Four tight nucleotide binding sites of chloroplast coupling factor 1.

We have examined the properties of the four tight nucleotide binding sites of reductively activated chloroplast coupling factor 1. Tight sites are here defined as those which retain bound nucleotides after passage of the chloroplast coupling factor 1 through Sephadex gel filtration centrifuge columns. Two of the sites, here called sites 4 and 5, have not been characterized in detail before. Site 4 has properties similar to those of site 1. It binds to ADP, ATP, and adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) tightly in the presence or absence of Mg2+. Bound ADP exchanges rapidly with medium ADP, but rapid exchange with ATP or AMP-PNP requires Mg2+. Site 4 may slowly hydrolyze bound ATP in the absence of medium nucleotides. Site 5 has properties similar to those of site 2. Tight binding of ATP and AMP-PNP requires Mg2+, but Mg29+)-ADP is not tightly bound. Site 5 does not hydrolyze bound ATP in the absence of medium nucleotides. Complete filling of all four tight nucleotide binding sites requires about one millimolar nucleotide, suggesting that low affinity binding sites are converted to tight binding via a nucleotide binding-induced conformational change.     

Structural mapping of nucleotide binding sites on chloroplast coupling factor

Fluorescence resonance energy transfer was used to measure the distances between three nucleotide binding sites on solubilized chloroplast coupling factor from spinach and between each nucleotide site and two tyrosine residues which are important for catalytic activity. The nucleotide energy donor was 1,N6-ethenoadenosine di- or triphosphate, and the nucleotide energy acceptor was 2'(3')-(trinitrophenyl)adenosine diphosphate. The tyrosine residues were specifically labeled with 7-chloro-4-nitro-2,1,3-benzoxadiazole, which served as an energy acceptor. The results obtained indicate the three nucleotide binding sites form a triangle with sides of 44, 48, and 36 A. (The assumption has been made in calculating these distances that the energy donor and acceptor rotate rapidly relative to the fluorescence lifetime.) Two of the nucleotide sites are approximately equidistant from each of the two tyrosines: one of the nucleotide sites is about 37 A and the other about 41 A from each tyrosine. The third nucleotide site is about 41 A from one of the tyrosines and greater than or equal to 41 A from the other tyrosine.     

Further characterization of nucleotide binding sites on chloroplast coupling factor one

The solubilized coupling factor from spinach chloroplasts (CF1) contains one nondissociable ADP/CF1 which exchanges slowly with medium ADP in the presence of Ca2+, Mg2+, or EDTA; medium ATP also exchanges in the presence of Ca2+ or EDTA, but it is hydrolyzed, and only ADP is found bound to CF1. The rate of ATP exchange with heat-activated CF1 is approximately 1000 times slower than the rate of ATP hydrolysis. In the presence of Mg2+, both latent CF1 and heat-activated CF1 bind one ATP/CF1, in addition to the ADP. This MgATP is not removed by dialysis, by gel filtration, or by the substrate CaATP during catalytic turnover; however, it is released when the enzyme is stored several days as an ammonium sulfate precipitate. The photoaffinity label 3'-O-[3-[N-(4-azido-2-nitrophenyl)amino]-propionyl]-ATP binds to the MgATP site, and photolysis results in labeling of the beta subunit of CF1. Equilibrium binding measurements indicate that CF1 has two identical binding sites for ADP with a dissociation constant of 3.9 microM (in addition to the nondissociable ADP site). When MgATP is bound to CF1, one ADP binding site with a dissociation constant of 2.9 microM is found. One ATP binding site is found in addition to the MgATP site with a dissociation constant of 2.9 microM. Reaction of CF1 with the photoaffinity label 3'-O-[3-[N-(4-azido-2-nitrophenyl)amino]propionyl]-ADP indicates that the ADP binding site which is not blocked by MgATP is located near the interface of alpha and beta subunits. No additional binding sites with dissociation constants less than 200 micro M are observed for MgATP with latent CF1 and for CaADP with heat-activated CF1. Thus, three distinct nucleotide binding sites can be identified on CF1, and the tightly bound ADP and MgATP are not at the catalytic site. The active site is either the third ADP and ATP binding site or a site not yet detected.     

