Interaction between the oligomycin sensitivity conferring protein and the F0 sector of the mitochondrial adenosinetriphosphatase complex: cooperative effect of the F1 sector

Beef heart mitchondrial oligomycin sensitivity conferring protein (OSCP) labeled with [14C]-N-ethylmaleimide ([14C]OSCP) at the only cysteine residue, Cys-118, present in the sequence [Ovchinnikov, Y. A., Modyanov, N. N., Grinkevich, V. A., Aldanova, N. A., Trubetskaya, O. E., Nazimov, I.V., Hundal, T., & Ernster, L. (1984) FEBS Lett. 166, 19-22] exhibits full biological activity in a reconstituted F0-F1 system [Dupuis, A., Issartel, J. P., Lunardi, J., Satre, M., & Vignais, P. V. (1985) Biochemistry 24, 728-733]. The binding parameters of [14C]OSCP with respect to the F0 sector of submitochondrial particles largely depleted of F1 and OSCP (AUA particles) have been explored. In the absence of added F1, a limited number of high-affinity OSCP binding sites were detected in the AUA particles (20-40 pmol/mg of particles); under these conditions, the low-affinity binding sites for OSCP were essentially not saturable. Addition of F1 to the particles promoted high-affinity binding for OSCP, with an apparent Kd of 5 nM, a value 16 times lower than the Kd relative to the binding of OSCP to F1 in the absence of particles. Saturation of the F1 and OSCP binding sites of AUA particles was attained with about 200 pmol of both F1 and OSCP added per milligram of particles. The oligomycin-dependent inhibition of F1-ATPase bound to AUA particles was assayed as a function of bound OSCP. At subsaturating concentrations of F1, the dose-effect curves were rectilinear until inhibition of ATPase activity by oligomycin was virtually complete, and maximal inhibition was obtained for an OSCP to F1 ratio of 1 (mol/mol).(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectral properties of fluorescent derivatives of the oligomycin sensitivity conferring protein and analysis of their interaction with the F1 and F0 sectors of the mitochondrial ATPase complex

In order to study the kinetics and the nature of the interactions between the oligomycin sensitivity conferring protein (OSCP) and the F0 and F1 sectors of the mitochondrial ATPase complex, fluorescent derivatives of OSCP, which are fully biologically active, have been prepared by reaction of OSCP with the following fluorescent thiol reagents: 6-acryloyl-2-(dimethylamino)naphthalene (acrylodan), 2-(4-maleimidylanilino)naphthalene-6-sulfonic acid (Mal-ANS), N-(1-pyrenyl)maleimide (Mal-pyrene), 7-(diethylamino)-3-(4-maleimidylphenyl)-4-methylcoumarin (Mal-coumarin), and fluorescein 5-maleimide (Mal-fluorescein). The preparation of these derivatives was based on the previous finding that the single cysteinyl residue of OSCP, Cys 118, can be covalently modified by alkylating reagents without loss of biological activity [Dupuis, A., Issartel, J. P., Lunardi, J., Satre, M., & Vignais, P. V. (1985) Biochemistry 24, 728-733]. For all fluorescent probes used, except Mal-pyrene and Mal-fluorescein, the emission spectra of conjugated OSCP were blue-shifted relative to those of the corresponding mercaptoethanol adducts, indicating that the fluorophores attached to Cys 118 were located in a hydrophobic pocket. These results were consistent with the high quantum yields and the increased fluorescence lifetimes of conjugated OSCP compared to mercaptoethanol adducts in aqueous buffer. They also fit with quenching data obtained with potassium iodide which showed that the fluorophore is shielded from the aqueous medium when it is attached to Cys 118 of OSCP. Especially noticeable was the wide half-width of the OSCP-acrylodan emission peak compared to that of mercaptoethanol-acrylodan.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions between the oligomycin sensitivity conferring protein (OSCP) and beef heart mitochondrial F1-ATPase. 1. Study of the binding parameters with a chemically radiolabeled OSCP

