Large conformational changes of the epsilon subunit in the bacterial F1F0 ATP synthase provide a ratchet action to regulate this rotary motor enzyme.

The F(1)F(0) ATP synthase is the smallest motor enzyme known. Previous studies had established that the central stalk, made of the gamma and epsilon subunits in the F(1) part and c subunit ring in the F(0) part, rotates relative to a stator composed of alpha(3)beta(3)deltaab(2) during ATP hydrolysis and synthesis. How this rotation is regulated has been less clear. Here, we show that the epsilon subunit plays a key role by acting as a switch of this motor. Two different arrangements of the epsilon subunit have been visualized recently. The first has been observed in beef heart mitochondrial F(1)-ATPase where the C-terminal portion is arranged as a two-alpha-helix hairpin structure that extends away from the alpha(3)beta(3) region, and toward the position of the c subunit ring in the intact F(1)F(0). The second arrangement was observed in a structure determination of a complex of the gamma and epsilon subunits of the Escherichia coli F(1)-ATPase. In this, the two C-terminal helices are apart and extend along the gamma to interact with the alpha and beta subunits in the intact complex. We have been able to trap these two arrangements by cross-linking after introducing appropriate Cys residues in E. coli F(1)F(0), confirming that both conformations of the epsilon subunit exist in the enzyme complex. With the C-terminal domain of epsilon toward the F(0), ATP hydrolysis is activated, but the enzyme is fully coupled in both ATP hydrolysis and synthesis. With the C-terminal domain toward the F(1) part, ATP hydrolysis is inhibited and yet the enzyme is fully functional in ATP synthesis; i.e., it works in one direction only. These results help explain the inhibitory action of the epsilon subunit in the F(1)F(0) complex and argue for a ratchet function of this subunit.

Rotation of the c subunit oligomer in fully functional F1Fo ATP synthase.

The F(1)F(o)-type ATP synthase is the smallest motor enzyme known. Previous studies had established that the central gamma and epsilon subunits of the F(1) part rotate relative to a stator of alpha(3)beta(3) and delta subunits during catalysis. We now show that the ring of c subunits in the F(o) part moves along with the gamma and epsilon subunits. This was demonstrated by linking the three rotor subunits with disulfide bridges between cysteine residues introduced genetically at the interfaces between the gamma, epsilon, and c subunits. Essentially complete cross-linking of the gamma, epsilon, and c subunits was achieved by using CuCl(2) to induce oxidation. This fixing of the three subunits together had no significant effect on ATP hydrolysis, proton translocation, or ATP synthesis, and each of these functions retained inhibitor sensitivity. These results unequivocally place the c subunit oligomer in the rotor part of this molecular machine.

Observations of rotation within the F(o)F(1)-ATP synthase: deciding between rotation of the F(o)c subunit ring and artifact.

F(o)F(1)-ATP synthase mediates coupling of proton flow in F(o) and ATP synthesis/hydrolysis in F(1) through rotation of central rotor subunits. A ring structure of F(o)c subunits is widely believed to be a part of the rotor. Using an attached actin filament as a probe, we have observed the rotation of the F(o)c subunit ring in detergent-solubilized F(o)F(1)-ATP synthase purified from Escherichia coli. Similar studies have been performed and reported recently [Sambongi et al. (1999) Science 286, 1722-1724]. However, in our hands this rotation has been observed only for the preparations which show poor sensitivity to dicyclohexylcarbodiimde, an F(o) inhibitor. We have found that detergents which adequately disperse the enzyme for the rotation assay also tend to transform F(o)F(1)-ATP synthase into an F(o) inhibitor-insensitive state in which F(1) can hydrolyze ATP regardless of the state of the F(o). Our results raise the important issue of whether rotation of the F(o)c ring in isolated F(o)F(1)-ATP synthase can be demonstrated unequivocally with the approach adopted here and also used by Sambongi et al.

Cross-linking and electron microscopy studies of the structure and functioning of the Escherichia coli ATP synthase.

ATP synthase, also called F(1)F(o)-ATPase, catalyzes the synthesis of ATP during oxidative phosphorylation. The enzyme is reversible and is able to use ATP to drive a proton gradient for transport purposes. Our work has focused on the enzyme from Escherichia coli (ECF(1)F(o)). We have used a combination of methods to study this enzyme, including electron microscopy and chemical cross-linking. The utility of these two approaches in particular, and the important insights they give into the structure and mechanism of the ATP synthase, are reviewed.

