to other membranous activities under fermentative conditions.     

Chloroplastic ATP synthase optimizes the trade-off between photosynthetic CO2 assimilation and photoprotection during leaf maturation

In the present study, we studied the role of chloroplastic ATP synthase in photosynthetic regulation during leaf maturation. We measured gas exchange, chlorophyll fluorescence, P700 redox state, and the electrochromic shift signal in mature and immature leaves. Under high light, the immature leaves displayed high levels of non-photochemical quenching (NPQ) and P700 oxidation ratio, and higher values for proton motive force (pmf) and proton gradient (ΔpH) across the thylakoid membranes but lower values for the activity of chloroplastic ATP synthase (g_H⁺) than the mature leaves. Furthermore, g_H⁺ was significantly and positively correlated with CO₂ assimilation rate and linear electron flow (LEF), but negatively correlated with pmf and ΔpH. ΔpH was significantly correlated with LEF and the P700 oxidation ratio. These results indicated that g_H⁺ was regulated to match photosynthetic capacity during leaf maturation, and the formation of pmf and ΔpH was predominantly regulated by the alterations in g_H⁺. In the immature leaves, the high steady-state ΔpH increased lumen acidification, which, in turn, stimulated photoprotection for the photosynthetic apparatus via NPQ induction and photosynthetic control. Our results highlighted the importance of chloroplastic ATP synthase in optimizing the trade-off between CO₂ assimilation and photoprotection during leaf maturation.     

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.     

ATP synthesis catalyzed by the ATPase complex from Rhodospirillum rubrum reconstituted into phospholipid vesicles together with bacteriorhodopsin

The coupling factor ATPase complex extracted by Triton X-100 from the photosynthetic bacterium Rhodospirillum rubrum could be incorporated into phospholipid vesicles after removal of the Triton. Vesicles reconstituted with this F₀ · F₁-type ATPase together with bacteriorhodopsin were found to catalyze, in the light, net ATP synthesis which was inhibited by the energy transfer inhibitors oligomycin and N,N-dicyclohexylcarbodiimide as well as by uncouplers. In vesicles reconstituted with the crude ATPase up to 50% of the observed rate of phosphorylation was independent on light and bacteriorhodopsin and insensitive to the above-listed inhibitors. This dark activity was, however, completely blocked by the adenylate kinase inhibitor, p¹,p⁵-di(adenosine-5′)pentaphosphate, which did not affect at all the net light-dependent phosphorylation nor the ATP-³²P_i exchange reaction. Vesicles reconstituted with the purified ATPase catalyzed only the light- and bacteriorhodopsin-dependent diadenosine pentaphosphate-insensitive phosphorylation. The rate of this photophosphorylation was found to be proportional to the amount of ATPase and bacteriorhodopsin, and linear for at least 20 min of illumination. These results indicate that the purified ATPase contains the complete assembly of subunits required to transduce electrochemical gradient energy into chemical energy.     

Photophosphorylation and the chemiosmotic perspective

Photophosphorylation was discovered in chloroplasts by D. Arnon and coworkers, and in bacterial ‘chromatophores’ (intercytoplasmic membranes) by A. Frenkel. Initial low rates were amplified by adding electron-carrying compounds such as FMN, later shown to support the ‘pseudocyclic’ electron flow. ATP synthesis, and coupling to electron flow, was detected accompanying linear electron flow from H₂O to either NADP⁺ or ferricyanide. Another pattern of electron flow supporting photophosphorylation was that of a cycle around Photosystem I (PS I). Isolation and analysis of the ATP synthase showed, as with mitochondrial and bacterial analogues, an intrinsic membrane complex (CF₀) and an extrinsic complex (CF₁). CF₁ is a latent ATPase, activated additively by the high-energy state of the thylakoids, and by reduction of a disulfide bond on the gamma subunit. Once reduced, ATP synthesis occurs at lower energy levels. The search for an ‘intermediate’ linking electron flow and ATP synthesis led to the discovery of post-illumination ATP synthesis by thylakoids, where turnover occurs in the dark. Once interpreted by P.Mitchell's chemiosmotic hypothesis, this led to the discovery of light-driven proton uptake into the thylakoid lumen, with accompanying Cl⁻ intake and Mg²⁺ and K⁺ output. Chemiosmosis was confirmed in several ways, including ATP synthesis in the dark due to an acid-to-base transition of thylakoids, and photophosphorylation accomplished in artificial lipid vesicles containing both the proton-pumping bacterial rhodopsin and a mitochondrial ATPase complex. The now generally accepted chemiosmotic interpretation is able to clarify some other aspects of photosynthesis as well. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of the Key Step for Light-driven Hydrogen Evolution in Green Algae