Upon treatment of beef heart mitochondrial oligomycin sensitivity conferring protein (OSCP) with [14C]-N-ethylmaleimide ( [14C]NEM) or dithiobis(nitro[14C] benzoate), 1 mol of either SH reagent was incorporated per mol of OSCP. Radiolabeling occurred at the level of the only cysteine residue, Cys-118, present in the OSCP sequence reported by Ovchinnikov et al. [Ovchinnikov, Y. A., Modyanov, N. N., Grinkevich, V. A., Aldanova, N. A., Trubetskaya, O. E., Nazimov, I. V., Hundal, T., & Ernster, L. (1984) FEBS Lett. 166, 19-22]; it did not alter the biological activity of OSCP tested in a reconstituted F0-F1 system that catalyzed oligomycin-sensitive ATPase activity or ATP-Pi exchange. The parameters of [14C]NEM-OSCP binding to isolated beef heart mitochondrial F1 were assessed by equilibrium dialysis. Addition of trace amounts of Tween 20 prevented unspecific adsorption of OSCP. The binding curves showed that each F1 possesses a high-affinity OSCP binding site (Kd = 0.08 microM) and two low-affinity OSCP binding sites (Kd = 6-8 microM). Binding of OSCP to the high-affinity site on F1 is probably responsible for the ability of OSCP to confer oligomycin sensitivity to F1 in the ATPase complex.     

Mapping of nucleotide-depleted mitochondrial F1-ATPase with 2-azido-[alpha-32P]adenosine diphosphate. Evidence for two nucleotide binding sites in the beta subunit

Photolabeling of nucleotide binding sites in nucleotide-depleted mitochondrial F1 has been explored with 2-azido [alpha-32P]adenosine diphosphate (2-N3[alpha-32P] ADP). Control experiments carried out in the absence of photoirradiation in a Mg2+-supplemented medium indicated the presence of one high affinity binding site and five lower affinity binding sites per F1. Similar titration curves were obtained with [3H]ADP and the photoprobe 3'-arylazido-[3H]butyryl ADP [( 3H]NAP4-ADP). Photolabeling of nucleotide-depleted F1 with 2-N3[alpha-32P]ADP resulted in ATPase inactivation, half inactivation corresponding to 0.6-0.7 mol of photoprobe covalently bound per mol F1. Only the beta subunit was photolabeled, even under conditions of high loading with 2-N3[alpha-32P]ADP. The identification of the sequences labeled with the photoprobe was achieved by chemical cleavage with cyanogen bromide and enzymatic cleavage by trypsin. Under conditions of low loading with 2-N3[alpha-32P]ADP, resulting in photolabeling of only one vacant site in F1, covalently bound radioactivity was located in a peptide fragment of the beta subunit spanning Pro-320-Met-358 identical to the fragment photolabeled in native F1 (Garin, J., Boulay, F., Issartel, J.-P., Lunardi, J., and Vignais, P. V. (1986) Biochemistry 25, 4431-4437). With a heavier load of photoprobe, leading to nearly 4 mol of photoprobe covalently bound per mol F1, an additional region of the beta subunit was specifically labeled, corresponding to a sequence extending from Gly-72 to Arg-83. The isolated beta subunit also displayed two binding sites for 2-N3-[alpha-32P]ADP. When F1 was first photolabeled with a low concentration of NAP4-ADP, leading to the covalent binding of 1.5 mol of NAP4-ADP/mol F1, with the bound NAP4-ADP distributed equally between the alpha and beta subunits, a subsequent photoirradiation in the presence of 2-N3[alpha-32P]ADP resulted in covalent binding of the 2-N3[alpha-32P]ADP to both alpha and beta subunits. It is concluded that each beta subunit in mitochondrial F1 contains two nucleotide binding regions, one of which belongs to the beta subunit per se, and the other to a subsite shared with a subsite located on a juxtaposed alpha subunit. Depending on the experimental conditions, the subsite located on the alpha subunit is either accessible or masked. Unmasking of the subsite in the three alpha subunits of mitochondrial F1 appears to proceed by a concerted mechanism.     

Photolabeling of mitochondrial F1-ATPase by an azido derivative of the oligomycin-sensitivity conferring protein

An azido derivative of the oligomycin sensitivity conferring protein (OSCP) was prepared by alkylation with the bifunctional reagent p-azido phenacyl bromide. Azido-OSCP was fully biologically active in the dark. Upon photoirradiation of a mixture of beef heart mitochondrial F1-ATPase and azido-OSCP, the resulting covalent photoproducts were separated by polyacrylamide gel electrophoresis in the presence of Na dodecyl sulfate and characterized by an immunochemical procedure. OSCP was found to react with the alpha and the beta subunits of F1 with strong preference for the alpha subunit.     

Structure of beef heart mitochondrial F1-ATPase. Arrangement of subunits as disclosed by cross-linking reagents and selective labeling by radioactive ligands.