The gammaepsilon-c subunit interface in the ATP synthase of Escherichia coli. cross-linking of the epsilon subunit to the c subunit ring does not impair enzyme function, that of gamma to c subunits leads to uncoupling.

Mutants with a cysteine residue in the gamma subunit at position 207 and the epsilon subunit at position 31 were expressed in combination with a c-dimer construct, which contains a single cysteine at position 42 of the second c subunit. These mutants are called gammaY207C/cc'Q42C and epsilonE31C/cc'Q42C, respectively. Cross-linking of epsilon to the c subunit ring was obtained almost to completion without significant effect on any enzyme function, i.e. ATP hydrolysis, ATP synthesis, and ATP hydrolysis-driven proton translocation were all close to that of wild type. The gamma subunit could also be linked to the c subunit ring in more than 90% yield, but this affected coupling. Thus, ATP hydrolysis was increased 2. 5-fold, ATP synthesis was dramatically decreased, and ATP hydrolysis-driven proton translocation was abolished, as measured by the 9-amino-6-chloro-2-methoxyacridinequenching method. These results for epsilonE31C/cc'Q42C indicate that the c subunit ring rotates with the central stalk element. That the gamma-epsilon cross-linked enzyme retains ATPase activity also argues for a gammaepsilon-c subunit rotor. However, the uncoupling induced by cross-linking of gamma to the c subunit ring points to important conformational changes taking place in the gammaepsilon-c subunit interface during this. Blocking these structural changes by cross-linking leads to a proton leak within the F(0).

The second stalk: the delta-b subunit connection in ECF1F0.

The ATP synthase F1F0 is the smallest molecular motor yet studied. ATP hydrolysis drives the rotary motion of the primary stalk subunits gamma and epsilon relative to the alpha 3 beta 3 part of F1. Evidence is reviewed to show that the delta and b subunits provide a second stalk that can act as a stator to facilitate these rotational movements.

Trapping of conformations of the Escherichia coli F1 ATPase by disulfide bond formation. A state of the enzyme with all three catalytic sites of equal and low affinity for nucleotides.

A mutant of Escherichia coli F1F0-ATPase, alphaS411C/betaY331W/betaE381C/gammaC87S, has been generated. CuCl2 treatment of this mutant led to cross-linking between alpha and beta subunits in yields of up to 90%. This cross-linking across non-catalytic site interfaces inhibited ATP hydrolysis activity. In the absence of cross-linking, MgATP bound in catalytic sites of the mutant with three different affinities of 0.1 microM, 6 microM and 60 microM, respectively, values that are comparable to wild-type. For MgADP, there was one tight site (0.34 microM) and two sites of lower affinity (each 27 microM), again comparable to wild-type enzyme. After cross-linking all three catalytic sites bound MgATP or MgADP with the same relatively low affinity (approximately 60 microM). Thus cross-linking fixed all three catalytic sites in the same conformation. Trypsin cleavage experiments showed that cross-linking fixed the epsilon subunit in the ATP+EDTA conformation.

Rotation of a gamma-epsilon subunit domain in the Escherichia coli F1F0-ATP synthase complex. The gamma-epsilon subunits are essentially randomly distributed relative to the alpha3beta3delta domain in the intact complex.

A triple mutant of Escherichia coli F1F0-ATP synthase, alphaQ2C/alphaS411C/epsilonS108C, has been generated for studying movements of the gamma and epsilon subunits during functioning of the enzyme. It includes mutations that allow disulfide bond formation between the Cys at alpha411 and both Cys-87 of gamma and Cys-108 of epsilon, two covalent cross-links that block enzyme function (Aggeler, R., and Capaldi, R. A. (1996) J. Biol. Chem. 271, 13888-13891). A cross-link is also generated between the Cys at alpha2 and Cys-140 of the delta subunit, which has no effect on functioning (Ogilvie, I., Aggeler, R., and Capaldi, R. A. (1997) J. Biol. Chem. 272, 16652-16656). CuCl2 treatment of the mutant alphaQ2C/alphaS411C/epsilonS108C generated five major cross-linked products. These are alpha-gamma-delta, alpha-gamma, alpha-delta-epsilon, alpha-delta, and alpha-epsilon. The ratio of alpha-gamma-delta to the alpha-gamma product was close to 1:2, i.e. in one-third of the ECF1F0 molecules the gamma subunit was attached to the alpha subunit at which the delta subunit is bound. Also, 20% of the epsilon subunit was present as a alpha-delta-epsilon product. With regard to the delta subunit, 30% was in the alpha-gamma-delta, 20% in the alpha-delta-epsilon, and 50% in the alpha-delta products when the cross-linking was done after incubation in ATP + MgCl2. The amounts of these three products were 40, 22, and 38%, respectively, in experiments where Cu2+ was added after preincubation in ATP + Mg2+ + azide. The delta subunit is fixed to, and therefore identifies, one specific alpha subunit (alphadelta). A distribution of the gamma and epsilon subunits, which is essentially random with respect to the alpha subunits, can only be explained by rotation of gamma-epsilon relative to the alpha3beta3 domain in ECF1F0.