Investigation of quercetin binding sites on chloroplast coupling factor 1.

The quercetin binding sites on spinach chloroplast coupling factor 1 (CF1) have been investigated using direct and competitive binding, stopped-flow, temperature-jump, and fluorescence resonance energy transfer measurements. It was found that 8-anilino-1-naphthalensulfonic acid (ANS) competes with quercetin binding at two sites on the solubilized enzyme which are distinct from the two tight nucleotide binding sites and the 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) reactive site. The bimolecular association of quercetin with CF1 is too fast to measure directly and is followed by two slower conformational changes. The distances from the tight nucleotide sites to the quercetin-ANS sites were estimated as 40-48 A by fluorescence resonance energy transfer using 1,N6-ethenoadenosine diphosphate and 1,N6-ethenoadenylyl imidodiphosphate as donors and quercetin as the acceptor. The distance from the quercetin-ANS site to the NBD-C1 reactive site was found to be about 30 A using ANS as a donor and NBD-C1 reacted with a tyrosine group on CF1 as the energy acceptor. A model is proposed for the relative location of these sites on CF1.     

Specificity of nucleotide binding sites in isolated chloroplast coupling factor (CF1).

The binding of various nucleotides to chloroplast coupling factor CF1 was studied by two dialysis techniques. It was found that the number of nucleoside diphosphate sites and their specificities for the base moiety is dependent on the magnesium concentration. In the presence of 50 micrometer added MgCl2, the protein has a single strong site/mol protein with Kd = 0.5 micrometer for ADP and high specificity (Kd greater than 20 micrometer for epsilonADP, GDP, CDP). In the presence of 5 mM MgCl2, the protein has two independent tight ADP sites (Kd = 0.4 micrometer) of low specificity (Kd approximately 0.8, 2, and 2 micrometer, respectively for episilonADP, GDP, and CDP). These results are compared with the specificity of the partial reactions for photophosphorylation.     

Characterization of nucleotide binding sites on the membrane-bound chloroplast ATP synthase (coupling factor CF1)

Phosphorylation of ADP and nucleotide exchange by membrane-bound coupling factor CF₁ are very fast reactions in the light, so that a direct comparison of both reactions is difficult. By adding substrate ADP and phosphate to illuminated thylakoids together with the uncoupler FCCP, the phosphorylation time is limited and the amount of ATP formed can be reduced to less than 1 ATP per enzyme. Low concentrations of medium nucleotides during illumination increase the amount of ATP formed during uncoupling presumably by binding to the tight nucleotide binding site (further designated as ‘site A’) with an affinity of 1 to 7 μM for ADP and ATP. ATP formation itself shows half-saturation at about 30 μM. Loosely bound nucleotides are exchanged upon addition of nucleotides with uncoupler (Schumann, J. (1984) Biochim. Biophys. Acta 766, 334–342). Release depends binding of nucleotides to a second site. The affinity of this site for ADP (in the presence of phosphate) is about 30 μM. It is assumed that phosphorylation and induction of exchange both occur on the same site (site B). During ATP hydrolysis, an ATP molecule is bound to site A, while on another site, ATP is hydrolyzed rapidly. The affinity of ADP for the catalytic site (70 μM) is in the same range as the observed Michaelis constant of ADP during phosphorylation; it is assumed that site B is involved in ATP hydrolysis. Site A exhibits some catalytic activity; it might be that site A is involved in ATP formation in a dual-site mechanism. For ATP hydrolysis, however, direct determination of exchange rates showed that the exchange rate of ATP bound to site A is about 30-times lower than ATP hydrolysis under the same conditions.     

Fluorescence energy transfer between ligand binding sites on chloroplast coupling factor 1.

The method of fluorescence energy transfer is used to measure the distance from the tight nucleotide binding sites to the 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reactive sites on solubilized spinach chloroplast coupling factor 1 (CF1). The fluorescent adenine nucleotide analogs 1,N-6-ethenoadenosine diphosphate and 1,N-6-ethenoadenylyl imidodiphosphate were used as donors and 4-nitrobenzo-2-oxa-1,3-diazole bound to a tyrosine group and to an amino group were used as acceptors of energy transfer. Using three different donor-acceptor pairs, the distance measured varied from 38 to 43 A assuming both donor sites are equidistant from the acceptor site. A model is proposed for the location of the tight nucleotide binding sites and the active site on the alpha and beta subunits of CF1.     