1. The following bifunctional reagents, dimethylsuberimidiate, dimethyladipimidate, methylmercaptobutyrimidate have been used to produce dimers between the neighboring subunits of beef heart F1-ATPase. 2. Treatment of beef heart F1-ATPase with dimethylsuberimidate or dimethyladipimidate resulted in the formation of four cross-linked products. Their molecular weights determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 11 500, 105 000, 95 000 and 80 000, respectively. The products of molecular weight 115 000 and 105 000 were predominant and could be detected at the early stage of the cross-linking reaction. Treatment of beef heart F1-ATPase with methylmercaptobutyrimidate resulted in the accumulation of the product of molecular weight 115 000 and in traces of products of lower molecular weight. When the cross-linked products obtained with methylmercaptobutyrimidate were cleaved by beta-mercaptoethanol, the original gel electrophoresis pattern was restored. 3. Cross-linking of beef heart F1-ATPase by dimethylsuberimidate, dimethyladipimidate and methylmercaptobutyrimidate was accompanied by a loss of the ATPase activity. Cleavage of the cross-linked products obtained with methylmercaptobutyrimidate did not restore the original ATPase activity. 4. Identification of subunits A and B in the products of molecular weight 115 000 and 105 000 was achieved by specific labeling of subunit A with N-[14C]ethylmaleimide and of subunit B by chloronitro [14C]benzooxodiazole. Both products were able to bind N-[14C]ethylmaleimide; only the 105 000 dalton product was able to bind chloronitro [14C]benzooxodiazole. 5. The product of molecular weight 115 000 obtained by treatment of beef heart ATPase with methylmercaptobutyrimidate could bind N-[14C]ethylmaleimide. Its cleavage, following N-[14C]ethylmaleimide binding, yielded one labeled peptide identified with subunit A by polyacrylamide gel electrophoresis. 6. The above results indicate that the product of molecular weight 115 000 is a dimer containing two subunits A and that the product of molecular weight 105 000 is a dimer containing one subunit A and one subunit B. It can therefore be concluded that, in beef heart F1-ATPase, the A subunits are close to each other and that subunit A is close to subunit B. In contrast the B sublnits are probably too far from each other to be cross-linked by dimethylsuberimidate, dimethyladipimidate or methylmercaptobutyrimidate.     

Mutations within the C-terminus of the gamma subunit of the photosynthetic F1-ATPase activate MgATP hydrolysis and attenuate the stimulatory oxyanion effect

Two highly conserved amino acid residues near the C-terminus within the gamma subunit of the mitochondrial ATP synthase form a "catch" with an anionic loop on one of the three beta subunits within the catalytic alphabeta hexamer of the F1 segment [Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628]. Forming the catch is considered to be an essential step in cooperative nucleotide binding leading to gamma subunit rotation. The analogous residues, Arg304 and Gln305, in the chloroplast F1 gamma subunit were changed to leucine and alanine, respectively. Each mutant gamma was assembled together with alpha and beta subunits from Rhodospirillum rubrum F1 into a hybrid photosynthetic F1 that carries out both MgATPase and CaATPase activities and ATP-dependent gamma rotation [Tucker, W. C., Schwarcz, A., Levine, T., Du, Z., Gromet-Elhanan, Z., Richter, M. L. and Haran, G. (2004) J. Biol. Chem. 279, 47415-47418]. Surprisingly, changing Arg304 to leucine resulted in a more than 2-fold increase in the kcat for MgATP hydrolysis. In contrast, changing Gln305 to alanine had little effect on the kcat but completely abolished the well-known stimulatory effect of the oxyanion sulfite on MgATP hydrolysis. The MgATPase activities of combined mutants with both residues substituted were strongly inhibited, whereas the CaATPase activities were inhibited, but to a lesser extent. The results indicate that the C-terminus of the photosynthetic F1 gamma subunit, like its mitochondrial counterpart, forms a catch with the alpha and beta subunits that modulates the nucleotide binding properties of the catalytic site(s). The catch is likely to be part of an activation mechanism, overcoming inhibition by free mg2+ ions, but is not essential for cooperative nucleotide exchange.     

Chemical modification of F1-ATPase by dicyclohexylcarbodiimide: application to analysis of the stoichiometry of subunits in Escherichia coli F1

N,N'-Dicyclohexylcarbodiimide (DCCD) covalently binds to the beta subunit of Escherichia coli F1-ATPase (BF1). The ATPase activity is fully inhibited when 1 mol of DCCD is bound/mol of BF1, in spite of the fact that BF1 contains several beta subunits [Satre, M., Lunardi, J., Pougeois, R., & Vignais, P.V. (1979) Biochemistry 18, 3134-3140]. Advantage was taken of the reactivity of DCCD with respect to BF1 to determine the exact stoichiometry of the beta subunits in BF1. Two methods were used. The first one was based on the fact that modification of the beta subunit by DCCD results in the disappearance of one negative charge, due to the binding of DCCD to a carboxyl group of the beta subunit. The nonmodified and the modified beta subunits were separated by electrofocusing, and the percentage of modified beta subunits was assessed as a function of the percentage of ATPase inactivation. The second method relied on direct comparison, after inactivation of BF1 by [14C]DCCD, of the specific radioactivities of the whole BF1 and the isolated beta subunits. Both methods indicate that each molecule of BF1 contains three beta subunits.     