Cross-linking of the delta subunit to one of the three alpha subunits has no effect on functioning, as expected if delta is a part of the stator that links the F1 and F0 parts of the Escherichia coli ATP synthase.

A mutant of the Escherichia coli F1F0-ATPase has been generated (alphaQ2C) in which the glutamine at position 2 of the alpha subunit has been replaced with a cysteine residue. Cu2+ treatment of ECF1 from this mutant cross-linked an alpha subunit to the delta subunit in high yield. Two different sites of disulfide bond formation were involved, i.e. between Cys90 (or the closely spaced Cys47) of alpha with Cys140 of delta, and between Cys2 of alpha and Cys140 of delta. Small amounts of other cross-linked products, including alpha-alpha, delta internal, and alpha-alpha-delta were obtained. In ECF1F0, there was no cross-linking between the intrinsic Cys of alpha and Cys140. Instead, the product generated between Cys2 of alpha and Cys140 of delta was obtained at near 90% yield. Small amounts of alpha-alpha and delta internal were present, and under high Cu2+ concentrations, alpha-alpha-delta was also formed. The ATPase activity of ECF1 and ECF1F0 was not significantly affected by the presence of these cross-links. When Cys140 of delta was first modified with N-ethylmaleimide in ECF1F0, an alpha-delta cross-link was still produced, although in lower yield, between Cys64 of delta and Cys2 of alpha. ATP hydrolysis-linked proton pumping of inner membranes from the mutant alpha2QC was only marginally affected by cross-linking of the alpha to the delta subunit. These results indicate that Cys140 and Cys64 of the delta subunit and Cys2 of the alpha subunit are in close proximity. This places the delta subunit near the top of the alpha-beta hexagon and not in the stalk region. As fixing the delta to the alpha by cross-linking does not greatly impair either the ATPase function of the enzyme, or coupled proton translocation, we argue that the delta subunit forms a portion of the stator linking F1 to F0.

The subunit delta-subunit b domain of the Escherichia coli F1F0 ATPase. The B subunits interact with F1 as a dimer and through the delta subunit.

The delta and b subunits are both involved in binding the F1 to the F0 part in the Escherichia coli ATP synthase (ECF1F0). The interaction of the purified delta subunit and the isolated hydrophilic domain of the b subunit (bsol) has been studied here. Purified delta binds to bsol weakly in solution, as indicated by NMR studies and protease protection experiments. On F1, i.e. in the presence of ECF1-delta, delta, and bsol interact strongly, and a complex of ECF1.bsol can be isolated by native gel electrophoresis. Both delta subunit and bsol are protected from trypsin cleavage in this complex. In contrast, the delta subunit is rapidly degraded by the protease when bound to ECF1 when bsol is absent. The interaction of bsol with ECF1 involves the C-terminal domain of delta as delta(1-134) cannot replace intact delta in the binding experiments. As purified, bsol is a stable dimer with 80% alpha helix. A monomeric form of bsol can be obtained by introducing the mutation A128D (Howitt, S. M., Rodgers, A. J.,W., Jeffrey, P. D., and Cox, G. B. (1996) J. Biol. Chem. 271, 7038-7042). Monomeric bsol has less alpha helix, i.e. only 58%, is much more sensitive to trypsin cleavage than dimer, and unfolds at much lower temperatures than the dimer in circular dichroism melting studies, indicating a less stable structure. The bsol dimer, but not monomer, binds to delta in ECF1. To examine whether subunit b is a monomor or dimer in intact ECF1F0, CuCl2 was used to induce cross-link formation in the mutants bS60C, bQ104C, bA128C, bG131C, and bS146C. With the exception of bS60C, CuCl2 treatment resulted in formation of b subunit dimers in all mutants. Cross-linking yield was independent of nucleotide conditions and did not affect ATPase activity. These results show the b subunit to be dimeric for a large portion of the C terminus, with residues 124-131 likely forming a pair of parallel alpha helices.

