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

Mutants of ECF(1)-ATPase were generated, containing cysteine residues in one or more of the following positions: alpha Ser-411, beta Glu-381, and epsilon Ser-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 epsilon Ser-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 beta E381C/epsilon S108C the beta-epsilon cross-link was obtained preferentially with Mg2+ and ADP + P-i (addition of MgCl2 + ATP) was present, while the alpha-epsilon cross-link product was strongly favored in the alpha S411C/epsilon S108C mutant in the Mg2+ ATP state (addition of MgCl2 + 5'-adenylyl-beta,gamma-imidodiphosphate). In the triple mutant alpha S411C/beta E381C/epsilon S108C, 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 cross-linked to the beta subunit in higher yield in Mg2+-ATP than in Mg(2+)-ADP, and when possible, i.e. in the triple mutant, always preferred the interaction with the beta over the alpha subunit.