F1 and F0 connections in the bovine mitochondrial ATP synthase: the role of the of alpha subunit N-terminus, oligomycin-sensitivity conferring protein (OCSP) and subunit d.

We have studied the functional effect of limited proteolysis by trypsin of the constituent subunits in the native and reconstituted F1F0 complex and isolated F1 of the bovine heart mitochondrial ATP synthase (EC 3.6.1.34). Chemical cross-linking of oligomycin-sensitivity conferring protein (OSCP) with other subunits of the ATP synthase and the consequent functional effects were also investigated. The results obtained show that the alpha subunit N-terminus is essential for the correct, functional connection of F1 to F0. The alpha-subunit N-terminus contacts OSCP which, in turn, contacts the F0I-PVP(b) and the F0-d subunits. The N-terminus of subunit alpha, OSCP, a segment of subunit d and the C-terminal and central region of F0I-PVP(b) subunits are peripherally located with respect to subunits gamma and delta which are completely shielded in the F1F0 complex against trypsin digestion. This qualifies the N-terminus of subunit alpha, OSCP, subunit d and F0I-PVP(b) as components of the lateral element of the stalk. These subunits, rather than being confined at one side of the complex which would leave most of the central part of the gamma subunit uncovered, surround the gamma and the delta subunits located in the central stalk.     

ATP synthase complex from bovine heart mitochondria. Subunit arrangement as revealed by nearest neighbor analysis and susceptibility to trypsin

The nearest neighbor relationships of bovine mitochondrial H(+)-ATPase subunits were investigated by the chemical cross-linking approach using the homobifunctional cleavable reagents dithiobis(succinimidyl propionate) and disuccinimidyl tartrate. Cross-linked proteins were resolved by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Individual subunits were detected by silver staining or by Western blotting and staining with subunit-specific antisera. Products larger than 80,000 daltons were not analyzed. Interactions between F1 subunits included cross-links between gamma and delta as well as gamma and epsilon subunits. Among F0 subunit interactions were observed cross-links of (i) coupling factor 6 (F6) with 8-, 20-, and 24-kDa proteins, (ii) oligomycin sensitivity-conferring protein (OSCP) with 24-kDa protein, and (iii) 20-kDa protein with 24-kDa protein. In addition, several cross-links among subunits involving F1 and F0 sectors were detected. These included cross-links between F6 and alpha, F6 and gamma, OSCP and alpha/beta, and 24-kDa protein and alpha/beta. Thus, OSCP, F6, and the 24-kDa protein were found to form cross-links with both F1 and F0 subunits. The surface accessibility of F0 subunits was investigated by subjecting aliquots of F0 to trypsin treatment. Our data demonstrated that the rate of degradation was in the order OSCP greater than 24-kDa protein greater than or equal to F6 greater than subunit 6. The degradation of subunits of F0 was prevented in intact or reconstituted F1-F0. Based on our present and previously published observations, a model of H(+)-ATPase has been proposed wherein OSCP, F6, and the 24-kDa protein are placed in the stalk region and the alpha and beta subunits of F1-ATPase have been extended down to the membrane surface to enclose the stalk segment.     

