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Oxidation of the α3(βD311C/R333C)3γ Subcomplex of the Thermophilic Bacillus PS3 F1-ATPase Indicates That Only Two β Subunits Can Exist in the Closed Conformation Simultaneously

1999; Elsevier BV; Volume: 274; Issue: 44 Linguagem: Inglês

10.1074/jbc.274.44.31366

1083-351X

Huimiao Ren, Chao Dou, Matthew S. Stelzer, William S. Allison,

Advanced NMR Techniques and Applications

In the crystal structure of the bovine heart mitochondrial F1-ATPase (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994)Nature 370, 621–628), the two liganded β subunits, one with MgAMP-PNP bound to the catalytic site (βT) and the other with MgADP bound (βD) have closed conformations. The empty β subunit (βE) has an open conformation. In βT and βD, the distance between the carboxylate of β-Asp315 and the guanidinium of β-Arg337 is 3.0–4.0 Å. These side chains are at least 10 Å apart in βE. The α3(βD311C/R333C)3γ subcomplex of TF1 with the corresponding residues substituted with cysteine has very low ATPase activity unless it is reduced prior to assay or assayed in the presence of dithiothreitol. The reduced subcomplex hydrolyzes ATP at 50% the rate of wild-type and is rapidly inactivated by oxidation by CuCl2 with or without magnesium nucleotides bound to catalytic sites. Titration of the subcomplex with iodo[14C]acetamide after prolonged treatment with CuCl2 in the presence or absence of 1 mm MgADP revealed nearly two free sulfhydryl groups/mol of enzyme. Therefore, one pair of introduced cysteines is located on a β subunit that exists in the open or partially open conformation even when catalytic sites are saturated with MgADP. Since V max of ATP hydrolysis is attained when three catalytic sites of F1are saturated, the catalytic site that binds ATP must be closing as the catalytic site that releases products is opening. In the crystal structure of the bovine heart mitochondrial F1-ATPase (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994)Nature 370, 621–628), the two liganded β subunits, one with MgAMP-PNP bound to the catalytic site (βT) and the other with MgADP bound (βD) have closed conformations. The empty β subunit (βE) has an open conformation. In βT and βD, the distance between the carboxylate of β-Asp315 and the guanidinium of β-Arg337 is 3.0–4.0 Å. These side chains are at least 10 Å apart in βE. The α3(βD311C/R333C)3γ subcomplex of TF1 with the corresponding residues substituted with cysteine has very low ATPase activity unless it is reduced prior to assay or assayed in the presence of dithiothreitol. The reduced subcomplex hydrolyzes ATP at 50% the rate of wild-type and is rapidly inactivated by oxidation by CuCl2 with or without magnesium nucleotides bound to catalytic sites. Titration of the subcomplex with iodo[14C]acetamide after prolonged treatment with CuCl2 in the presence or absence of 1 mm MgADP revealed nearly two free sulfhydryl groups/mol of enzyme. Therefore, one pair of introduced cysteines is located on a β subunit that exists in the open or partially open conformation even when catalytic sites are saturated with MgADP. Since V max of ATP hydrolysis is attained when three catalytic sites of F1are saturated, the catalytic site that binds ATP must be closing as the catalytic site that releases products is opening. BH-MF1, and RL-MF1, the F1-ATPases from the thermophilic Bacillus PS3, bovine heart mitochondria, and rat liver mitochondria, respectively dithiothreitol trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid adenosine 5′-(β,γ-imino)triphosphate The proton-translocating F0F1-ATP synthases couple proton electrochemical gradients to condensation of ADP with Pi in energy-transducing