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J Biol Chem, Vol. 274, Issue 44, 31366-31372, October 29, 1999

Oxidation of the ₃(D311C/R333C)₃ Subcomplex of the Thermophilic Bacillus PS3 F₁-ATPase Indicates That Only Two  Subunits Can Exist in the Closed Conformation Simultaneously^*

Huimiao Ren, Chao Dou^§, Matthew S. Stelzer, and William S. Allison^¶

From the Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093-0506

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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  subunits, one with MgAMP-PNP bound to the catalytic site (_T) and the other with MgADP bound (_D) have closed conformations. The empty  subunit (_E) has an open conformation. In _T and _D, the distance between the carboxylate of -Asp³¹⁵ and the guanidinium of -Arg³³⁷ is 3.0-4.0 Å. These side chains are at least 10 Å apart in _E. The ₃(D311C/R333C)₃ 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  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 F₁ are saturated, the catalytic site that binds ATP must be closing as the catalytic site that releases products is opening.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The proton-translocating F₀F₁-ATP synthases couple proton electrochemical gradients to condensation of ADP with P_i 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 ₃₃ stoichiometry. When removed from the membrane, F₁ is an ATPase containing six nucleotide binding sites. Three are catalytic sites that are predominantly on  subunits at / interfaces. The three other sites, called noncatalytic sites, are located predominantly on  subunits at different / interfaces (1-3).

The crystal structures of the bovine heart and rat liver mitochondrial F₁-ATPases as well as that of the ₃₃ subcomplex of the TF₁-ATPase have been determined (4-6). 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 (4). One, designated _T, contains MgAMP-PMP, another, designated _D, contains MgADP and the third catalytic site, designated _E, 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 _T and _D are essentially identical. In contrast, these residues are arranged differently in _E. In the crystals of the ₃₃ subcomplex of TF₁ that form in media free of Mg²⁺ and nucleotides, the  subunits are in closed conformations corresponding to the liganded  subunits of the mitochondrial enzymes. The  subunits in the ₃₃ structure are in open conformations corresponding to _E of BH-MF₁ (5).

In the crystals of the rat liver F₁-ATPase (RL-MF₁), which form in media containing ATP, P_i, 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⁺ (6). One catalytic site contains ADP alone, whereas the other two contain ADP and P_i. Since Mg²⁺ is not associated with ADP and P_i 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 _D and _T and the open conformation of _E in BH-MF₁. In the crystal structure of RL-MF₁, the conformations of all three  subunits resemble the closed conformation of _D and _T in the crystal structure of BH-MF₁. Bianchet et al. (6) suggest that the open conformation of _E 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  subunits plays a central role in catalysis, whereas the BH-MF₁ structure with two closed  subunits and one open  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  subunits that do not contribute to catalytic sites in BH-MF₁ that have side chains within interaction distance in _D and _T but are considerably distant from each other in _E. For example, the carboxylate of -Asp³¹⁵ is within 3.0 and 3.7 Å of the guanidinium of -Arg³³⁷, in _T and _D, respectively, whereas these side chains are 10.3 Å apart in _E. Other pairs of amino acid residues that meet these criteria are -Ala¹⁵⁸/-Tyr³¹¹; -Met¹⁶⁷/-Phe¹⁸³, and -Lys¹⁷⁵/-Ser²⁰³. Double mutants of the ₃₃ subcomplex of TF₁ (7) were prepared in which residues corresponding to -Asp³¹⁵/-Arg³³⁷, -Ala¹⁵⁸/-Tyr³¹¹, -Met¹⁶⁷/-Phe¹⁸³, and -Lys¹⁷⁵/-Ser²⁰³ of BH-MF₁ were 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 ₃(D311C/R333C)₃ double mutant corresponding to the -Asp³¹⁵/-Arg³³⁷ pair in BH-MF₁ met these criteria.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- 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. The Escherichia coli strains and the plasmids used to prepare the wild-type and mutant ₃₃ subcomplexes were described by Matsui and Yoshida (7). The purified enzyme subcomplexes were stored as suspensions in 75% ammonium sulfate at 4 °C.