Structural and functional studies of cross-linked Go protein subunits

The guanine nucleotide binding proteins (G proteins) that couple hormone and other receptors to a variety of intracellular effector enzymes and ion channels are heterotrimers of alpha, beta, and gamma subunits. One way to study the interfaces between subunits is to analyze the consequences of chemically cross-linking them. We have used 1,6-bismaleimidohexane (BMH), a homobifunctional cross-linking reagent that reacts with sulfhydryl groups, to cross-link alpha to beta subunits of Go and Gi-1. Two cross-linked products are formed from each G protein with apparent molecular masses of 140 and 122 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both bands formed from Go reacted with anti-alpha o and anti-beta antibody. The mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is anomalous since the undenatured, cross-linked proteins have the same Stokes radius as the native, uncross-linked alpha beta gamma heterotrimer. Therefore, each cross-linked product contains one alpha and one beta subunit. Activation of Go by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) does not prevent cross-linking of alpha to beta gamma, consistent with an equilibrium between associated and dissociated subunits even in the presence of GTP gamma S. The same cross-linked products of Go are formed in brain membranes reacted with BMH as are formed in solution, indicating that the residues cross-linked by BMH in the pure protein are accessible when Go is membrane bound. Analysis of tryptic peptides formed from the cross-linked products indicates that the alpha subunit is cross-linked to the 26-kDa carboxyl-terminal portion of the beta subunit. The cross-linked G protein is functional, and its alpha subunit can change conformation upon binding GTP gamma S. GTP gamma S stabilizes alpha o to digestion by trypsin (Winslow, J.W., Van Amsterdam, J.R., and Neer, E.J. (1986) J. Biol. Chem. 261, 7571-7579) and also stabilizes the alpha subunit in the cross-linked product. Cross-linked G o can be ADP-ribosylated by pertussis toxin. This ADP-ribosylation is inhibited by GTP gamma S with a concentration dependence that is indistinguishable from that of the control, uncross-linked G o. These two kinds of experiments indicate that alpha o is able to change its conformation even though it cannot separate completely from beta gamma. Thus, although dissociation of the subunits accompanies activation of G o in solution, it is not obligatory for a conformational change to occur in the alpha subunit.     

Efficient reconstitution of mitochondrial energy-transfer reactions from depleted membranes and F1-ATPase as a function of the amount of bound oligomycin sensitivity-conferring protein (OSCP)

Pig heart mitochondrial membranes depleted of F1 and OSCP by various treatments were analyzed for their content in alpha and beta subunits of F1 and in OSCP using monoclonal antibodies. Membrane treatments and conditions of rebinding of F1 and OSCP were optimized to reconstitute efficient NADH- and ATP-dependent proton fluxes, ATP synthesis and oligomycin-sensitive ATPase activity. F1 and OSCP can be rebound independently to depleted membranes but to avoid unspecific binding of F1 to depleted membranes (ASUA) which is not efficient for ATP synthesis, F1 must be rebound before the addition of OSCP. The rebinding of OSCP to depleted membranes reconstituted with F1 inhibits the ATPase activity of rebound F1, while it restores the ATP-driven proton flux measured by the quenching of ACMA fluorescence. The rebinding of OSCP also renders the ATPase activity of bound F1 sensitive to uncouplers. The rebinding of OSCP alone or F1 alone, does not modify the NADH-dependent proton flux, while the rebinding of both F1 and OSCP controls this flux, inducing an inhibition of the rate of NADH oxidation. Similarly, oligomycin, which seals the F0 channel even in the absence of F1 and OSCP, inhibits the rate of NADH oxidation. OSCP is required to adjust the fitting of F1 to F0 for a correct channelling of protons efficient for ATP synthesis. All reconstituted energy-transfer reactions reach their optimal value for the same amount of OSCP. This amount is consistent with a stoichiometry of two OSCP per F1 in the F0-F1 complex.     

Role of the ATP synthase alpha-subunit in conferring sensitivity to tentoxin.

Tentoxin, produced by phytopathogenic fungi, selectively affects the function of the ATP synthase enzymes of certain sensitive plant species. Binding of tentoxin to a high affinity (K(i) approximately 10 nM) site on the chloroplast F(1) (CF(1)) strongly inhibits catalytic function, whereas binding to a second, lower affinity site (K(d) > 10 microM) leads to restoration and even stimulation of catalytic activity. Sensitivity to tentoxin has been shown to be due, in part, to the nature of the amino acid residue at position 83 on the catalytic beta subunit of CF(1). An aspartate in this position is required, but is not sufficient, for tentoxin inhibition. By comparison with the solved structure of mitochondrial F(1) [Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628], Asp83 is probably located at an interface between alpha and beta subunits on CF(1) where residues on the alpha subunit could also participate in tentoxin binding. A hybrid core F(1) enzyme assembled with beta and gamma subunits of the tentoxin-sensitive spinach CF(1), and an alpha subunit of the tentoxin-insensitive photosynthetic bacterium Rhodospirillum rubrum F(1) (RrF(1)), was stimulated but not inhibited by tentoxin [Tucker, W. C., Du, Z., Gromet-Elhanan, Z. and Richter, M. L. (2001) Eur. J. Biochem. 268, 2179-2186]. In this study, chimeric alpha subunits were prepared by introducing short segments of the spinach CF(1) alpha subunit from a poorly conserved region which is immediately adjacent to beta-Asp83 in the crystal structure, into equivalent positions in the RrF(1) alpha subunit using oligonucleotide-directed mutagenesis. Hybrid enzymes containing these chimeric alpha subunits had both the high affinity inhibitory tentoxin binding site and the lower affinity stimulatory site. Changing beta-Asp83 to leucine resulted in loss of both inhibition and stimulation by tentoxin in the chimeras. The results indicate that tentoxin inhibition requires additional alpha residues that are not present on the RrF(1) alpha subunit. A structural model of a putative inhibitory tentoxin binding pocket is presented.     

