Analysis of ionic channels by a flash spectrophotometric technique applicable to thylakoid membranes: CF0, the proton channel of the chloroplast ATP synthase,and, for comparison,gramicidin

Summary We previously introduced a flash spectrophotometric method to analyze proton conduction by CF₀ in vesicles derived from thylakoid membranes (H. Lill, S. Engelbrecht, G. Schönknecht & W. Junge, 1986,Eur. J. Biochem. 160:627–634). The unit conductance of CF₀, as revealed by this technique, was orders of magnitude higher than that theoretically expected for a hydrogen-bonded chain. We scrutinized the validity of this method. Small vesicles were derived from thylakoids by EDTA treatment. The intrinsic electric generators in the membrane were stimulated by short flashes of light and the relaxation of the voltage via ionic channels was measured through electrochromic absorption changes of intrinsic pigments. The voltage decay was stimulated by a statistical model. As the vesicle-size distribution had only a minor influence, the simulation required only two fit parameters, the first proportional to the unit conductance of an active channelG, and the second denoting the average number of active channels per vesiclen. This technique was applied to CF₀, the proton channel of the chloroplast ATP synthase, and to gramicidin, serving as a standard. For both channels we found the above two fit parameters physically meaningful. They could be independently varied in predictable wasy, i.e.n by addition of known inhibitors of F₀-type proton channels andG via the temperature. for gramicidin, the unit conductance (2.7 pS) was within the range described in the literature. This established the competence of this method for studies on the mechanism of proton conduction by CF₀, whose conductance so far has not been accessible to other, more conventional approaches. The time-averaged unit conductance of CF₀ was about 1 pS, equivalent to the turnover of 6×10⁵ H⁺/(CF₀·sec) at 100 mV driving force.     

Interaction of pyrophosphate with catalytic and noncatalytic sites of chloroplast ATP synthase

The effect of pyrophosphate (PP_i) on labeled nucleotide incorporation into noncatalytic sites of chloroplast ATP synthase was studied. In illuminated thylakoid membranes, PP_i competed with nucleotides for binding to noncatalytic sites. In the dark, PP_i was capable of tight binding to noncatalytic sites previously vacated by endogenous nucleotides, thereby preventing their subsequent interaction with ADP and ATP. The effect of PP_i on ATP hydrolysis kinetics was also elucidated. In the dark at micromolar ATP concentrations, PP_i inhibited ATPase activity of ATP synthase. Addition of PP_i to the reaction mixture at the step of preliminary illumination inhibited high initial activity of the enzyme, but stimulated its activity during prolonged incubation. These results indicate that the stimulating effect of PP_i light preincubation with thylakoid membranes on ATPase activity is caused by its binding to ATP synthase noncatalytic sites. The inhibition of ATP synthase results from competition between PP_i and ATP for binding to catalytic sites. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 7, pp. 956–962.     

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.     

Regulation of Proton Flow and ATP Synthesis in Chloroplasts

The chloroplast ATP synthase is strictly regulated so that it is very active in the light (rates of ATP synthesis can be higher than 5 mol/min/mg protein), but virtually inactive in the dark. The subunits of the catalytic portion of the ATP synthase involved in activation, as well as the effects of nucleotides are discussed. The relation of activation to proton flux through the ATP synthase and to changes in the structure of enzyme induced by the proton electrochemical gradient are also presented. It is concluded that the and subunits of CF₁ play key roles in both regulation of activity and proton translocation.     

Interaction of ADP and ATP with noncatalytic sites of isolated and membrane-bound chloroplast coupling factor CF<Subscript>1</Subscript>

This study of ATP and ADP binding to noncatalytic sites of membrane-bound CF1 (ATP synthase) revealed two noncatalytic sites with different specificities and affinities for nucleotides. One of these is characterized by a high affinity and specificity to ADP (Kd=2.6+/-0.3 microM). However, a certain increase in ADP apparent dissociation constant at high ATP/ADP ratio in the medium allows a possibility that ATP binds to this site as well. The other site displays high specificity to ATP. When the ADP-binding site is vacant, it shows a comparatively low affinity for ATP, which greatly increases with increasing ADP concentration accompanied by filling of the ADP-binding site. The reported specificities of these two sites are independent of thylakoid membrane energization, since both in the dark and in the light the ratios of ATP/ADP tightly bound to the noncatalytic sites were very close. The difference in noncatalytic site affinity for ATP and ADP is shown to depend on the amount of delta subunit in a particular sample. Thylakoid membrane ATP synthase, with stoichiometric content of delta-subunit (one delta-subunit per CF1 molecule), showed the maximal difference in ADP and ATP affinities for the noncatalytic sites. For CF1, with substoichiometric delta subunit values, this difference was less, and after delta subunit removal it decreased still more.     