Construction of Plasmids Containing Mutant Genes-- Plasmid pKK223-3, which carries genes encoding the , , and  subunits of TF₁, was used for both mutagenesis and gene expression. Expression plasmids were constructed using polymerase chain reaction with the Quick Change^TM site-directed mutagenesis kit from Stratagene. The plasmids were purified using the Wizard^TM 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.

Enzyme Preparation and Assays-- The wild-type and mutant subcomplexes were expressed and purified as described previously (7, 8). 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 (9). Preparations treated in this manner are designated CDTA-treated subcomplexes.

The ₃(D311C/R333C)₃ mutant subcomplex was reduced as follows. Ammonium sulfate precipitates of the subcomplex were collected by centrifugation and dissolved in 50 mM Tris-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 (9) 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 (10).

Other Analytical Methods-- Protein concentrations were determined by the method of Bradford (11). 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. (12).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Comparison of the ATPase Activities of the ₃(A160C/Y307C)₃, ₃(Q177C/S205C)₃, ₃(Q169C/F185C)₃, and ₃(D311C/R333C)₃ Subcomplexes-- Table I shows the distances in the crystal structure of the ₃₃ subcomplex of TF₁ (5) 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 _T, _D, and _E in the crystal structure of MF₁ (4) are also tabulated.

View this table: [in this window] [in a new window]   Table I Distances between side chains in the crystal structures of the 33 subcomplex of TF1 (5) and between corresponding residues in the crystal structure of BH-MF1 (4) that were substituted with cysteine in the 33 subcomplex of TF1

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 ₃₃ subcomplex (8). The A160C/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 ₃(A160C/Y307C)₃ subcomplex in the presence or absence of DTT. Defective assembly was not responsible for the lack of ATPase activity. A normal pattern of , , and  subunits was observed in a 3:3:1 ratio after the mutant subcomplex was submitted to SDS-polyacrylamide gel electrophoresis.

The ₃(Q177C/S205C)₃ 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 mg¹ min¹. 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 ₃₃ subcomplex by a factor of 3-4 (8).

The specific activity of the ₃(Q169C/F185C)₃ double mutant was 7 µmol of ATP hydrolyzed mg¹ min¹ 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 mg¹ min¹. The ATPase activity of this double mutant was inactivated by 2 mM o-iodosobenzoate with a pseudo-first order rate constant of 1.4 × 10² min^-1. It was inactivated much more slowly by 2 mM 5,5'-dithiobis-(2-nitrobenzoic acid). The rate of inactivation of the ₃(Q169C/F185C)₃ subcomplex by o-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 ₃(Q169C/F185C)₃ subcomplex could not be reactivated by treatment with 10 mM DTT.

Stabilization of the Reduced ₃(D311C/R333C)₃ Subcomplex during Assay-- Fig. 1A shows that the isolated ₃(D311C/R333C)₃ subcomplex has negligible ATPase activity unless the assay medium is supplemented with DTT or another thiol. Whereas traces a and c of Fig. 1A 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. 1B shows that after reduction as described under "Experimental Procedures" the ₃(D311C/R333C)₃ subcomplex was inactivated during assay unless DTT or 0.1 mM EDTA was included in the assay medium. Trace a in Fig. 1B illustrates assay of the enzyme in the absence of DTT or EDTA. Traces b and c of Fig. 1B 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 ₃(D311C/R333C)₃ subcomplex. Since the presence of EDTA in the assay medium does not affect ATPase activity of the isolated enzyme, illustrated by trace c of Fig. 1A, the isolated ₃(D311C/R333C)₃ subcomplex exists in the oxidized form.