Topography of oligomycin sensitivity conferring protein in the mitochondrial adenosinetriphosphatase-ATP synthase

The topographical organization of oligomycin sensitivity conferring protein (OSCP) in the mitochondrial adenosinetriphosphatase (ATPase)-ATP synthase complex has been studied. The accessibility of OSCP to monoclonal antibodies has been qualitatively visualized by using the protein A-gold electron microscopy immunocytochemistry or quantitatively estimated by immunotitration of OSCP in depolymerized or intact membranes. Besides, OSCP cannot be labeled by 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID) which selectively labels the hydrophobic core of membrane proteins. These observations demonstrate an external location of OSCP on the inner face of the inner mitochondrial membrane. The position of OSCP relative to other peptides of the complex has been analyzed by cross-linking experiments using either zero length N-(ethoxycarbonyl)-2-ethoxydihydroquinoline or 11-A span dimethyl suberimidate cross-linkers in the ATPase-ATP synthase complex. The OSCP cross-linked products were identified either by immunocharacterization with anti-alpha, anti-beta, or anti-OSCP monoclonal antibodies or by their molecular weight. OSCP was cross-linked with either the alpha- or beta-subunits of F1 or to a subunit of Mr 24 000. Other types of cross-linking were obtained by the labeling of OSCP with [cysteamine-35S]-N-succinimidyl 3-[[2-((2-nitro-4-azidophenyl)amino)ethyl]dithio]propionate ([35S]SNAP) and reconstitution of SNAP-OSCP with F1 in urea-treated submitochondrial particles. Under these conditions, OSCP is found to be adjacent to two other peptides of molecular weight close to 30 000. A comparison is made between the topology and the organization of the b-subunit of Escherichia coli and OSCP, suggesting an analogy between OSCP and the hydrophilic part of the b-subunit.     

Structural characterization of the interaction of the delta and alpha subunits of the Escherichia coli F1F0-ATP synthase by NMR spectroscopy

A critical point of interaction between F(1) and F(0) in the bacterial F(1)F(0)-ATP synthase is formed by the alpha and delta subunits. Previous work has shown that the N-terminal domain (residues 3-105) of the delta subunit forms a 6 alpha-helix bundle [Wilkens, S., Dunn, S. D., Chandler, J., Dahlquist, F. W., and Capaldi, R. A. (1997) Nat. Struct. Biol. 4, 198-201] and that the majority of the binding energy between delta and F(1) is provided by the interaction between the N-terminal 22 residues of the alpha- and N-terminal domain of the delta subunit [Weber, J., Muharemagic, A., Wilke-Mounts, S., and Senior, A. E. (2003) J. Biol. Chem. 278, 13623-13626]. We have now analyzed a 1:1 complex of the delta-subunit N-terminal domain and a peptide comprising the N-terminal 22 residues of the alpha subunit by heteronuclear protein NMR spectroscopy. A comparison of the chemical-shift values of delta-subunit residues with and without alpha N-terminal peptide bound indicates that the binding interface on the N-terminal domain of the delta subunit is formed by alpha helices I and V. NOE cross-peak patterns in 2D (12)C/(12)C-filtered NOESY spectra of the (13)C-labeled delta-subunit N-terminal domain in complex with unlabeled peptide verify that residues 8-18 in the alpha-subunit N-terminal peptide are folded as an alpha helix when bound to delta N-terminal domain. On the basis of intermolecular contacts observed in (12)C/(13)C-filtered NOESY experiments, we describe structural details of the interaction of the delta-subunit N-terminal domain with the alpha-subunit N-terminal alpha helix.     

