Characterization of nucleotide binding sites on chloroplast coupling factor 1.

A study of the equilibrium binding of ADP, 1,N6-ethenoadenosine diphosphate, adenylyl imidodiphosphate, and 1,N6-ethenoadenylyl imidodiphosphate to solubilized spinach chloroplast coupling factor 1 (CF1) has been carried out. All four nucleotides were found to bind to two apparently identical "tight" sites, with characteristic dissociation contants generally less than 10 muM. The binding to these "tight" sites is similar in the presence of Mg2+ and Ca2+, is stronger in 0.1 M NaC1 than in 20 mM Tris-C1, and is only slightly altered by heat activation. The slow rate of association of ADP and 1,N6-ethenoadenosine diphosphate at these sites rules out the possibility that they are catalytic sites for ATPase activity on the solubilized enzyme. A third tight site for adenylyl imidodiphosphate was found on the heat-activated enzyme. The dissociation constant for this interaction (7.6 muM) is similar to the adenylyl imidodiphosphate competitive inhibition constant for ATPase activity (4 muM). ADP, which inhibits ATPase activity but is not a strong competitive inhibitor, binds only weakly at a third site (dissociation constant greater than 70 muM). One mole of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reacted per mole of CF1 prevents ADP and adenylyl imidodiphosphate binding at the "catalytic" site and abolishes the ATPase activity. A model is proposed in which the "tight" nucleotide binding sites act as allosteric conformational switches for the ATPase activity of solubilizedCF1.     

Structural mapping of chloroplast coupling factor

Fluorescence resonance energy transfer measurements have been used to investigate the spatial relationships between the nucleotide binding sites and the gamma-subunit of the H+-ATPase from chloroplasts and the orientation of these sites with respect to the membrane surface. Fluorescent maleimides reacted covalently at specific sulfhydryl sites on the gamma-subunit served as energy donors. One sulfhydryl site can be labeled only under energized conditions on the thylakoid membrane surface (light site). The two gamma-sulfhydryls exposed after catalytic activation served as a second donor site (disulfide site). In one set of experiments, the nucleotide analogue 2'(3')-(trinitrophenyl)adenosine triphosphate, selectively bound at each of the three nucleotide binding sites of the solubilized coupling factor, was used as an energy acceptor; in another, octadecylrhodamine with its acyl chain inserted in the vesicle bilayer and the rhodamine fluorophore exposed along the membrane surface was the energy acceptor. The distance between the sulfhydryl and disulfide sites was also obtained by sequentially labeling the sites with coumarin (donor) and fluorescein (acceptor) maleimide derivatives, respectively. The results indicate that all three nucleotide sites are approximately equal to 50 A from the light-labeled gamma-sulfhydryl. Two of the nucleotide sites are very far from the gamma-disulfide (greater than 74 A), while the third site, which binds nucleotides reversibly under all conditions, is 62 A from this sulfhydryl. The light-labeled sulfhydryl and disulfide sites are about 42-47 A apart. Finally, the distance of closest approach between the membrane surface of the reconstituted system and the gamma-disulfide is 31 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural organization of chloroplast coupling factor

Fluorescence resonance energy transfer measurements have been used to construct spatial maps for the accessible sulfhydryl of the gamma subunit (dark site) and the essential tyrosine residue of the beta subunits relative to previously mapped sites on the H+-ATPase from chloroplasts. The extent of energy transfer was measured between a coumarinylmaleimide derivative reacted covalently at the dark site and acceptor species selectively bound at the gamma-disulfide and the three nucleotide binding sites of the solubilized coupling factor complex. The nucleotide energy acceptor was 2'(3')-(trinitrophenyl)adenosine triphosphate, and the gamma-disulfide site was labeled with fluoresceinylmaleimide. The dark-site sulfhydryl also was labeled with pyrenylmaleimide which served as an energy donor for 7-chloro-4-nitro-2,1,3-benzoxadiazole reacted at the beta-tyrosine sites. Similar measurements were also made with pyrenylmaleimide covalently attached to the gamma-sulfhydryl accessible only under energized conditions on the thylakoid membrane surface (light site). The observed transfer efficiencies indicate that the dark-site sulfhydryl is approximately 45 A from all three nucleotide sites and 41-46 A from the gamma-disulfide site. The average distances separating the essential beta-tyrosines and the light- and dark-site sulfhydryls are 38 and 42 A, respectively. (In calculating these distances, random orientation of the donor-acceptor dipoles was assumed.) The results are consistent with a previously described structural model of the intact enzyme and can be used to gain insight into the overall structural organization or alpha-, beta-, and gamma-polypeptides within the coupling factor.     