H+ transport and coupling by the F0 sector of the ATP synthase: Insights into the molecular mechanism of function

The F₀ sector of the ATP synthase complex facilitates proton translocation through the membrane, and via interaction with the F₁ sector, couples proton transport to ATP synthesis. The molecular mechanism of function is being probed by a combination of mutant analysis and structural biochemistry, and recent progress on theEscherichia coli F₀ sector is reviewed here. TheE. coli F₀ is composed of three types of subunits (a, b, andc) and current information on their folding and organization in F₀ is reviewed. The structure of purified subunitc in chloroform-methanol-H₂O resembles that in native F₀, and progress in determining the structure by NMR methods is reviewed. Genetic experiments suggest that the two helices of subunitc must interact as a functional unit around an essential carboxyl group as protons are transported. In addition, a unique class of suppressor mutations identify a transmembrane helix of subunita that is proposed to interact with the bihelical unit of subunitc during proton transport. The role of multiple units of subunitc in coupling proton translocation to ATP synthesis is considered. The special roles of Asp61 of subunitc and Arg210 of subunita in proton translocation are also discussed.     

Kinetic studies of ATP synthase: The case for the positional change mechanism

The mitochondrial ATP synthases shares many structural and kinetic properties with bacterial and chloroplast ATP synthases. These enzymes transduce the energy contained in the membrane's electrochemical proton gradients into the energy required for synthesis of high-energy phosphate bonds. The unusual three-fold symmetry of the hydrophilic domain, F₁, of all these synthases is striking. Each F₁ has three identical subunits and three identical subunits as well as three additional subunits present as single copies. The catalytic site for synthesis is undoubtedly contained in the subunit or an , interface, and thus each enzyme appears to contain three identical catalytic sites. This review summarizes recent isotopic and kinetic evidence in favour of the concept, originally proposed by Boyer and coworkers, that energy from the proton gradient is exerted not directly for the reaction at the catalytic site, but rather to release product from a single catalytic site. A modification of this binding change hypotheses is favored by recent data which suggest that the binding change is due to a positional change in all three subunits relative to the remaining subunits of F₁ and F₀ and that the vector of rotation is influenced by energy. The positional change, or rotation, appears to be the slow step in the process of catalysis and it is accelerated in all F₁F₀ ATPases studied by substrate binding and by the proton gradient. However, in the mammalian mitochondrial enzyme, other types of allosteric rate regulation not yet fully elucidated seem important as well.     

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.     

Covalent Binding of 1,N 6-ethenoadenosine diphosphate to catalytic and noncatalytic sites of chloroplast ATP synthase

Catalytic and noncatalytic sites of the chloroplast coupling factor (CF₁) were selectively modified by incubation with the dialdehyde derivative of fluorescent adenosine diphosphate analog 1,N⁶-ethenoadenosine diphosphate. The modified CF₁ was reconstituted with EDTA-treated thylakoid membranes of chloroplasts. The effects of light-induced transmembrane proton gradient and phosphate ions on the fluorescence of 1,N⁶-ethenoadenosine diphosphate, covalently bound to the catalytic sites of ATP synthase, were studied. Quenching of fluorescence of covalently bound 1,N⁶-ethenoadenosine diphosphate was observed under illumination of thylakoid membranes with saturating white light. Addition of inorganic phosphate to the reaction mixture in the dark increased the fluorescence of the label. Quenching reappeared under repeated illumination; however, addition of phosphate ions had no effect on the fluorescence yield in this case. When 1,N⁶-ethenoadenosine diphosphate was covalently bound to noncatalytic sites of ATP synthase, no similar fluorescence changes were observed. The relation between the observed changes of 1,N⁶-ethenoadenosine diphosphate fluorescence and the mechanism of energy-dependent structural changes in the catalytic site of ATP synthase is discussed.     