Demonstration of two exchangeable non-catalytic and two cooperative catalytic sites in isolated bovine heart mitochondrial F1, using the photoaffinity labels [2-3H]8-azido-ATP and [2-3H]8-azido-ADP

The photoreactive nucleotides [2-3H]8-azido-ATP and [2-3H]8-azido-ADP could be used to label the nucleotide binding sites on isolated mitochondrial F1-ATPase to a maximum of 4 mol of nucleotide per mol F1, also when the F1 was depleted of tightly bound nucleotides. At a photolabel concentration of 300-1000 microM, label was found on both alpha and beta subunits in a typically 1:3 ratio, independent of the total amount bound. Under these conditions the covalent binding of two nucleotides is needed for full inactivation (Wagenvoord, R.J., Van der Kraan, I. and Kemp, A. (1977) Biochim. Biophys. Acta 460, 17-24). At lower concentrations of [2-3H]8-azido-ATP (20 microM), it was found that covalent binding of only 1 mol of nucleotide per mole F1 was required for complete inactivation to take place indicating catalytic site cooperativity in the mechanism of ATP hydrolysis. Under those conditions, radioactivity was only found on the beta subunits, which would indicate that the catalytic site is located on a beta subunit and that a second site is located on the alpha/beta interface. It is found that four out of the six nucleotide binding sites are exchangeable and can be labelled with 8-azido-AT(D)P, i.e., two catalytic sites and two non-catalytic sites.     

ATP synthase complex from bovine heart mitochondria. Passive H+ conduction through F0 does not require oligomycin sensitivity-conferring protein

Oligomycin sensitivity-conferring protein (OSCP) is a water-soluble subunit of bovine heart mitochondrial H(+)-ATPase (F1-F0). In order to investigate the requirement of OSCP for passive proton conductance through mitochondrial F0, OSCP-depleted membrane preparations were obtained by extracting purified F1-F0 complexes with 4.0 M urea. The residual complexes, referred to as UF0, were found to be deficient with respect to OSCP, as well as alpha, beta, and gamma subunits of F1-ATPase, but had a full complement of coupling factor 6 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting techniques. These UF0 complexes had no intrinsic ATPase activity and were able to bind nearly the same amount of F1-ATPase in the presence of either OSCP or NH4+ ions alone, or a combination of the two. However, the preparations exhibited an absolute dependency on OSCP for conferral of oligomycin sensitivity to membrane-bound ATPase. The passive proton conductance in UF0 proteoliposomes was measured by time-resolved quenching of 9-amino-6-chloro-2-methoxyacridine or 9-aminoacridine fluorescence following a valinomycin-induced K(+)-diffusion potential. The data clearly establish that OSCP is not a necessary component of the F0 proton channel nor is its presence required for conductance blockage by the inhibitors oligomycin or dicyclohexylcarbodiimide. Furthermore, OSCP does not prevent or block passive H+ leakage. Comparisons of OSCP with the F1-F0 subunits from Escherichia coli and chloroplast lead us to suggest that mitochondrial OSCP is, both structurally and functionally, a hybrid between the beta and delta subunits of the prokaryotic systems.     

ATP synthase complex from beef heart mitochondria. Role of the thiol group of the 25-kDa subunit of Fo in the coupling mechanism between Fo and F1

In order to assess the role of thiol groups in the Fo part of the ATP synthase in the coupling mechanism of ATP synthase, we have treated isolated Fo, extracted from beef heart Complex V with urea, with thiol reagents, primarily with diazenedicarboxylic acid bis-(dimethylamide) (diamide) but also with Cd2+ and N-ethylmaleimide. FoF1 ATP synthase was reconstituted by adding isolated F1 and the oligomycin-sensitivity-conferring-protein (OSCP) to Fo. The efficiency of reconstitution was assessed by determining the sensitivity to oligomycin of the ATP hydrolytic activity of the reconstituted enzyme. Contrary to Cd2+, incubation of diamide with Fo, before the addition of F1 and OSCP, induced a severe loss of oligomycin sensitivity, due to an inhibited binding of F1 to Fo. This effect was reversed by dithiothreitol. Conversely, if F1 and OSCP were added to Fo before diamide, no effect could be detected. These results show that F1 (and/or OSCP) protects Fo thiols from diamide and are substantiated by the finding that the oligomycin sensitivity of ATP hydrolysis activity of isolated Complex V was also unaltered by diamide. Gel electrophoresis of FoF1 ATP synthase, reconstituted with diamide-treated Fo, revealed that the loss of oligomycin sensitivity was directly correlated with diminution of band Fo 1 (or subunit b). Concomitantly a band appeared of approximately twice the molecular weight of subunit Fo 1. As this protein contains only 1 cysteine residue (Walker, J. E., Runswick, M. J., and Poulter, L. (1987) J. Mol. Biol. 197, 89-100), the effect of diamide is attributed to the formation of a disulfide bridge between two of these subunits. These results offer further evidence for the proposal, based on aminoacid sequence and structural analysis, that subunit Fo 1 of mammalian Fo is involved in the binding with F1 (Walker et al. (1987]. N-Ethylmaleimide affects oligomycin sensitivity to a lesser extent than diamide, suggesting that the mode of action of these reagents (and the structural changes induced in Fo) is different.     