View larger version (7K): [in this window] [in a new window]   Fig. 1.   The effects of DTT and EDTA in the assay buffer on hydrolysis of 2 mM ATP by the isolated reduced and carboxamidomethylated 3(D311C/R333C)3 subcomplex. The isolated and reduced enzyme subcomplexes were prepared as described under "Experimental Procedures." Carboxamidomethylated enzyme was prepared by incubating the reduced enzyme at 1 mg/ml in 50 mM Tris-Cl, pH 8.0, containing 0.2 mM DTT and 2 mM iodoacetamide for 10 h. Excess iodoacetate and DTT were removed by passing the reaction mixture through a centrifuge column of Sephadex G-50 equilibrated with 50 mM Tris-Cl, pH 8.0, containing 100 µM EDTA. Samples (3 µg each) were assayed. The numbers at the end of each trace represent specific ATPase activity at the final rate. A, the isolated 3(D311C/R333C)3 subcomplex; B, the reduced 3(D311C/R333C)3 subcomplex; C, the carboxamidomethylated 3(D311C/R333C)3 subcomplex. The different traces represent assays with no additions (a), assays in the presence of 10 mM DTT (b), and assays in the presence of 100 µM EDTA (c).

Iodoacetate Inactivates the Reduced Form of the ₃(D311C/R333C)₃ Subcomplex-- Following reduction of the ₃(D311C/R333C)₃ 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 ₃(D311C/R333C)₃ 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  subunit wounds the enzyme, and modification of both Cys³¹¹ and Cys³³³ in the same  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 -Cys¹⁹³ that is resistant to modification by iodoacetate.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Inactivation of the reduced 3(D311C/R333C)3 subcomplex by iodoacetate in the presence and absence of bound nucleotides. Samples, 100 µl each of CDTA-treated 3(D311C/R333C)3 subcomplex at 1 mg/ml in 50 mM Tris-Cl, pH 8.0, containing 0.1 mM EDTA, were incubated with 1 mM DTT for 30 min. After additions were made (none (), 3 µM ADP plus 2 mM MgCl2 (), 9 µM ADP plus 2 mM MgCl2 (), and 2 mM ADP plus 2 mM MgCl2 ()), the samples were incubated an additional 5 min. Then iodoacetate was added to a final concentration of 4 mM to initiate inactivation. At the times indicated, 6 µg were removed from the reaction mixtures and assayed with 2 mM ATP using the regeneration system in the presence of 10 mM DTT.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Correlation of inactivation of 3(D311C/R333C)3 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 3(D311C/R333C)3 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 (9) 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.

Complete carboxymethylation lowered the capacity of the ₃(D311C/R333C)₃ 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 mM MgCl₂ 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 ₃(D311C/R333C)₃ 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.

Carboxamidomethylation Converts the Reduced ₃(D311C/R333C)₃ Subcomplex to a Slightly More Active Form-- Modification of the ATPase activity of the ₃(D311C/R333C)₃ 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. 1C. Trace a of Fig. 1C 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. 1B.

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.

Prolonged Oxidation of the Reduced ₃(D311C/R333C)₃ Subcomplex Fails to Promote Formation of Disulfide Bonds in All Three  Subunits-- The reduced ₃(D311C/R333C)₃ 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  subunits containing disulfide bonds on conversion of the reduced to the oxidized form, the reduced ₃(D311C/R333C)₃ 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  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  subunit that does not occur during carboxamidomethylation with iodoacetamide.

View this table: [in this window] [in a new window]   Table II Labeling of the reduced and oxidized forms of the 3(D311C/R333C)3 double mutant with iodo[3H]acetate and iodo[14C]acetamide The reduced 3(D311C/R333C)3 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 (9) 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 µM EDTA. Samples (3 µl each of the second effluents) were taken to determine protein concentration (11), and 10 µl samples of the second effluents were submitted to liquid scintillation counting.