Significance of the epsilon subunit in the thiol modulation of chloroplast ATP synthase

To understand the regulatory function of the gamma and epsilon subunits of chloroplast ATP synthase in the membrane integrated complex, we constructed a chimeric FoF1 complex of thermophilic bacteria. When a part of the chloroplast F1 gamma subunit was introduced into the bacterial FoF1 complex, the inverted membrane vesicles with this chimeric FoF1 did not exhibit the redox sensitive ATP hydrolysis activity, which is a common property of the chloroplast ATP synthase. However, when the whole part or the C-terminal alpha-helices region of the epsilon subunit was substituted with the corresponding region from CF1-epsilon together with the mutation of gamma, the redox regulation property emerged. In contrast, ATP synthesis activity did not become redox sensitive even if both the regulatory region of CF1-gamma and the entire epsilon subunit from CF1 were introduced. These results provide important features for the regulation of FoF1 by these subunits: (1) the interaction between gamma and epsilon is important for the redox regulation of FoF1 complex by the gamma subunit, and (2) a certain structural matching between these regulatory subunits and the catalytic core of the enzyme must be required to confer the complete redox regulation mechanism to the bacterial FoF1. In addition, a structural requirement for the redox regulation of ATP hydrolysis activity might be different from that for the ATP synthesis activity.     

Mutagenesis Studies of the F1F0 ATP Synthase b Subunit Membrane Domain

A homodimer of b subunits constitutes the peripheral stalk linking the F₁ and F₀ sectors of the Escherichia coli ATP synthase. Each b subunit has a single-membrane domain. The constraints on the membrane domain have been studied by systematic mutagenesis. Replacement of a segment proximal to the cytoplasmic side of the membrane had minimal impact on F₁F₀ ATP synthase. However, multiple substitutions on the periplasmic side resulted in defects in assembly of the enzyme complex. These mutants had insufficient oxidative phosphorylation to support growth, and biochemical studies showed little F₁F₀ ATPase and no detectable ATP-driven proton pumping activity. Expression of the b _N2A,T6A,Q10A subunit was also oxidative phosphorylation deficient, but the b _N2A,T6A,Q10A protein was incorporated into an F₁F₀ complex. Single amino acid substitutions had minimal reductions in F₁F₀ ATP synthase function. The evidence suggests that the b subunit membrane domain has several sites of interaction contributing to assembly of F₀, and that these interactions are strongest on the periplasmic side of the bilayer.     

Interaction of Sulfite with the Noncatalytic and Catalytic Sites of Chloroplast Coupling Factor CF1

The interaction between sulfite, an efficient Mg²⁺-dependent F₁-ATPase activator, and chloroplast CF₁-ATPase was studied. The sulfite anion was shown to inhibit ADP and ATP binding to the noncatalytic sites of CF₁. The stimulating activity of sulfite persists when all noncatalytic sites are nucleotide-occupied. Phosphate, a competing candidate for binding to CF₁ catalytic sites, suppresses this activity. These results support the suggestion that the stimulation of Mg²⁺-dependent ATPase activity of CF₁ is caused by sulfite binding to its catalytic sites.     

F0F1 ATP synthase of Streptomycetes: Modulation of activity and oligomycin resistance by protein Ser/Thr kinases

Inverted membrane vesicles of Gram-positive actinobacteria Streptomyces fradiae, S. lividans, and S. avermitilis have been prepared and membrane-bound F₀F₁ ATP synthase has been biochemically characterized. It has been shown that the ATPase activity of membrane-bound F₀F₁ complex is Mg²⁺-dependent and moderately stimulated by high concentrations of Ca²⁺ ions (10–20 mM). The ATPase activity is inhibited by N,N′-dicyclohexylcarbodiimide and oligomycin A, typical F₀F₁ ATPase inhibitors that react with the membrane-bound F₀ complex. The assay of biochemical properties of the F₀F₁ ATPases of Streptomycetes in all cases showed the presence of ATPase populations highly susceptible and insensitive to oligomycin A. The in vitro labeling and inhibitory assay showed that the inverted phospholipid vesicles of S. fradiae contained active membrane-bound Ser/Thr protein kinase(s) phosphorylating the proteins of the F₀F₁ complex. Inhibition of phosphorylation leads to decrease of the ATPase activity and increase of its susceptibility to oligomycin. The in vivo assay confirmed the enhancement of actinobacteria cell sensitivity to oligomycin after inhibition of endogenous phosphorylation. The sequencing of the S. fradiae genes encoding oligomycin-binding A and C subunits of F₀F₁ ATP synthase revealed their close phylogenetic relation to the genes of S. lividans and S. avermitilis.     