Assessment of the number of nucleotide binding sites on chloroplast coupling factor 1 by the continuous variation method

The method of continuous variation (Job plot analysis) and difference absorbance spectroscopy were used to investigate the binding of 2'(3')-(trinitrophenyl)-ADP and -ATP to chloroplast coupling factor 1 (CF1). Experiments performed at a low total concentration (30 microM) of nucleotide and enzyme binding sites (assuming three or four binding sites per CF1) could be interpreted in terms of approximately three nucleotide binding sites per CF1. At higher total concentrations (100 and 400 microM), the number of apparent binding sites increased to almost four. Computer-generated Job plots, using a protein-ligand complex formation scheme of n independent, nonequivalent binding sites, gave good fits to the experimental data at all concentrations when four binding sites were modeled. The dissociation constant of the fourth site was estimated to be approximately 20 microM. Additional nucleotide binding sites were not directly observed by this method and, if they exist, have very weak binding affinities (dissociation constants greater than approximately 1 mM).     

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.     

Specificity of nucleotide binding sites in isolated chloroplast coupling factor (CF1)

The binding of various nucleotides to chloroplast coupling factor CF₁ was studied by two dialysis techniques. It was found that the number of nucleoside diphosphate sites and their specificities for the base moiety is dependent on the magnesium concentration. In the presence of 50 μM added MgCl₂, the protein has a single strong site/mol protein with K_d = 0.5 μM for ADP and high specificity (K_d > 20 μM for ?ADP, GDP, CDP). In the presence of 5 mM MgCl₂, the protein has two independent tight ADP sites (K_d = 0.4 μM) of low specificity (K_d ≈ 0.8, 2, and 2 μrmM, respectively for ?ADP, GDP, and CDP). These results are compared with the specificity of the partial reactions for photophosphorylation.     

Kinetics of nucleotide binding to chloroplast coupling factor (CF1).

Studies of the kinetics of association and dissociation of the formycin nucleotides FTP and FDP with CF1 were carried out using the enhancement of formycin fluorescence. The protein used, derived from lettuce chloroplasts by chloroform induced release, contains only 4 types of subunit and has a molecular weight of 280 000. In the presence of 1.25 mM MgCl2, 1 mol of ATP or FTP is bound to the latent enzyme, with Kd = 10(-7) or 2 . 10(-7), respectively. The fluorescence emission (lambdamax 340 nm) of FTP is enhanced 3-fold upon binding, and polarization of fluorescence is markedly increased. The fluorescence changes have been used to follow FTP binding, which behaves as a bimolecular process with k1 = 2.4 . 10(4) M-1 . s-1. FTP is displaced by ATP in a process apparently involving unimolecular dissociation of FTP with K-1 = 3 . 10(-3) S-1. The ratio of rates is comparable to the equilibrium constant and no additional steps have been observed. The protein has 3 sites for ADP binding. Rates of ADP binding are similar in magnitude to those for FTP. ADP and ATP sites are at least partly competitive with one another. The kinetics of nucleotide binding are strikingly altered upon activation of the protein as an ATPase. The rate of FTP binding increases to at least 10(6) M-1 . s-1. This suggests that activation involves lowering of the kinetic barriers to substrate and product binding-dissociation and has implications for the mechanism of energy transduction in photophosphorylation.     