Inhibition of chloroplast coupling factor by naphthylglyoxal

The trypsin-activated Ca²⁺ -ATPase of spinach chloroplast membranes was completely inhibited by treatment with naphthylglyoxal, a fluorescent compound that should bind covalently to arginine residues. The inhibition followed apparent first-order kinetics. The apparent order of reaction with respect to inhibitor concentration gave values near unity, suggesting that inactivation is a consequence of modifying one arginine residue per active site. Partial protection against naphthylglyoxal was afforded by ADP and ATP, with either less or no protection by other nucleotide bases. At inhibition levels less than complete, the K_m for ATP was not affected but the V_max of the enzyme was diminished. The light-dependent exchange of tightly bound nucleotides on the membrane-bound enzyme was not inhibited by naphthylglyoxal treatment, indicating significant retention of the conformational response of the enzyme to the membrane high-energy state. Using [³H]naphthylglyoxal, the extent of inhibition was a linear function of the amount of naphthylglyoxal bound up to 60% inhibition. The curves extrapolated to 2 mol naphthylglyoxal bound, associated with complete inhibition of ATPase. The radioactive naphthylglyoxal was distributed equally between α- and β-subunits.     

Integrated functioning of the chloroplast coupling factor

This review is focused on some functional characteristics of the chloroplast coupling factor. The structure of the enzyme and the putative role of its subunits are recalled. An attempt is made to discriminate the driving force and the activator effects of the electrochemical proton gradient. Respective roles of delta pH, delta phi, external and internal pH are discussed with regard to mechanistic implications. The hypothesis of a functional switch of the enzyme between two states with better efficiency either in ATP synthesis or in ATP hydrolysis is also examined. A brief survey is made on some problems complicating quantitative studies of energy coupling, such as localized chemiosmosis, delta pH and delta phi computations, and scalar ATPases. The main data on the enzyme activation and the energy-dependent release of tightly bound nucleotides are summarized. The arguments for and against the catalytic competence of theses nucleotides are reviewed. Lastly, some prevailing models of the catalytic mechanism are presented. The relevance of nucleotides binding change events in this process is discussed.     

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.     

Cytoplasmic inheritance of chloroplast coupling factor 1 subunits

An analysis of interspecific hybrids of Nicotiana spp. in which one of the parents was sensitive to tentoxin showed that this sensitivity was transmitted only through the female parent. Since tentoxin acts by selectively binding to the alpha,beta subunit complex of chloroplast coupling factor 1, the gene(s) specifying either one or both of these subunits is located in the cytoplasm.     

Delta subunit of chloroplast coupling factor 1 inhibits proton leakage through coupling factor O

The ATP synthase of chloroplasts consists of a proton-conducting portion, CF0, and a catalytic portion, CF1. The smaller subunits of CF1, in particular delta, may play a key role in the coupling of proton transport to ATP synthesis. Purified subunit delta, when added to partially CF1-depleted thylakoid membranes, can restore photophosphorylation (Engelbrecht, S., and Junge, W. (1987) Eur. J. Biochem. 172, 213-218). We report here that it does so by blocking proton conduction through CF0. Thylakoids were CF1-depleted by incubation in hypoosmolar NaCl/EDTA solutions. Variation of the NaCl concentrations and of the incubation times not only changed the overall degree of CF1 depletion but also the subunit composition of solubilized CF1, namely CF1 containing delta and CF1(-delta). This was quantified by immunoelectrophoresis and by fast protein liquid chromatography. Proton conduction was measured by flash spectrophotometry by using standard electrochromic and pH-indicating absorption changes. The removal of integral CF1 was correlated with high electric conductance of thylakoid membranes, an increased extent of rapid proton leakage, and loss of ATP synthesis activity, which exceeded the percentual loss of CF1. The removal of predominantly CF1(-delta) resulted in comparatively lesser effects on the loss of ATP synthesis and on the extent and velocity of proton leakage. On the same line, addition of integral CF1 and of purified delta diminished the electric leak in CF1-depleted thylakoids. Both approaches, the controlled removal of CF1 and CF1(-delta), respectively, and addition of delta and CF1 showed that delta can act as a "stopcock" to the exposed proton channel CF0.     