Comparative Mg(2+)-dependent sequential covalent binding stoichiometries of 3'-O-(4-benzoyl)benzoyl adenosine 5'-diphosphate of MF1, TF1, and the alpha 3 beta 3 core complex of TF1. The binding change motif is independent of the F1 gamma delta epsilon subunits.

Three F1 preparations, the beef heart (MF1) and thermophilic bacterium (TF1) holoenzymes, and the alpha 3 beta 3 "core" complex of TF1 reconstituted from individually expressed alpha and beta subunits, were compared as to their kinetic and binding stoichiometric responses to covalent photoaffinity labeling with BzATP and BzADP (+/- Mg2+). Each enzyme displayed an enhanced pseudo-first order rate of photoinhibition and one-third of the sites covalent binding to a catalytic site for full inhibition, plus, but not minus Mg2+. Titration of near stoichiometric [MgBzADP]/[F1] ratios during photolysis disclosed two sequential covalent binding patterns for each enzyme; a high affinity binding corresponding to unistoichiometric covalent association concomitant with enzyme inhibition, followed by a low affinity multisite-saturating covalent association. Thus, in the absence of the structural asymmetry inducing gamma delta epsilon subunits of the holoenzyme, the sequential binding of nucleotide at putative catalytic sites on the alpha 3 beta 3 complex of any F1 appears sufficient to effect binding affinity changes. With MF1, final covalent saturation of BzADP-accessible sites was achieved with 2 mol of BzADP/mol of enzyme, but with TF1 or its alpha 3 beta 3 complex, saturation required 3 mol of BzADP/mol of enzyme. Such differential final labeling stoichiometries could arise because of the endogenous presence of 1 nucleotide already bound to one of the 3 potential catalytic sites on normally prepared MF1, whereas TF1, possessing no endogenous nucleotide, has 3 vacant BzADP-accessible sites. Kinetics measurements revealed that regardless of the incremental extent of inhibition of the TF1 holoenzyme by BzADP during photolysis, the two higher apparent Km values (approximately 1.5 x 10(-4) and approximately 10(-3) M, respectively) of the progressively inactivated incubation are unchanged relative to fully unmodified enzyme. As reported for BzATP (or BzADP) and MF1 (Ackerman, S.H., Grubmeyer, C., and Coleman, P.S. (1987) J. Biol. Chem. 262, 13765-13772), this supports the fact that the photocovalent inhibition of F1 is a one-hit one-kill phenomenon. Isoelectric focusing gels revealed that [3H]BzADP covalently modifies both TF1 and MF1 exclusively on the beta subunit, whether or not Mg2+ is present. A single 19-residue [3H]BzADP-labeled peptide was resolved from a tryptic digest of MF1, and this peptide corresponded with the one believed to contain at least a portion of the beta subunit catalytic site domain (i.e. beta Ala-338----beta Arg-356).     

Cross-linking study of the quaternary fine structure of mitochondrial F1-ATPase

A method has been developed for exploring the quaternary fine structure of oligomeric proteins by crosslinking studies and applied to bovine heart mitochondrial F1-ATPase. The F1 was first labeled with 1-fluoro-2,4-dinitro-[14C]benzene, subsequently reduced with sodium hydrosulfite, and finally cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Gel electrophoresis in the chemically modified protein in the presence of sodium dodecyl sulfate and mercaptoethanol showed the existence of a 105-115-kilodalton molecular species in addition to the five monomeric subunits of F1. This cross-linked species could be alpha 2, alpha beta, or beta 2. Isolation of the cross-linked species and titration with 5,5'-dithiobis-(2-nitrobenzoic acid) showed the absence of sulfhydryl group. Therefore, the cross-linked species must be the dimer beta 2. After digestion of the purified beta 2 with pepsin, a single radioactive peptide was isolated. Determination of the amino acid sequence of this peptide and comparison of its radioactivity with the total radioactivity on beta-subunits show that it was formed exclusively by cross-linking Lys162 of one beta-subunit with Glu199 of another beta-subunit. The observation that two beta-subunits can be cross-linked by a rigid phenylenediamine bridge of 5.7- or 4.3-A length is difficult to reconcile with the widely assumed structure of F1 with the alpha- and beta-subunits occupying alternate corners of a planar hexagon, but is consistent with the structure in which a triangular set of three beta-subunits sits above a triangular set of three alpha-subunits in a staggered conformation.