Formation of MgADP-Fluoroaluminate Complexes in the Reduced and Oxidized ₃(D311C/R333C)₃ Subcomplex-- Previous studies have shown that the MgADP-fluoroaluminate complex forms slowly when Al³⁺ and F were added to wild-type and mutant ₃₃ subcomplexes of TF₁ after loading a single catalytic site with MgADP (13, 14). 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 III compares the rates of formation of MgADP-fluoroaluminate complexes in catalytic sites of the reduced and oxidized ₃(D311C/R333C)₃ or wild-type ₃₃ 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 of inhibitory fluoroaluminate complexes in the reduced mutant subcomplex and the wild-type subcomplex were about 10-fold faster when Al³⁺ and F were added after prior exposure of the enzymes to 200 µM ADP plus Mg²⁺ rather than stoichiometric ADP plus Mg²⁺. This suggests that the wild-type and reduced mutant subcomplexes display similar positive cooperativity between catalytic sites. In contrast, the rate of formation of the inhibitory MgADP-fluoroaluminate complex is the same when Al³⁺ and F are added to the oxidized mutant enzyme, indicating no cooperativity between catalytic sites. Whereas the presence of 2 mM P_i inhibited formation of the inhibitory fluoroaluminate complex when MgADP was bound to a single catalytic site, 2 mM P_i stimulated the rate of formation of MgADP-fluoroaluminate complexes when the wild-type and reduced mutant subcomplexes were treated with 200 µM ADP plus 2 mM Mg²⁺ before adding Al³⁺ and F. In contrast, the presence of 2 mM P_i inhibited formation of the MgADP-fluoroaluminate complex when Al³⁺ and F were added to the oxidized subcomplex after exposure to 200 µM ADP plus 2 mM Mg²⁺.

View this table: [in this window] [in a new window]   Table III Rates of formation of MgADP-fluoroaluminate complexes in active sites of the oxidized and reduced 3(D311C/R333C)3 subcomplexes under various conditions The rates of formation (kform) are the pseudo-first order rate constants for irreversible inactivation observed after adding Al3+ and F to the TF1 subcomplexes in the presence of 200 µM ADP plus 2 mM Mg2+ or with stoichiometric MgADP bound to a catalytic site as described previously (13, 14). To load a single catalytic site with MgADP, the isolated mutant subcomplex at 1 mg/ml in 50 mM Tris-Cl, pH 8.0, was incubated with stoichiometric ADP in the presence of 1 mM Mg2+ for 30 min, at which time the solution was passed through a centrifuge column of Sephadex G-50 (9) that was equilibrated with the same buffer. The reduced 3(D311C/R333C)3 subcomplex used in these experiments was prepared by incubating the isolated enzyme with or without MgADP bound to a single catalytic site with 5 mM DTT for 30 min before making subsequent additions. The oxidized 3(D311C/R333C)3 subcomplex was prepared by treating the isolated enzyme with 1 mM CDTA for 30 min and passing it through a Sephadex G-50 centrifuge column (9) that was equilibrated with 50 mM Tris-Cl, pH 8.0. Assays were conducted in the presence of 10 mM DTT.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The proximity of the side chains of -Asp³¹⁵ and -Arg³³⁷ in _T and _D, but not in _E, in the crystal structure of BH-MF₁ implies that these residues may participate in functionally important electrostatic interactions during catalysis. However, substitution of the corresponding residues in the ₃₃ subcomplex of TF₁ with cysteine does not severely impair ATP hydrolysis. The reduced ₃(D311C/R333C)₃ subcomplex hydrolyzes 2 mM ATP at about 50% the rate exhibited by the wild-type subcomplex. Furthermore, carboxamidomethylation of the introduced cysteines slightly enhances ATP hydrolysis. Nevertheless, it is clear that the side chains of these residues must be free to change position during catalysis. The mutant subcomplex is rapidly inactivated by several reagents that promote oxidation of adjacent cysteine side chains in proteins to disulfide bonds. This presumably locks affected  subunits in the closed conformation. Perception of the consequences of locking a  subunit in the disulfide form can be realized from inspection of Fig. 4. The different positions of the side chains of -Asp³¹⁵ and -Arg³³⁷ in the nucleotide binding domain of _T and _E in the crystal structure of BH-MF₁ are illustrated. It is clear that the nucleotide binding domains, shown in yellow, are folded very differently in _T and _E. To highlight the different conformations of _T and _E, helices B and C, which contain components of catalytic sites are illustrated in cyan and magenta, respectively. Transition from the closed conformation of _T to the open conformation of _E moves the carboxylate of -Asp³¹⁵ from the guanidinium of -Arg³³⁷ by more than 7 Å.