Structural changes in the gamma and epsilon subunits of the Escherichia coli F1F0-type ATPase during energy coupling.

Structural changes in the Escherichia coli ATP synthase (ECF1F0) occur as part of catalysis, cooperativity and energy coupling within the complex. The gamma and epsilon subunits, two major components of the stalk that links the F1 and F0 parts, are intimately involved in conformational coupling that links catalytic site events in the F1 part with proton pumping through the membrane embedded F0 section. Movements of the gamma subunit have been observed by electron microscopy, and by cross-linking and fluorescence studies in which reagents are bound to Cys residues introduced at selected sites by mutagenesis. Conformational changes and shifts of the epsilon subunit related to changes in nucleotide occupancy sites have been followed by similar approaches.

Conformational changes in the Escherichia coli ATP synthase (ECF1F0) monitored by nucleotide-dependent differences in the reactivity of Cys-87 of the gamma subunit in the mutant betaGlu-381 --> Ala.

Cys-87, one of two intrinsic cysteines of the gamma subunit of the Escherichia coli ATP synthase (ECF1F0), is in a short segment of this subunit that binds to the bottom domain of a beta subunit close to a glutamate (Glu-381). Cys-87 was unreactive to maleimides under all conditions in wild-type ECF1 and ECF1F0 but became reactive when Glu-381 of beta was replaced by a cysteine or alanine. The reactivity of Cys-87 with maleimides was nucleotide-dependent, occurring with ATP or ADP + EDTA in catalytic sites, in the presence of AMP.PNP + Mg2+ but not with ADP + Mg2+ bound, whether Pi was present or not, and not when nucleotide binding sites were empty. Binding of N-ethylmaleimide had no effect, whereas 7-diethyl-amino-3-(4'-maleimidylphenyl)-4-methylcoumarin increased the ATPase activity of ECF1 more than 2-fold by reaction with Cys-87. In ECF1F0, these reagents inhibited activity. The nucleotide dependence of the reaction of Cys-87 of the gamma subunit depended on the presence of the epsilon subunit. In epsilon subunit-free ECF1, maleimides reacted with Cys-87 under all nucleotide conditions, including when catalytic sites were empty. These results are discussed in terms of nucleotide-dependent movements of the gamma subunit during functioning of the F1F0-type ATPase.

Nucleotide-dependent movement of the epsilon subunit between alpha and beta subunits in the Escherichia coli F1F0-type ATPase.

Mutants of ECF1-ATPase were generated, containing cysteine residues in one or more of the following positions: alphaSer-411, betaGlu-381, and epsilonSer-108, after which disulfide bridges could be created by CuCl2 induced oxidation in high yield between alpha and epsilon, beta and epsilon, alpha and gamma, beta and gamma (endogenous Cys-87), and alpha and beta. All of these cross-links lead to inhibition of ATP hydrolysis activity. In the two double mutants, containing a cysteine in epsilonSer-108 along with either the DELSEED region of beta (Glu-381) or the homologous region in alpha (Ser-411), there was a clear nucleotide dependence of the cross-link formation with the epsilon subunit. In betaE381C/epsilonS108C the beta-epsilon cross-link was obtained preferentially when Mg2+ and ADP + Pi (addition of MgCl2 + ATP) was present, while the alpha-epsilon cross-link product was strongly favored in the alphaS411C/epsilonS108C mutant in the Mg2+ ATP state (addition of MgCl2 + 5'-adenylyl-beta,gamma-imidodiphosphate). In the triple mutant alphaS411C/betaE381C/epsilonS108C, the epsilon subunit bound to the beta subunit in Mg2+-ADP and to the alpha subunit in Mg2+-ATP, indicating a significant movement of this subunit. The gamma subunit cross-linked to the beta subunit in higher yield in Mg2+-ATP than in Mg2+-ADP, and when possible, i.e. in the triple mutant, always preferred the interaction with the beta over the alpha subunit.

Conformational changes in the gamma and epsilon subunits are integral to the functioning of the Escherichia coli H(+)-pumping ATPase (ECF1F0).