Under anaerobic conditions, several species of green algae perform a light-dependent hydrogen production catalyzed by a special group of [FeFe] hydrogenases termed HydA. Although highly interesting for biotechnological applications, the direct connection between photosynthetic electron transport and hydrogenase activity is still a matter of speculation. By establishing an in vitro reconstitution system, we demonstrate that the photosynthetic ferredoxin (PetF) is essential for efficient electron transfer between photosystem I and HydA1. To investigate the electrostatic interaction process and electron transfer between PetF and HydA1, we performed site-directed mutagenesis. Kinetic analyses with several site-directed mutagenesis variants of HydA1 and PetF enabled us to localize the respective contact sites. These experiments in combination with in silico docking analyses indicate that electrostatic interactions between the conserved HydA1 residue Lys³⁹⁶ and the C terminus of PetF as well as between the PetF residue Glu¹²² and the N-terminal amino group of HydA1 play a major role in complex formation and electron transfer. Mapping of relevant HydA1 and PetF residues constitutes an important basis for manipulating the physiological photosynthetic electron flow in favor of light-driven H₂ production.     

Cardiolipin affects the supramolecular organization of ATP synthase in mitochondria

F₁F₀ ATP synthase forms dimers that tend to assemble into large supramolecular structures. We show that the presence of cardiolipin is critical for the degree of oligomerization and the degree of order in these ATP synthase assemblies. This conclusion was drawn from the statistical analysis of cryoelectron tomograms of cristae vesicles isolated from Drosophila flight-muscle mitochondria, which are very rich in ATP synthase. Our study included a wild-type control, a cardiolipin synthase mutant with nearly complete loss of cardiolipin, and a tafazzin mutant with reduced cardiolipin levels. In the wild-type, the high-curvature edge of crista vesicles was densely populated with ATP synthase molecules that were typically organized in one or two rows of dimers. In both mutants, the density of ATP synthase was reduced at the high-curvature zone despite unchanged expression levels. Compared to the wild-type, dimer rows were less extended in the mutants and there was more scatter in the orientation of dimers. These data suggest that cardiolipin promotes the ribbonlike assembly of ATP synthase dimers and thus affects lateral organization and morphology of the crista membrane.     

Singlet oxygen inhibits ATPase and proton translocation activity of the thylakoid ATP synthase CF1CFo

Singlet oxygen (¹O₂) produced in plants during photosynthesis has a strong damaging effect not only on both photosystems but also on the whole photosynthetic machinery. This is also applicable for the adenosine triphosphate (ATP) synthase. Here we describe the impact of ¹O₂ generated by the photosensitizer Rose Bengal on the ATP hydrolysis and ATP-driven proton translocation activity of CF1CFo. Both activities were reduced dramatically within 1 min of exposure. Interestingly, it is shown that oxidized thylakoid ATP synthase is more susceptible to ¹O₂ than CF1CFo in its reduced state, a new insight on the mechanism of ¹O₂ interaction with the γ subunit.     

Effects of light on nitrogen and carbon uptake during a Prorocentrum minimum bloom

During a 4-week period in late spring 1998 an extensive Prorocentrum minimum (Pavillard) Schiller bloom developed in several tributaries of the Chesapeake Bay. Experiments were carried out in one of these tributaries using ¹³C and ¹⁵N isotopic techniques to characterize C and N uptake as a function of irradiance during the course of this bloom. Uptake rates of N substrates (NO₃⁻, NH₄⁺, urea, and an amino acid mixture) and C substrates (bicarbonate and urea) were measured. For each N substrate, short-term uptake rates (0.5 h) were not substantially different over the irradiance range measured, suggesting that N uptake of this dinoflagellate was not strongly light-dependent over this time scale. Dark uptake rates of all N substrates ranged between 35 and 113% of light uptake rates. Over the duration of the P. minimum bloom, however, total ambient N uptake rates increased with increasing natural irradiance. Uptake of bicarbonate showed typical light-dependent photosynthetic characteristics and the measured photosynthetic parameters suggested that at least on the short time scale (0.5 h), P. minimum cells were adapted to high light. Rates of C uptake from the substrate urea were minimal, <1% of total C uptake from photosynthesis, but doubled over the course of the bloom, and like N uptake, were not strongly light-dependent on the short time scale (0.5 h). Significant N dark uptake by P. minimum was likely to have been important by providing N sources over the daily scale to sustain the bloom.     