View larger version (54K): [in this window] [in a new window]   Fig. 4.   Comparison of the locations of the side chains of -Asp315 and -Arg337 in the crystal structure of BH-MF1. This figure was constructed from the coordinates of BH-MF1 (4) using the software program provided by Roger Sayle (Glaxo Wellcome Research and Development, Greenford, UK).The nucleotide binding domains of T and E are shown in yellow. The COOH-terminal -helical domains are shown in blue. The NH2-terminal -barrel domains are not shown.

The finding that nearly two of the introduced cysteines can be derivatized with iodoacetamide after prolonged exposure of the ₃(D311C/R333C)₃ subcomplex to oxidizing conditions in the presence or absence of saturating MgADP suggests that disulfide bonds are only formed in two of the three  subunits. This is consistent with the results of a study recently reported by Tsunoda et al. (15) who used computer modeling of the crystal structure of BH-MF₁ to show that it is not possible for three  subunits to exist in the closed conformation simultaneously.

It is interesting that carboxymethylation of the introduced cysteines of the ₃(D311C/R333C)₃ double mutant inactivates, rather than stimulates, ATPase activity as observed when they are carboxamidomethylated. Maximal inactivation is observed only after carboxymethylation of all six of the introduced sulfhydryl groups. This suggests that the diminution of ATPase activity that increases with increasing carboxymethylation is caused by charge repulsion of carboxymethylated cysteines on the same  subunit. The observation that bound MgADP protects against carboxymethylation with iodoacetate, whereas it has no effect on carboxamidomethylation with iodoacetamide, is consistent with this premise. Liganding of catalytic sites forces the introduced cysteine side chains together in a given  subunit, thus making it more difficult to modify both of them with the negatively charged carboxymethyl group.

In the crystal of the ()₃ subcomplex of TF₁ deduced by Shirakihara et al. (5), the three  subunits have identical, open conformations. As shown here, treatment of the ₃(D311C/R333C)₃ double mutant with CuCl₂ in the absence of bound nucleotides leads to disulfides in two  subunits. Therefore, two  subunits exist in closed or partly closed conformations in the absence of liganding catalytic sites with nucleotides in the presence of Mg²⁺. This asymmetry must be induced by the coiled-coil composed of the amino and carboxyl termini of the  subunit within the central cavity of the ()₃ hexamer. That two  subunits can exist in the closed conformation in the absence of bound nucleotides was also demonstrated by Tsunoda et al. (15). In the crystal structure of BH-MF₁, the side chain of Ile³⁹⁰ in _T is in contact with the side chain of Ile³⁹⁰ in _D. Computer modeling reported by Tsunoda et al. (15) showed that this contact only occurs when two  subunits are in the closed conformation. The residue in TF₁ corresponding to -Ile³⁹⁰ of BH-MF₁ is -Ile³⁸⁶. Treatment of the ₃(I386C)₃ subcomplex of TF₁ with 0.25 µM CuCl₂ in the presence of MgATP, MgADP, or MgADP and NH₃ inactivated the enzyme and was accompanied by cross-linking of two  subunits. In the absence of nucleotides plus Mg²⁺, inactivation and cross-linking occurred more slowly. This indicates that two  subunits are at least partly closed in the absence of ligation of catalytic sites with MgATP or MgADP.

The demonstration that disulfide bonds are formed in two  subunits upon oxidation of the ₃(D311C/R333C)₃ double mutant in the absence of bound nucleotides and that two  subunits are cross-linked on oxidation of the ₃(I386C)₃ subcomplex in the absence of bound nucleotides has important mechanistic implications. These findings are consistent with earlier observations, suggesting that the heterogeneous affinities of catalytic sites of F₁-ATPases for MgATP reflect intrinsic asymmetry of catalytic sites dictated by the position of the coiled-coil of the  subunit within the central cavity of the ()₃ hexamer rather than negative cooperativity of binding (16, 17). Three K_d values of about 2 nM, 0.2 µM, and 34 µM were detected when quenching of the tryptophan fluorescence of the ₃(Y341W)₃ subcomplex was titrated with MgADP (17). According to the results presented here, the two low K_d values represent binding of MgADP to  subunits in closed conformations, whereas the K_d of 34 µM represents binding of MgADP to the open catalytic site.