ATP synthesis and ATP hydrolysis by F1F0-type ATPases involve conformational changes transmitted from the catalytic site regions to the proton channel, a distance of more than 100 A. Our studies focus attention on the gamma and epsilon subunits that provide a part of the stalk region in the energy-coupling process within the complex. There are conformational changes in the gamma subunit, and translocations of the epsilon unit, linked to nucleotide-binding changes in catalytic sites, which might be expected to alter the interaction of this subunits with c subunits and, hence, be linked to proton translocation.

Arrangement of the epsilon subunit in the Escherichia coli ATP synthase from the reactivity of cysteine residues introduced at different positions in this subunit.

ECF1F0 has been purified from three mutants in which a Cys has been incorporated by site-directed mutagenesis in the epsilon subunit: these mutants are epsilon S10C, epsilon H38C and epsilon S108C, respectively. ECF1F0 from the mutant epsilon S10C had a 2-fold higher activity than wild-type enzyme, due to altered association of the epsilon subunit with the rest of the complex, and yet showed normal proton pumping function. The other two mutants had ATPase activities similar to wild-type enzyme. The introduced Cys was exposed for reaction with maleimides in epsilon S10C and epsilon S108C. In epsilon H38C, the introduced Cys reacted readily with N-ethylmaleimide in isolated ECF1, but was unavailable for reaction with this or other maleimides in ECF1F0. When this Cys at position 38 in the epsilon subunit was reacted with various maleimides in isolated ECF1 and then the ECF1 bound back to F0, the interaction between the two parts was perturbed. While ECF1F0 reconstituted with unmodified ECF1 functioned normally, enzyme with maleimide-reacted Cys-38 showed much reduced proton pumping, had only around 50% of the DCCD inhibition of unmodified or wild-type enzyme, and had a much higher LDAO activation (as much as 8.3-fold, c.f. 4-fold for wild type). Nucleotide-dependent conformational changes have been observed previously, in studies of ECF1 from the mutants epsilon S10C and epsilon S108C. Identical nucleotide-dependent structural changes were observed in cross-linking experiments with tetrafluorophenylazide maleimides when the intact ECF1F0 from these mutants was examined. Taken together, the Cys reactivity data and cross-linking results provide the orientation of the epsilon subunit in the enzyme complex.

Disulfide bond formation between the COOH-terminal domain of the beta subunits and the gamma and epsilon subunits of the Escherichia coli F1-ATPase. Structural implications and functional consequences.

A set of mutants of the Escherichia coli F1F0-type ATPase has been generated by site-directed mutagenesis as follows: beta E381C, beta S383C, beta E381C/epsilon S108C, and beta S383C/epsilon S108C. Treatment of ECF1 isolated from any of these mutants with CuCl2 induces disulfide bond formation. For the single mutants, beta E381C and beta S383C, a disulfide bond is formed in essentially 100% yield between a beta subunit and the gamma subunit, probably at Cys87 based on the recent structure determination of F1 (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628). In the double mutants, two disulfide bonds are formed, again in essentially full yield, one between beta and gamma, the other between a beta and the epsilon subunit via Cys108. The same two cross-links are produced with CuCl2 treatment of ECF1F0 isolated from either of the double mutants. These results show that the parts of gamma around residue 87 (a short alpha-helix) and the epsilon subunit interact with different beta subunits. The yield of covalent linkage of beta to gamma is nucleotide dependent and highest in ATP and much lower with ADP in catalytic sites. The yield of covalent linkage of beta to epsilon is also nucleotide dependent but in this case is highest in ADP and much lower in ATP. Disulfide bond formation between either beta and gamma, or beta and epsilon inhibits the ATPase activity of the enzyme in proportion to the yield of the cross-linked product. Chemical modification of the Cys at either position 381 or 383 of the beta subunit inhibits ATPase activity in a manner that appears to be dependent on the size of the modifying reagent. These results are as expected if movements of the catalytic site-containing beta subunits relative to the gamma and epsilon subunits are an essential part of the cooperativity of the enzyme.

Coupling between catalytic sites and the proton channel in F1F0-type ATPases.

F1F0-type ATPases catalyse both ATP-driven proton translocation and proton-gradient-driven ATP synthesis. Recent cryoelectronmicroscopy and low-resolution X-ray studies provide a first glimpse at the structure of this complicated membrane-bound enzyme. The F1 part is roughly globular and linked to the membrane-intercalated F0 part by a narrow stalk domain, which contains the gamma-, delta- and epsilon-subunits along with domains of the b-subunit of the F0 part. Here, we review evidence that conformational and positional changes in the gamma- and epsilon-subunits provide the coupling between catalytic sites and proton translocation within the F1F0 complex.