Lack of site-specificity of spinach chloroplast coupling factor 1

The irreversible inhibition of chloroplast phosphorylation by either sulfate anions, or N-ethylmaleimide, is energy dependent. Chloroplasts must first be illuminated in the presence of the inhibitors and a mediator of electron flow, for the subsequent phosphorylation to show any inhibition. Both inhibitors affect the chloroplast coupling factor 1.Electron transport only through Photosystem I can be used to activate either of these inhibitions. The subsequent inhibition in a second light reaction is the same whether ATP synthesis is supported by Photosystem I, or by Photosystem II electron transport. The reverse experiment, activating inhibition by electron transport only through Photosystem II, is possible in the case of sulfate. Again, the inhibition is expressed whether Photosystem II or Photosystem I electron flow supports ATP synthesis. We conclude that the two electron transport regions probably generate the same high energy state which is able to activate all members of a functionally uniform coupling factor population. These enzyme molecules must catalyze phosphorylation coupled to electron transport through either region of the chain. The results tend to discredit models requiring a separate group of coupling factor molecules unique to each part of the chain.     

Isolation and properties of chloroplast coupling factor from wheat

1. Wheat chloroplast coupling factor (CF1) was extracted with a modification of the chloroform extraction method of Younis et al. (J. Biol. Chem. 252, 1814--1818, 1977). A one-step purification on an 8--25% sucrose gradient yielded a CF1 which was at least 98% pure as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. 2. Inclusion of proteolysis inhibitors during extraction and purification consistently gave a CF1 containing all five subunits. Selective loss of the sigma and epsilon subunits was observed when proteolysis inhibitors were omitted. 3. Proteolysis inhibitors prevented the release of wheat CF1 from the thylakoid by the low-ionic-strength wash method of Strotmann et al. (Biochim. Biophys. Acta, 314, 202--210, 1973). The enzyme extracted with chloroform and low ionic strength were compared by electrophoresis and no evidence of a difference in molecular weight of any subunit was observed. This suggests that a proteolytic event is not required for release of wheat CF1 by the low-ionic-strength method, even though release is inhibited by proteolysis inhibitors. 4. The gamma subunit of wheat CF1 probably contains at least one internal disulfide bridge, as the electrophoretic mobility of this subunit is lower in the presence of reducing agent than in its absence. 5. Wheat CF1 was viewed by the electron microscope after negative staining. Discrete particles, many appearing hexagonal, were observed at high magnifications. Markham rotational analysis confirmed that the enzyme has sixfold symmetry in at least one of its orientations.     

Interaction of oxyanions with thioredoxin-activated chloroplast coupling factor 1

Interaction between F(1)-ATPase activity stimulating oxyanions and noncatalytic sites of coupling factor CF(1) was studied. Carbonate, borate and sulfite anions were shown to inhibit tight binding of [14C]ATP and [14C]ADP to CF(1) noncatalytic sites. The demonstrated change of their inhibitory efficiency in carbonate-borate-sulfite order coincides with the previously found change in efficiency of these anions as stimulators of CF(1)-ATPase activity [Biochemistry (Mosc.) 43 (1978) 1206-1211]. Inhibition of tight nucleotide binding to noncatalytic sites was accompanied by stimulation of nucleotide binding to catalytic sites. This suggests that stimulation of CF(1)-ATPase activity is caused by interaction between oxyanions and noncatalytic sites. A most efficient stimulator of CF(1)-ATPase activity, sulfite oxyanion, appeared to be a competitive inhibitor with respect to ATP and a partial noncompetitive inhibitor with respect to ADP. The inhibition weakened with increasing time of CF(1) incubation with sulfite and nucleotides. Sulfite is believed to inhibit fast reversible interaction between nucleotides and noncatalytic sites and to produce no effect on subsequent tight binding of nucleotides. A possible mechanism of the oxyanion-stimulating effect is discussed.     

Chemical cross-linking studies of chloroplast coupling factor 1.