membranes. The F0 moiety is a membrane-embedded protein complex that mediates proton translocation. F1 is a peripheral membrane protein complex composed of five different subunits with α3β3γδε stoichiometry. When removed from the membrane, F1 is an ATPase containing six nucleotide binding sites. Three are catalytic sites that are predominantly on β subunits at α/β interfaces. The three other sites, called noncatalytic sites, are located predominantly on α subunits at different α/β interfaces (1Weber J. Senior A.E. Biochim. Biophys. Acta. 1997; 1319: 19-58Crossref PubMed Scopus (396) Google Scholar, 2Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1606) Google Scholar, 3Allison W.S. Acc. Chem. Res. 1998; 31: 819-826Crossref Scopus (48) Google Scholar). The crystal structures of the bovine heart and rat liver mitochondrial F1-ATPases as well as that of the α3β3 subcomplex of the TF1-ATPase have been determined (4Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2764) Google Scholar, 5Shirakihara Y. Leslie A.G.W. Abrahams J.P. Walker J.E. Ueda T. Sekimoto Y. Kambara M. Saika K. Kagawa Y. Yoshida M. Structure. 1997; 5: 825-836Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 6Bianchet M.A. Hullihen J. Pedersen P.L. Amzel L.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11065-11070Crossref PubMed Scopus (224) Google Scholar). In the crystals of the bovine heart enzyme (BH-MF1),1 which form in media containing AMP-PNP, ADP, Mg2+, and N3−, noncatalytic sites are homogeneously liganded with MgAMP-PNP, whereas catalytic sites are heterogeneously liganded (4Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2764) Google Scholar). One, designated βT, contains MgAMP-PMP, another, designated βD, contains MgADP and the third catalytic site, designated βE, is empty. Except for small differences in the regions of the terminal phosphates of bound nucleotides, the arrangements of functional amino acid side chains in catalytic sites in βT and βD are essentially identical. In contrast, these residues are arranged differently in βE. In the crystals of the α3β3 subcomplex of TF1 that form in media free of Mg2+ and nucleotides, the α subunits are in closed conformations corresponding to the liganded α subunits of the mitochondrial enzymes. The β subunits in the α3β3 structure are in open conformations corresponding to βE of BH-MF1 (5Shirakihara Y. Leslie A.G.W. Abrahams J.P. Walker J.E. Ueda T. Sekimoto Y. Kambara M. Saika K. Kagawa Y. Yoshida M. Structure. 1997; 5: 825-836Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). In the crystals of the rat liver F1-ATPase (RL-MF1), which form in media containing ATP, Pi, and EDTA, noncatalytic sites are homogeneously occupied with MgATP. In contrast to the crystals of BH-MF1, all three catalytic sites of RL-MF1 contain bound nucleotides in the absence of Mg2+ (6Bianchet M.A. Hullihen J. Pedersen P.L. Amzel L.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11065-11070Crossref PubMed Scopus (224) Google Scholar). One catalytic site contains ADP alone, whereas the other two contain ADP and Pi. Since Mg2+ is not associated with ADP and Pi bound to catalytic sites of RL-MF1, side chains in catalytic sites that interact with the Mg2+ ion in the crystal structure of BH-MF1 are arranged differently in the crystal structure of RL-MF1. However, these differences are minor compared with the difference between the closed conformations of βD and βT and the open conformation of βE in BH-MF1. In the crystal structure of RL-MF1, the conformations of all three β subunits resemble the closed conformation of βD and βT in the crystal structure of BH-MF1. Bianchet et al. (6Bianchet M.A. Hullihen J. Pedersen P.L. Amzel L.