Substrate binding-induced alteration of nucleotide binding site properties of chloroplast coupling factor 1

Two adenine nucleotide binding sites of chloroplast coupling factor 1 (CF1) were shown previously to switch their properties after exposure of the enzyme to Mg2(+)-ATP or Ca2(+)-ATP (Shapiro, A. B., and McCarty, R. E. (1988) J. Biol. Chem. 263, 14160-14165). The change in binding properties was monitored by fluorescence resonance energy transfer between Lucifer Yellow vinyl sulfone covalently bound to one alpha subunit and trinitrophenyl-ATP (TNP-ATP) tightly bound to nucleotide binding site 1. When the nucleotide binding properties of sites 1 and 3 switch during catalysis, site 3, which is nearer Lucifer Yellow than site 1, switches its nucleotide binding properties with site 1, allowing TNP-ATP to become tightly bound near Lucifer Yellow. The smaller separation allows energy transfer to occur, resulting in decreased Lucifer Yellow fluorescence. In this paper, we show that adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) bound to CF1 and caused nucleotide binding sites 1 and 3 to switch properties, but was not hydrolyzed. Using AMP-PNP, we also found that relaxation of the properties of the sites to the precatalysis state after removal of substrate occurred in the absence of hydrolysis of the last bound nucleotide. When Mg2+ was omitted during exposure of CF1 to ATP, there was very little hydrolysis or nucleotide site switching. When Mg2+ was added to a very low concentration which was more than stoichiometric with CF1, however, site switching occurred at its maximal level with virtually no increase in ATP hydrolysis. These results support a model in which binding of substrate Mg2(+)-ATP, not hydrolysis, causes the putative catalytic sites to switch properties, in agreement with the alternating site catalytic cooperativity hypothesis (Boyer, P. D. (1989) FASEB J. 3, 2164-2178). TNP-ATP, the fluorescence acceptor, did not cause nucleotide site switching when incubated with CF1 in the presence of EDTA to eliminate free Mg2+. Two possible additional nucleotide binding sites were detected, in addition to the three well characterized sites. At least one of these sites was close to the Lucifer Yellow site, judging by the amount of energy transfer caused by partial occupancy with TNP-ATP.     

Amino acid sequence of the nucleotide binding region of chloroplast coupling factor 1

The labeling of chloroplast coupling factor 1 by 3'-O-(4-benzoyl)benzoyl-ATP (BzATP) was studied. When the enzyme was incubated with approximately 10 microM BzATP and 6 mM MgCl2 at pH 7.9 for approximately 20 min and passed through two Sephadex G-50 centrifuge columns, three BzATP molecules were bound per coupling factor molecule. Photolysis of radioactive enzyme-bound BzATP followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that the BzATP bound primarily to the beta-polypeptide. If unbound BzATP was not removed by centrifuge columns prior to photolysis, significant labeling of the alpha-polypeptide also occurred. After photolysis, the BzATP-labeled enzyme was treated with trypsin, and two radioactive peptides were isolated by high-performance liquid chromatography on a C18 column. The two peptides were sequenced and found to correspond to amino acids 360-378 and 393-397 of the beta-polypeptide. For the sequence 360-378, two specific amino acids were found to be radioactive (Tyr-362 and Asp-369). This region of the polypeptide is highly conserved in several different species and probably corresponds to part of the nucleotide binding region of the catalytic site. In the case of amino acids 393-397, a very low level of radioactivity was found for all amino acids. The significance of this peptide for the binding of nucleotides to coupling factor 1 could not be established.     

Characterization of nucleotide-binding sites on the chloroplast coupling factor 1 using two photolabile analogs

Nucleotide-binding sites on the chloroplast coupling factor 1 (CF1) have been probed using two photoreactive ADP analogs: 2-azido-ADP (2-N3-ADP) and 2',3'-O-(4-benzoyl)benzoyl-ADP (Bz-ADP). Photolabeling of the isolated CF1 with 2-N3-ADP results in incorporation of the analog exclusively into the beta-subunit of the enzyme. The location of the nucleotide-binding site(s) within the beta-subunit of the CF1 was investigated using peptide mapping. Within the discrimination limits of this technique, it is concluded that the azido- and benzoyl-modified analogs both bind to the same conformation of the nucleotide-binding site(s) of soluble CF1. Bz-ADP, however, labels the binding site(s) on membrane-bound CF1 in a slightly different manner.     