Cross-linking reagents have been used to link covalently adjacent subunits of solubilized spinach chloroplast coupling factor 1, which is a latent ATPase. 1,5-Difluoro-2,4-dinitrobenzene, dimethyl-3,3'-dithiobispropionimidate, and dimethylsuberimidate are able to form bridges of 3 to 11 A between amino groups, and hydrogen peroxide and the o-phenanthroline-cupric ion complex catalyze the oxidation of intrinsic sulfhydryl groups. The five individual subunit bands (alpha, beta, gamma, delta, and epsilon) and several new aggregate bands can be separated by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The same four fastest moving aggregate bands, as characterized by their mobilities, migrate more slowly than the heaviest subunit band and appear with all of the cross-linkers employed. The subunit composition of the aggregate bands has been determined through the use of the reversible cross-linkers, dimethyldithiobispropionimidate, (o-phenanthroline)2Cu(II), and H2O2, and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis in which aggregates are separated in the first dimension, the disulfide cross-links are cleaved, and the individual subunits present in the aggregates are separated in the second dimension. The subunits are detected by Coomassie brilliant blue staining and by labeling some of the sulfhydryl groups of the gamma and epsilon subunits with radioactive N-ethylmaleimide. The results obtained indicate that the alpha and beta subunits can cross-link directly with each of the other subunits, that two beta subunits are adjacent, and that gamma epsilon, gamma epsilon 2, alpha delta, and beta delta aggregates are present. A minimal subunit stoichiometry consistent with these results is alpha 2 beta 2 gamma delta epsilon 2. A possible structural model of the coupling factor is derived from the data. Similar, but less extensive, experiments have been carried out with the heat-activated coupling factor (which is an ATPase); no differences in the spatial arrangement of subunits are detected from the two-dimensional gel electrophoresis analysis of the cross-linked aggregates.     

Lack of correlation between effects of tentoxin on chloroplast coupling factor and chloroplast ultrastructure

The mediation of tentoxin-induced chlorosis through inhibition of chloroplast coupling factor 1 (CF₁) ATPase activity was investigated through an examination of the effects of tentoxin on electrophoretically-separated CF₁ ATPases from sensitive and insensitive Nicotiana species. Sensitive species exhibited three major ATPases, only one of which was inhibited at some concentrations of tentoxin. Insensitive Nicotiana species showed the same three "isozymes"upon electrophoresis but none of the isozymes were tentoxin sensitive. CF₁ isolated from Zea mays L. cv. Pioneer 3541, which is insensitive to tentoxin in vivo based on lack of chlorosis, exhibited two ATPases, one of which was sensitive to tentoxin. The concentration/activity relationships between tentoxin and ATPase inhibition of the sensitive isozyme did not correlate well with the chlorosis induced at similar levels of tentoxin in vivo. Both Oenothera hookeri Torr. & Gray and the CF₁-deficient I iota mutant derived from it are sensitive to tentoxin as determined by loss of chlorophyll and ultrastructural changes typical of the tentoxin syndrome. These results support a mechanism of action different from inhibition of CF¹ for tentoxin-induced chlorosis.     

The site of synthesis of pea chloroplast coupling factor 1

Inhibitors of cytoplasmic polyribosome function and chloroplastpolyribosome function were used to study the site of synthesisof the five subunits of coupling factor I (CF₁) in Pisum sativum.The results of these in vivo experiments are presented as evidencefor the cytoplasmic synthesis of two subunits, C and D, andthe synthesis in the chloroplast of the other three, A, B andE. ¹ Supported in part by grant PCM-74-13534 from the NationalScience Foundation. (Received April 17, 1978; )     

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.     

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.     

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.     

Binding of the chloroplast coupling factor CF to lipid vesicles

The binding of the chloroplast coupling factor CF to lipid vesicles was analyzed by gel filtration. CF can be bound to vesicles made of chloroplast lipids but not of lecithin. The presence in the vesicle walls of a proteolipid subunit of the hydrophobic component of the coupling factor increases the binding of CF. The apparent binding constant and the maximum protein/lipid ratio are calculated. The Ca2+-ATPase activity of bound CF is markedly lower than that of dissolved CF. It is confirmed that the proteolipid is a N,N'-dicyclohexylcarbodiimide sensitive proton channel. The binding of CF on proteolipid vesicles decreases their proton permeability.