The findings reported here and those reported by Tsunoda et al. (15) demonstrating that only two  subunits exist in the closed conformation simultaneously in the presence of MgADP or MgATP are consistent with the crystal structure of BH-MF₁ reported by Abrahams et al. (4). However, they are inconsistent with the crystal structure of RL-MF₁ reported by Bianchet et al. (6) that indicates that all three  subunits are in the closed conformation in the absence of Mg²⁺ with two of them liganded with ADP and P_i and the third liganded with ADP only.

Evidence indicating that only two  subunits can exist in closed conformations simultaneously is also inconsistent with models proposed for ATP hydrolysis and synthesis that include conformational states of F₁ in which three catalytic sites are closed (1, 6). This does not mean that maximal rates of ATP hydrolysis are achieved when only two catalytic sites are saturated. Weber et al. have convincingly demonstrated that ATP hydrolysis catalyzed by E. coli F₁ achieves maximal velocity when three catalytic sites are saturated (18). The model illustrated in Fig. 5 is proposed to accommodate this finding with the observations presented here and by Tsunoda et al. (15), indicating that three  subunits of F₁ cannot exist in the closed conformation simultaneously. The model, which is modified from a scheme in Ref. 3, depicts a round of trisite ATP hydrolysis in which one  subunit (_E) is transiently in the open conformation and the two others (_D and _T) are in the closed conformation and liganded with MgATP. Binding of MgATP to _E causes 1) simultaneous movement of the  subunit in a counterclockwise direction; 2) propagation of a conformational signal from the catalytic site of _E to the catalytic site of _D (indicated by the curved arrow between _E and _D), and 3) opening of the catalytic site of _D, which is accompanied by ATP hydrolysis. The transition state for ATP hydrolysis is represented by [ATP]. During this process, the  subunit rotates 120°. This is necessary to overcome steric restrictions imposed by the  subunit pointed out by Tsunoda et al. (15) that prevent closing of three  subunits simultaneously. From computer modeling, they showed that the coiled-coil of the  subunit within the central cavity of the ()₃ hexamer allows simultaneous closing of only two  subunits. Therefore, in order to proceed from the initial state illustrated in Fig. 5, where _D and _T are in closed conformations, to the final state, where _T and _E are in closed conformations, the position of the coiled-coil of the  subunit within the central cavity must change as _E closes and _D opens. To be consistent with the parameters of rotational catalysis deduced by Noji et al. (19) and Yasuda et al. (20), the coiled-coil must rotate 120° in the counterclockwise direction in this process.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   A model for trisite ATP hydrolysis with simultaneous closing of one catalytic site and opening of another. The stippled circles represent  subunits liganded with MgATP. The hexagons represent unliganded  subunits in the open conformation. The ellipses represent  subunits that are in the process of converting between open and closed conformations. In the concerted process, E is converting from the open to the closed conformation, whereas D is converting from the closed to the open conformation.

	FOOTNOTES

^* This work was supported by NIGMS, National Institutes of Health, Grant GM16974.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The first two authors contributed equally to this work.

^§ Current address: Roche Diagnostics Corp., 9115 Hague Rd., Indianapolis, IN 46250.

^¶ To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093-0506. Tel.: 858-534-3057; Fax: 858-822-0079; E-mail: wsa@checfs2.ucsd.edu.

	ABBREVIATIONS

The abbreviations used are: TF₁, BH-MF₁, and RL-MF₁, the F₁-ATPases from the thermophilic Bacillus PS3, bovine heart mitochondria, and rat liver mitochondria, respectively; DTT, dithiothreitol; CDTA, trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid; AMP-PNP, adenosine 5'-(,-imino)triphosphate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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