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11065-11070Crossref PubMed Scopus (224) Google Scholar) suggest that the open conformation of βE in the crystal structure of BH-MF1 is the consequence of low concentrations of nucleotides in the crystallization medium. Extending this argument, they propose that the RL-MF1 structure with three closed β subunits plays a central role in catalysis, whereas the BH-MF1 structure with two closed β subunits and one open β subunit exists transiently during catalysis when products dissociate and substrates rebind. To determine which of these structures is more consistent with properties of F1-ATPases in solution, we took advantage of pairs of amino acid residues in β subunits that do not contribute to catalytic sites in BH-MF1 that have side chains within interaction distance in βD and βT but are considerably distant from each other in βE. For example, the carboxylate of β-Asp315 is within 3.0 and 3.7 Å of the guanidinium of β-Arg337, in βT and βD, respectively, whereas these side chains are 10.3 Å apart in βE. Other pairs of amino acid residues that meet these criteria are β-Ala158/β-Tyr311; β-Met167/β-Phe183, and β-Lys175/β-Ser203. Double mutants of the α3β3γ subcomplex of TF1 (7Matsui T. Yoshida M. Biochim. Biophys. Acta. 1995; 1231: 139-146Crossref PubMed Scopus (86) Google Scholar) were prepared in which residues corresponding to β-Asp315/β-Arg337, β-Ala158/β-Tyr311, β-Met167/β-Phe183, and β-Lys175/β-Ser203 of BH-MF1were substituted with cysteine. The aim of preliminary studies was to find a double mutant that was stable and also could be converted reversibly between an oxidized, inactive form and a reduced, active form. Of the four double mutants generated, only the α3(βD311C/R333C)3γ double mutant corresponding to the β-Asp315/β-Arg337 pair in BH-MF1 met these criteria. Biochemicals used in assays and buffer components were purchased from Sigma. Dithiothreitol, o-iodosobenzoic acid, iodoacetic acid, N-ethylmaleimide, iodoacetamide, DEAE-Sephacel, and Sephacryl S-300HR were also from Sigma. The radioactive reagents used were [3H]ADP from NEN Life Science Products; iodo[3H]acetate from American Radiolabeled Chemicals, and iodo[14C]acetamide from ICN. Aldrich supplied 5,5′-dithiobis-(2-nitrobenzoic acid) and sodium fluoride. Aluminum chloride was purchased from Fisher. Toronto Research Chemicals supplied [(1-trimethylammonium)methyl]methanethiosulfonate bromide. Toyopearl Butyl-650S resin was from TosoHaas. The oligonucleotides used for mutagenesis were purchased from Life Technology, Inc. TheEscherichia coli strains and the plasmids used to prepare the wild-type and mutant α3β3γ subcomplexes were described by Matsui and Yoshida (7Matsui T. Yoshida M. Biochim. Biophys. Acta. 1995; 1231: 139-146Crossref PubMed Scopus (86) Google Scholar). The purified enzyme subcomplexes were stored as suspensions in 75% ammonium sulfate at 4 °C. Plasmid pKK223-3, which carries genes encoding the α, β, and γ subunits of TF1, was used for both mutagenesis and gene expression. Expression plasmids were constructed using polymerase chain reaction with the Quick ChangeTM site-directed mutagenesis kit from Stratagene. The plasmids were purified using the WizardTM Plus miniprep kit from Promega. The first mutation was introduced into wild-type pKK223-3 by polymerase chain reaction and confirmed by sequence analysis. The resulting mutant plasmid was used as template for the second polymerase chain reaction substitution, which was subsequently confirmed. The resulting pKK223-3 mutant plasmids were expressed in E. coli strain JM103 (unc−). The primers used in the polymerase chain reactions are as follows with mismatched bases underlined: Y307C, 5′-CGATTCAAGCGATTTGCGTCCCGGCCG; Q177C, 5′-CACAACATCGCCTGTGAGCACGGCGG; S205C, 5′-GAGATGAAAGATTGCGGCGTCATCAGC; Q169C, 5′-GGTCTTGATCTGTGAGCTGATTCACAACAT; F185C, 5′-GGGATTTCCGTCTGTGCTGGCGTCGGC; D311C, 5′-CGTCCCGGCCTGCGACTATACGGACC; R333C, 5′-CGACGAACCTGGAGTGTAAGCTCGCGG and the corresponding complementary primers. The wild-type and mutant subcomplexes were expressed and purified as described previously (7Matsui T. Yoshida M. Biochim. Biophys. Acta. 1995; 1231: 139-146Crossref PubMed Scopus (86) Google Scholar,8Jault J.