The Oligomycin Axis of Mitochondrial ATP Synthase: OSCP and the Proton Channel

Oligomycin has long been known as an inhibitor of mitochondrial ATP synthase, putatively binding the F_o subunits 9 and 6 that contribute to proton channel function of the complex. As its name implies, OSCP is the oligomycin sensitivity-conferring protein necessary for the intact enzyme complex to display sensitivity to oligomycin. Recent advances concerning the structure and mechanism of mitochondrial ATP synthase have led to OSCP now being considered a component of the peripheral stator stalk rather than a central stalk component. How OSCP confers oligomycin sensitivity on the enzyme is unknown, but probably reflects important protein–protein interactions made within the assembled complex and transmitted down the stator stalk, thereby influencing proton channel function. We review here our studies directed toward establishing the stoichiometry, assembly, and function of OSCP in the context of knowledge of the organization of the stator stalk and the proton channel.     

F1F0 ATP synthase subunit c is a substrate of the novel YidC pathway for membrane protein biogenesis

The Escherichia coli YidC protein belongs to the Oxa1 family of membrane proteins that have been suggested to facilitate the insertion and assembly of membrane proteins either in cooperation with the Sec translocase or as a separate entity. Recently, we have shown that depletion of YidC causes a specific defect in the functional assembly of F1F0 ATP synthase and cytochrome o oxidase. We now demonstrate that the insertion of in vitro-synthesized F1F0 ATP synthase subunit c (F0c) into inner membrane vesicles requires YidC. Insertion is independent of the proton motive force, and proteoliposomes containing only YidC catalyze the membrane insertion of F0c in its native transmembrane topology whereupon it assembles into large oligomers. Co-reconstituted SecYEG has no significant effect on the insertion efficiency. Remarkably, signal recognition particle and its membrane-bound receptor FtsY are not required for the membrane insertion of F0c. In conclusion, a novel membrane protein insertion pathway in E. coli is described in which YidC plays an exclusive role.     

Activation of the CF0-CF1, ATP synthase from spinach chloroplasts by chloroplast lipids

The interactions of CF₀-CF₁ with different lipids were studied by following the stimulation of Mg-ATPase and of P_i-ATP exchange activities of reconstituted CF₀-CF₁ proteoliposomes. The following results were obtained: (1) Both P_i-ATP exchange and Mg-ATPase activities are stimulated by lipids. Furthermore, the inhibition of Mg-ATPase by N,N′-dicyclohexylcarbodiimide is dependent on the interactions of CF₀-CF₁ with lipids. (2) A polar lipid extract of thylakoid membranes stimulates Mg-ATPase activity of CF₀-CF₁ more efficiently than phospholipids. The relative effectiveness of Mg-ATPase stimulation is: chloroplast lipids > soybean phospholipids > phosphatidylcholine/phosphatidylserine (4: 1) > phosphatidylcholine. The rate of P_i-ATP exchange in chloroplast lipids CF₀-CF₁ proteoliposomes is, however, lower than in soybean lipids CF₀-CF₁ proteoliposomes, due to their higher permeability to protons. Addition of 10% phosphatidylserine to chloroplast lipids reduces their permeability to protons and stimulates P_i-ATP exchange. (3) The kinetic mechanism of ATPase stimulation by chloroplast lipids is by decreasing the K_m (ATP) and by increasing V_max in comparison to soybean lipid proteoliposomes. This may explain the low affinity for ATP and the slow turnover rate of the purified enzyme in artificial lipids in comparison to the native enzyme in chloroplast thylakoids. (4) Chloroplast lipids lacking monogalactosyldiacylglycerols only poorly activate CF₀-CF₁. A large stimulation of P_i-ATP exchange is obtained by a mixture of 60% monogalactosyldiacylglycerol and 40% of the rest of the chloroplast lipids, but not by mixtures of monogalactosyldiacylglycerol with phospholipids. Hydrogenation of the unsaturated fatty acids of monogalactosyldiacylglycerol inhibits the activation of CF₀-CF₁. (5) The results suggest that: (a) interactions of specific chloroplast lipids with CF₀-CF₁ activates the enzyme by increasing its turnover and its affinity for ATP; (b) specific requirements for CF₀-CF₁ activation are the presence of monogalactosyldiacylglycerols together with another chloroplast lipid component and of highly unsaturated fatty acids.