Kinetics of nucleotide binding to chloroplast coupling factor (CF1)

Studies of the kinetics of association and dissociation of the formycin nucleotides FTP and FDP with CF₁ were carried out using the enhancement of formycin fluorescence. The protein used, derived from lettuce chloroplasts by chloroform induced release, contains only 4 types of subunit and has a molecular weight of 280 000.In the presence of 1.25 mM MgCl₂, 1 mol of ATP or FTP is bound to the latent enzyme, with K_d = 10^?7 or 2 · 10^?7, respectively. The fluorescence emission (λ_max 340 nm) of FTP is enhanced 3-fold upon binding, and polarization of fluorescence is markedly increased. The fluorescence changes have been used to follow FTP binding, which behaves as a bimolecular process with K₁ = 2.4 · 10⁴ M^?1 · s^?1. FTP is displaced by ATP in a process apparently involving unimolecular dissociation of FTP with k_?1 = 3 · 10^?3 s^?1. The ratio of rates is comparable to the equilibrium constant and no additional steps have been observed.The protein has 3 sites for ADP binding. Rates of ADP binding are similar in magnitude to those for FTP. ADP and ATP sites are at least partly competitive with one another.The kinetics of nucleotide binding are strikingly altered upon activation of the protein as an ATPase. The rate of FTP binding increases to at least 10⁶ M^?1 · s^?1. This suggests that activation involves lowering of the kinetic barriers to substrate and product binding-dissociation and has implications for the mechanism of energy transduction in photophosphorylation.     

Rotation of nucleotide sites is not required for the enzymatic activity of chloroplast coupling factor 1

New heterobifunctional photoaffinity cross-linking reagents, 6-maleimido-N-(4-benzoylphenyl)hexanamide, 12-maleimido-N-(4-benzoylphenyl)dodecanamide, and 12-[14C]maleimido-N-(4-benzoylphenyl)dodecanamide, were synthesized to investigate the mechanism of ATP hydrolysis by chloroplast coupling factor 1. These reagents react with sulfhydryl groups on the gamma-polypeptide. Subsequent photolysis cross-links the gamma-polypeptide covalently to alpha- and beta-polypeptides. The cross-linkers prevent major movements of the gamma-polypeptide with respect to the alpha- and beta-polypeptides but are sufficiently long to permit some flexibility in the enzyme structure. When approximately 50% of the gamma-polypeptide was cross-linked to alpha- and beta-polypeptides, a 7% loss in ATPase activity was observed for the longer cross-linker and a 12% loss for the shorter. These results indicate that large movements of alpha- and beta-polypeptides with respect to the gamma-polypeptide are not essential for catalysis. In particular, rotation of the polypeptide chains to create structurally equivalent sites during catalysis is not a required feature of the enzyme mechanism.     

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.     

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 (CF(1)) 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 CF(1) with Mg(2+) 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 x 10(-1) min(-1) and k(-2)(ATP) congruent with 10(-3) min(-1), respectively. As follows from the study, the noncatalytic sites of CF(1) are not homogeneous. One of them retains the major part of endogenous ADP after CF(1) precipitation with ammonium sulfate. Its other two sites can bind both ADP and ATP but have different kinetic parameters and different affinity for nucleotides.     

Characterization of three-subunit chloroplast coupling factor

The delta- and epsilon-polypeptides were removed from chloroplast coupling factor 1 (CF1). The resulting enzyme, CF1(-delta, epsilon), is a stable active ATPase containing only alpha-, beta-, and gamma-polypeptides. The dependence of the steady-state kinetics of ATP hydrolysis catalyzed by CF1(-delta, epsilon) on the concentrations of ATP and ADP was found to be essentially the same as by activated CF1. Nucleotide binding studies with CF1(-delta, epsilon) revealed three binding sites: a nondissociable ADP site (site 1), a tight MgATP binding site (site 2), and a site that binds ADP and ATP with a dissociation constant in the micromolar range (site 3). Similar results have been obtained with CF1. For both CF1 and CF1(-delta, epsilon), the binding of MgATP at site 2 is tight only in the presence of Mg2+. Fluorescence resonance energy transfer was used to map distances between the gamma-sulfhydryl ("dark" site) and gamma-disulfide and between the gamma-sulfhydryl and the three nucleotide sites. These distances are within 5% of the corresponding distances on CF1. These results indicate that removal of the delta- and epsilon-polypeptides from CF1 does not cause significant changes in the structure, kinetics, and nucleotide binding sites of the enzyme.