-M. Matsui T. Jault F.M. Kaibara C. Muneyuki E. Yoshida M. Kagawa Y. Allison W.S. Biochemistry. 1995; 34: 16412-16418Crossref PubMed Scopus (68) Google Scholar). Unless stated otherwise, stock solutions of the enzyme subcomplexes were prepared by the following procedure. After centrifugation of the ammonium sulfate suspensions, the pellets were dissolved in 50 mm Tris-Cl, pH 8.0, containing 1 mm CDTA. The solutions were incubated for 1 h at 23 °C, at which time they were passed through 1-ml centrifuge columns of Sephadex G-50 equilibrated with 50 mm Tris-Cl, pH 8.0, containing 0.1 mm EDTA (9Penefsky H.S. J. Biol. Chem. 1977; 252: 2891-2899Abstract Full Text PDF PubMed Google Scholar). Preparations treated in this manner are designated CDTA-treated subcomplexes. The α3(βD311C/R333C)3γ mutant subcomplex was reduced as follows. Ammonium sulfate precipitates of the subcomplex were collected by centrifugation and dissolved in 50 mmTris-HCl, pH 8.0, containing 5 mm CDTA and 10 mm DTT. After incubating for 2 h, the resulting solution was passed through a centrifuge column of Sephadex G-50 equilibrated with 50 mm Tris-HCl, pH 8.0, containing 0.1 mm EDTA. After reduction, ATPase activity did not decline for at least 2 h. When the reduced enzyme was subsequently treated with iodoacetate or iodoacetamide, the Sephadex G-50 centrifuge columns (9Penefsky H.S. J. Biol. Chem. 1977; 252: 2891-2899Abstract Full Text PDF PubMed Google Scholar) were equilibrated with 50 mm Tris-HCl, pH 8.0, containing 0.1 mm EDTA plus 0.2 mm DTT. ATPase activity was determined spectrophotometrically with 2 mm ATP plus 3 mm Mg2+ using ATP regeneration with phosphoenolpyruvate and pyruvate kinase coupled to NADH oxidation by lactate dehydrogenase under conditions described earlier (10Jault J.-M. Allison W.S. J. Biol. Chem. 1994; 269: 319-325Abstract Full Text PDF PubMed Google Scholar). Protein concentrations were determined by the method of Bradford (11Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). Endogenous nucleotides bound to the enzyme subcomplexes were determined by high pressure liquid chromatography using ion pairing with tetrabutyl ammonium hydrogen sulfate as described by Bullough et al. (12Bullough D.A. Brown E.L. Saario J.D. Allison W.S. J. Biol. Chem. 1988; 263: 14053-14060Abstract Full Text PDF PubMed Google Scholar). Table I shows the distances in the crystal structure of the α3β3 subcomplex of TF1 (5Shirakihara Y. Leslie A.G.W. Abrahams J.P. Walker J.E. Ueda T. Sekimoto Y. Kambara M. Saika K. Kagawa Y. Yoshida M. Structure. 1997; 5: 825-836Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar) between side chains of amino acid residues that were substituted with cysteine in the four double mutants examined in this study. The distances between the corresponding side chains in βT, βD, and βE in the crystal structure of MF1 (4Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2764) Google Scholar) are also tabulated.Table IDistances between side chains in the crystal structures of the α3 β3 subcomplex of TF1 (5Shirakihara Y. Leslie A.G.W. Abrahams J.P. Walker J.E. Ueda T. Sekimoto Y. Kambara M. Saika K. Kagawa Y. Yoshida M. Structure. 