Formation of ATP by the adenosine triphosphatase complex from spinach chloroplasts reconstituted together with bacteriorhodopsin

The energy-linked ATPase complex has been isolated from spinach chloroplasts. This protein complex contained all the subunits of the chloroplast coupling factor (CF₁) as well as several hydrophobic components. When the activated complex was reconstituted with added soybean phospholipids, it catalyzed the exchange of radioactive inorganic phosphate with ATP. Sonication of the complex into proteoliposomes together with bacteriorhodopsin yielded vesicles that catalyzed light-dependent ATP formation. Both the ³²P_i-ATP exchange reactions and ATP formation were sensitive to uncouplers such as 3-tert-butyl-5,2′-dichloro-4′-nitrosalicylanilide, bis-(hexafluoroacetonyl)acetone and carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone, that act to dissipate a proton gradient. The energy transfer inhibitors dicyclohexylcarbodiimide, triphenyltin chloride and 2-β-d-glucopyranosyl-4,6′-dihydroxydihydrochalcone were also effective inhibitors of both reactions.     

Energy-linked activities in reconstituted yeast adenosine triphosphatase proteoliposome. Adenosine triphosphate formation coupled with electron flow between ascorbate and ferricyanide.

(1) Conditions are described wherein the yeast oligomycin-sensitive adenosine triphosphatase (ATPase) complex can be reconstituted together with phospholipids to yield extremely high rates of ATP-³²P_j exchange. The vesicles so formed exhibit proton uptake upon addition of Mg²⁺-ATP and a relatively slow decay of the proton gradient. (2) The stimulation of ATP-³²P_i exchange by valinomycin + K⁺ reported previously (Ryrie, I. J. (1975) Arch. Biochem. Biophys. 168, 704–711) is apparently not simply due to a diffusion potential. The findings suggest that an electroimpelled, valinomycin-dependent migration of K⁺ may occur together with the electrogenic movements of protons during ATP hydrolysis and synthesis to establish optimal energized conditions for ATP-³²P_i exchange. (3) An artificial oxidative phosphorylation system in the reconstituted vesicles is described: [³²P]ATP formation from ADP and ³²P_i is shown to be linked with electron flow between external ascorbate and internal ferricyanide where a permeable proton carrier, such as phenazine methosulfate, is used to establish a proton gradient. That the yeast ATPase is capable of net ATP synthesis has also been demonstrated in a light-dependent reaction using ATPase proteoliposomes reconstituted together with bacteriorhodopsin.     

Singlet oxygen affects the activity of the thylakoid ATP synthase and has a strong impact on its γ subunit

Singlet oxygen is reported to have the most potent damaging effect upon the photosynthetic machinery. Usually this reactive oxygen molecule acts in concert with other ROS types under stressful conditions. To understand the specific role of singlet oxygen we took advantage of the conditional flu mutant of Arabidopsis thaliana. In flu, the negative feedback loop is abolished, which blocks chlorophyll biosynthesis in the dark. Therefore high amounts of free protochlorophyllide accumulate during darkness. If flu gets subsequently illuminated, free protochlorophyllide acts as a photosensitiser leading almost exclusively to high amounts of ¹O₂. Analysing the thylakoid protein pattern by using 2D PAGE and subsequent MALDI-TOF analysis, we could show, in addition to previous described effects on photosystem II, that singlet oxygen has a massive impact on the thylakoid ATP synthase, especially on its γ subunit. Additionally, it could be shown that the activity of the ATP synthase is reduced upon singlet oxygen exposure and that the rate of non-photochemical quenching is affected in flu mutants exposed to ¹O₂.     

Functional characterization of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> with alterations in the <Emphasis Type="Italic">atpE</Emphasis> gene

The FUD17 strain of Chlamydomonas reinhardtii is a photosynthesis-deficient, acetate-requiring mutant with a defect in the chloroplast atpE gene, which codes for the ε subunit of the chloroplast ATP synthase. In this work, the FUD17 mutant was examined in relation to other known ATP synthase mutants as an initial step toward using this strain to generate altered versions of the atpE gene for site-directed mutagenesis of the ε subunit. The FUD17 strain grows well and is normally pigmented in the dark (heterotrophic conditions), but cannot grow autotrophically in the light, even when media are supplemented with acetate. Under heterotrophic conditions, it shows no accumulation of the ε subunit, and much lower levels of the α and β subunits of the chloroplast ATP synthase. FUD17 shows no light-dependent oxygen evolution and shows a strong, light-dependent alteration in its chlorophyll fluorescence. These results show that FUD17 possesses similar characteristics to other ATP synthase mutants and fails to express an assembled ATP synthase complex on its thylakoid membrane. A preliminary attempt at site-directed mutagenesis is described which produced a slightly truncated form of the ε subunit, which is expressed normally in the cell. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.