TFPACD, a novel bifunctional reagent for reacting with DCCD sites in proteins: studies using Escherichia coli ATP synthase.

A novel cross-linker, 1-[6-(4-azido-2,3,5,6-tetrafluorobenzamido)hexyl]-3-cyclohexylc arbodiimide (TFPACD), has been synthesized and tested by reaction with the Escherichia coli ATP Synthase (ECF1F0). The reagent has a carbodiimide as one reactive group, which is shown to react with ECF1F0 in a similar way to 1,3-dicyclohexylcarbodiimide (DCCD) and modify the beta subunit of the ECF1 part and the c subunit of the F0 part. Reaction with both the ECF1 and F0 parts of the complex inhibited ATPase activity. The second reactive group in the reagent is the photoactivatable tetrafluorophenylazide moiety. Subsequent UV photolysis of TFPACD--modified ECF1 and ECF1F0 led to generation of cross-linked products in significant yields, one between beta and alpha subunits; the second, dimers of the c subunit of the F0 part.

The gamma subunit of the Escherichia coli F1-ATPase can be cross-linked near the glycine-rich loop region of a beta subunit when ADP + Mg2+ occupies catalytic sites but not when ATP + Mg2+ is bound.

A mutant of the Escherichia coli F1-ATPase, gamma S8C, has been reacted with a novel bifunctional reagent, N-maleimido-N'-(4-azido-2,3,5,6-tetrafluorobenzamido) cystamine (TFPAM-SS1). Modification of Cys-8 via the maleimide, followed by photolysis to convert the azido group to a reactive nitrene, led to cross-linking of the gamma subunit to a beta subunit. When this cross-linking was conducted with ADP + Mg2+ in catalytic sites, the predominant cross-linked product had a M(r) of 108,000. If cross-linking was done with uncleaved ATP + Mg2+ in catalytic sites, cross-linked products of 102,000 and 84,000 were formed. Cross-linking under both conditions led to inhibition of ATPase activity. TFPAM-SS1 could be cleaved by using reducing agents to break the disulfide bond that links the malemide and tetrafluorophenylazide moieties. Cleavage of this disulfide bond after formation of 102,000 and 84,000 species led to full recovery of ATPase activity. When the 108-kDa cross-linked product was cleaved, full activity was not restored, presumably because of insertion of the tetrafluorophenylazide into a functionally important site on the beta subunit. After cleavage of the disulfide bond, the free thiols could be reacted with [14C]N-ethylmaleimide, thereby radioactively tagging the sites of insertion of the tetrafluorophenylnitrene moiety. In this way, the site of cross-linking from Cys-8 of gamma to the beta subunit in the presence of ADP + Mg2+ was localized to within the sequence Val 145-Lys-155, which contains the glycine-rich loop. This loop region is a part of the catalytic site of the enzyme.

ATP hydrolysis-linked structural changes in the N-terminal part of the gamma subunit of Escherichia coli F1-ATPase examined by cross-linking studies.

A mutant of Escherichia coli F1-ATPase (ECF1) in which the serine residue in position 8 of the gamma subunit has been replaced by a cysteine residue (gamma S8C) has been used to study nucleotide-dependent cross-linking of the gamma subunit to a beta subunit. When examined in the presence of ADP+Mg2+, either supplied directly or as produced during catalytic turnover of ATP+Mg2+, the main cross-linked product generated using the heterobifunctional, photoactivatable, cross-linker tetrafluorophenylazide maleimide-6 had a M(r)(app) of 108,000. When ATP hydrolysis was inhibited, either by cold or by reaction with sodium azide, or when ATP hydrolysis was prevented by the use of adenyl-5'-yl beta,gamma-imidodiphosphate, the main cross-linked products were species with M(r)(app) of 102,000 and 84,000. The nucleotide-dependent switching from one cross-linking pattern to another could only be observed when the epsilon subunit was bound to ECF1; it was not seen in ECF1*, an enzyme preparation missing delta and epsilon subunits, but was observed in preparations selectively depleted of the delta subunit. We conclude that the changes detected in these cross-linking experiments are occurring during the hydrolysis of ATP when the beta-gamma phosphate bond is cleaved and that they are related to the coupling of ATP hydrolysis to proton translocation.