1997; 5: 825-836Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar) and between corresponding residues in the crystal structure of BH-MF1 (4Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2764) Google Scholar) that were substituted with cysteine in the α3 β3 γ subcomplex of TF1Adjacent residuesβTβDβEÅTF1β-Ala160/β-Tyr3077.9TF1β-Gln177/β-Ser20513.3TF1β-Gln169/β-Phe1858.0TF1β-Asp311/β-Arg33314.7MF1β-Ala158/β-Tyr3113.64.212.3MF1β-Lys175/β-Ser2034.83.811.8MF1β-Met167/β-Phe1834.84.810.6MF1β-Asp315/β-Arg3373.03.710.3 Open table in a new tab The effects of DTT on the ATPase activities of the purified double mutant subcomplexes were determined. Each isolated enzyme subcomplex was treated with CDTA before assays were performed. This procedure removes endogenous MgADP from a catalytic site of the purified, wild-type α3β3γ subcomplex (8Jault J.-M. Matsui T. Jault F.M. Kaibara C. Muneyuki E. Yoshida M. Kagawa Y. Allison W.S. Biochemistry. 1995; 34: 16412-16418Crossref PubMed Scopus (68) Google Scholar). The βA160C/Y307C double mutant was isolated in an inactive form that could not be activated by treatment with DTT before assay or by including DTT in the assay medium. Lauryl dimethylamine oxide, which stimulates ATPase activity of the wild-type and certain other mutant subcomplexes did not activate the α3(βA160C/Y307C)3γ subcomplex in the presence or absence of DTT. Defective assembly was not responsible for the lack of ATPase activity. A normal pattern of α, β, and γ subunits was observed in a 3:3:1 ratio after the mutant subcomplex was submitted to SDS-polyacrylamide gel electrophoresis. The α3(βQ177C/S205C)3γ double mutant had negligible ATPase activity in the absence of activation with dithiothreitol. When 10 mm DTT was included in the assay medium, its specific activity for hydrolyzing 2 mm ATP increased to 0.8 μmol of ATP mg−1 min−1. ATPase activity was stimulated about 10-fold when 0.03% lauryl dimethylamine oxide was present in the assay medium. Lauryl dimethylamine oxide stimulates the ATPase activity of the wild-type α3β3γ subcomplex by a factor of 3–4 (8Jault J.-M. Matsui T. Jault F.M. Kaibara C. Muneyuki E. Yoshida M. Kagawa Y. Allison W.S. Biochemistry. 1995; 34: 16412-16418Crossref PubMed Scopus (68) Google Scholar). The specific activity of the α3(βQ169C/F185C)3γ double mutant was 7 μmol of ATP hydrolyzed mg−1 min−1 in the absence of activation with DTT. Prior treatment with DTT or the inclusion of 10 mm DTT in the assay medium did not stimulate ATPase activity of this mutant. Surprisingly, including lauryl dimethylamine oxide in the assay medium in the concentration range of 0.01–0.06% lowered the ATPase activity to less than 0.07 μmol of ATP hydrolyzed mg−1 min−1. The ATPase activity of this double mutant was inactivated by 2 mm o-iodosobenzoate with a pseudo-first order rate constant of 1.4 × 10−2 min–1. It was inactivated much more slowly by 2 mm5,5′-dithiobis-(2-nitrobenzoic acid). The rate of inactivation of the α3(βQ169C/F185C)3γ subcomplex byo-iodosobenzoate or 5,5′-dithiobis-(2-nitrobenzoic acid) was attenuated by ADP or ATP with or without Mg2+ present. After oxidation with o-iodosobenzoate, the α3(βQ169C/F185C)3γ subcomplex could not be reactivated by treatment with 10 mm DTT. Fig. 1 A shows that the isolated α3(βD311C/R333C)3γ subcomplex has negligible ATPase activity unless the assay medium is supplemented with DTT or another thiol. Whereas traces a andc of Fig. 1 A were obtained in the absence of thiols, trace b shows the time-dependent activation of ATPase activity observed when the assay medium contained 10 mm DTT. Fig. 1 B shows that after reduction as described under “Experimental Procedures” the α3(βD311C/R333C)3γ subcomplex was inactivated during assay unless DTT or 0.1 mm EDTA was included in the assay medium. Trace a in Fig. 1 Billustrates assay of the enzyme in the absence of DTT or EDTA.Traces b and c of Fig. 1 B represent assays conducted in the presence of 10 mm DTT or 100 μm EDTA, respectively. The dependence of protection against oxidation on EDTA concentration revealed that maximal protection was achieved with 50 μm EDTA. Other experiments indicated that a contaminant in the MgCl2 was responsible for oxidation in the absence of EDTA. Therefore, 100 μm EDTA was included in the assay medium in experiments with the reduced α3(βD311C/R333C)3γ subcomplex. Since the presence of EDTA in the assay medium does not affect ATPase activity of the isolated enzyme, illustrated bytrace c of Fig. 1 A, the isolated α3(βD311C/R333C)3γ subcomplex exists in the oxidized form. Following reduction of the α3(βD311C/R333C)3γ subcomplex with 1 mm DTT for 30 min, the addition of iodoacetate to 4 mm led to 80% inactivation of ATPase activity within 10 min followed by slower inactivation, illustrated in Fig.2. The addition of increasing concentrations of ADP plus Mg2+ to the reduced subcomplex prior to adding iodoacetate slowed the rate of inactivation in both phases. Fig. 3 correlates the mol of [3H]acetate incorporated per mol of the α3(βD311C/R333C)3γ subcomplex with the extent of inactivation observed during inactivation of the reduced, mutant enzyme with iodo[3H]acetate. It is clear from Fig.3 that multiple hits are required to cause full inactivation. To reconcile this unusual stoichiometry, it is possible that modification of a single cysteine in a given β subunit wounds the enzyme, and modification of both Cys311 and Cys333 in the same β subunit wounds the enzyme to a greater extent. The introduced cysteines appear to be the only residues that were appreciably carboxymethylated. The wild-type subcomplex was not inactivated when treated with iodo[3H]acetate under the same conditions and incorporated no more than 0.3 mol of reagent/mol of enzyme in the absence of nucleotides and much less than that in the presence of MgADP. The wild-type subcomplex, like the mutant, contains α-Cys193 that is resistant to modification by iodoacetate.Figure 3Correlation of inactivation of α3(βD311C/R333C)3γ subcomplex by iodo[3H]acetate with the amount of reagent covalently incorporated. DTT was added to a final concentration of 1 mm to 500 μl of the CDTA-treated α3(βD311C/R333C)3γ subcomplex at 1 mg/ml in 50 mm Tris-Cl, 100 μm EDTA, pH 8.0. This solution was then incubated at 23 °C for 30 min, at which time [3H]iodoacetate (14 cpm/pmol) was added to a final concentration of 4 mm. Samples (5 μl each) of the reaction mixture were assayed for residual ATPase activity at the times indicated in the presence of 10 mm DTT using the ATP regeneration system. At the same intervals, 50-μl samples of the reaction mixture were withdrawn and passed through 1-ml centrifuge columns of Sephadex G50 (9Penefsky H.S. J. Biol. Chem. 1977; 252: 2891-2899Abstract Full Text PDF PubMed Google Scholar) that were equilibrated with 50 mm Tris-HCl, pH 8.0, containing 0.1 mm EDTA. The effluents were submitted to liquid scintillation counting to determine incorporation of [3H]acetate.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Complete carboxymethylation lowered the capacity of the α3(βD311C/R333C)3γ subcomplex to bind ADP in the presence of Mg2+. This was established as follows. After carboxymethylation of the nucleotide-depleted mutant subcomplex by treatment with 4 mm iodoacetate in the presence of 1 mm DTT for 16 h, the modified enzyme was passed through a 1-ml centrifuge column of Sephadex G-50 equilibrated with 50 mm Tris-Cl, pH 8.0, to remove excess iodoacetate and DTT. The gel-filtered subcomplex was then incubated with 200 μm [3H]ADP plus 2 mmMgCl2 for 30 min, at which time it was passed through two successive centrifuge columns of Sephadex G-50. Determination of radioactivity and protein concentration in the second effluent showed that the carboxymethylated α3(βD311C/R333C)3γ subcomplex retained 0.2 mol of [3H]ADP/mol. When treated under the same conditions, the wild-type subcomplex retained 0.9 mol of [3H]ADP/mol. Modification of the ATPase activity of the α3(βD311C/R333C)3γ subcomplex by sulfhydryl reagents other than iodoacetate was also explored. Treatment of the reduced subcomplex with N-ethylmaleimide or [(1-trimethylammonium)methyl]methanethiosulfonate bromide, a reagent that derivatizes cysteine residues with a quarternary ammonium cation, using the same protocol described for iodoacetate, caused slower inactivations that were partially protected by MgATP or MgADP. In contrast, carboxamidomethylation of the subcomplex with excess iodoacetamide in the presence of DTT converted the enzyme to a form that no longer required DTT or EDTA in the assay medium to retain constant activity. This is illustrated in Fig.1 C. Trace a of Fig. 1 C represents ATP hydrolysis by the reduced, mutant subcomplex in the absence of DTT or EDTA after treating it exhaustively with iodoacetamide. In the absence of carboxamidomethylation, the reduced enzyme is inactivated during assay in the absence of DTT or EDTA as illustrated by trace a of Fig. 1 B. The rate of conversion of the reduced mutant subcomplex to a more active form by iodoacetamide was not affected by MgADP. When the subcomplex was treated with 4 mm iodoacetamide in the presence of 1 mm DTT, 50% conversion was observed within 2 min. Maximal conversion occurred within 60 min. The addition of ADP with or without MgCl2 during treatment with iodoacetamide did not affect the rate of conversion. The reduced α3(βD311C/R333C)3γ subcomplex is rapidly inactivated in the presence of CuCl2, tetrathionate,o-iodosobenzoate, 5,5′-dithiobis-(2-nitrobenzoic acid), or H2O2. To determine the number of β subunits containing disulfide bonds on conversion of the reduced to the oxidized form, the reduced α3(βD311C/R333C)3γ subcomplex was inactivated with CuCl2 in the presence or absence of MgADP. In each case, complete inactivation was observed within 5 min. The reaction mixtures were then incubated an additional 2 h to allow further oxidation. After removing CuCl2, free ADP, and Mg2+, inactivated enzyme was treated with iodo[3H]acetate or iodo[14C]acetamide for 16 h to derivatize free sulfhydryl groups. These experiments are summarized in Table II. The results obtained when iodo[14C]acetamide was used to monitor residual free sulfhydryl groups, strongly indicate that disulfide bonds are formed in only two β subunits during inactivation. In this case, nearly two sulfhydryl groups were derivatized after inactivating the enzyme with CuCl2. The slightly lower values of free sulfhydryl groups titrated with iodo[3H]acetate may reflect charge repulsion encountered during carboxymethylation of Cys311 and Cys333 in the same β subunit that does not occur during carboxamidomethylation with iodoacetamide.Table IILabeling of the reduced and oxidized forms of the α3 (βD311C/R333C)3 γ double mutant with iodo[3H]acetate and iodo[14 C]acetamideLabeling reagentReduced mutantOxidized mutantOxidized mutant plus MgADPmol/molIodo[3H]acetate6.3 ± 0.21.5 ± 0.11.2 ± 0.1Iodo[14C]acetamide6.1 ± 0.22.1 ± 0.11.7 ± 0.1The reduced α3(βD311C/R333C)3γ subcomplex was prepared in 50 mm Tris-Cl, pH 8.0, containing 100 μm EDTA as described under “Experimental Procedures.” The oxidized subcomplex was prepared by treating the reduced subcomplex at 1 mg/ml with 200 μm CuCl2 in the presence or absence of 1 mm ADP plus 2 mm MgCl2 for 2 h, at which time the reaction mixture was passed through a centrifuge column of Sephadex G-50 column (9Penefsky H.S. J. Biol. Chem. 1977; 252: 2891-2899Abstract Full Text PDF PubMed Google Scholar) equilibrated with 50 mm Tris-Cl, pH 8.0, containing 100 μm EDTA. ATPase activity was completely inactivated within 5 min of adding CuCl2. Aliquots of the reduced and oxidized preparations at 1 mg/ml in Tris-Cl, pH 8.0, containing 100 μm EDTA were treated with 2 mm iodo[3H]acetate or iodo[14C]acetamide for 16 h to derivatize free sulfhydryl groups. Unreacted iodo[3H]acetate and iodo[14C]acetamide were removed from the samples by passing them through two consecutive centrifuge columns of Sephadex G-50 equilibrated with Tris-Cl, pH 8.0, containing 100 μmEDTA. Samples (3 μl each of the second effluents) were taken to determine protein concentration (11Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar), and 10 μl samples of the second effluents were submitted to liquid scintillation counting. Open table in a new tab The reduced α3(βD311C/R333C)3γ subcomplex was prepared in 50 mm Tris-Cl, pH 8.0, containing 100 μm EDTA as described under “Experimental Procedures.” The oxidized subcomplex was prepared by treating the reduced subcomplex at 1 mg/ml with 200 μm CuCl2 in the presence or absence of 1 mm ADP plus 2 mm MgCl2 for 2 h, at which time the reaction mixture was passed through a centrifuge column of Sephadex G-50 column (9Penefsky H.S. J. Biol. Chem. 1977; 252: 2891-2899Abstract Full Text PDF PubMed Google Scholar) equilibrated with 50 mm Tris-Cl, pH 8.0, containing 100 μm EDTA. ATPase activity was completely inactivated within 5 min of adding CuCl2. Aliquots of the reduced and oxidized preparations at 1 mg/ml in Tris-Cl, pH 8.0, containing 100 μm EDTA were treated with 2 mm iodo[3H]acetate or iodo[14C]acetamide for 16 h to derivatize free sulfhydryl groups. Unreacted iodo[3H]acetate and iodo[14C]acetamide were removed from the samples by passing them through two consecutive centrifuge columns of Sephadex G-50 equilibrated with Tris-Cl, pH 8.0, containing 100 μmEDTA. Samples (3 μl each of the second effluents) were taken to determine protein concentration (11Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar), and 10 μl samples of the second effluents were submitted to liquid scintillation counting. Previous studies have shown that the MgADP-fluoroaluminate complex forms slowly when Al3+ and F− were added to wild-type and mutant α3β3γ subcomplexes of TF1after loading a single catalytic site with MgADP (13Dou C. Grodsky N.B. Matsui T. Yoshida M. Allison W.S. Biochemistry. 1997; 36: 3719-3727Crossref PubMed Scopus (33) Google Scholar, 14Grodsky N.B. Dou C. Allison W.S. Biochemistry. 1998; 37: 1007-1014Crossref PubMed Scopus (16) Google Scholar). In contrast, when MgADP was loaded onto two catalytic sites or when the subcomplex was incubated with excess ADP plus Mg2+, MgADP-fluoroaluminate complexes formed rapidly in two catalytic sites after the addition of Al3+ and F−. This suggests that MgADP-fluoroaluminate complexes are formed cooperatively in two catalytic sites. Table IIIcompares the rates of formation of MgADP-fluoroaluminate complexes in catalytic sites of the reduced and oxidized α3(βD311C/R333C)3γ or wild-type α3β3γ subcomplexes under various conditions. The reduced, mutant subcomplex formed fluoroaluminate complexes somewhat faster than wild-type enzyme when Al3+and F− were added to the subcomplexes containing stoichiometric MgADP or in the presence of excess ADP and Mg2+. The rates of formation