Nucleotide - dependent Movement of the e Subunit between a and b Subunits in the Escherichia coli F 1 F 0 - type ATPase

Mutants of ECF1-ATPase were generated, containing cysteine residues in one or more of the following positions: aSer-411, bGlu-381, and eSer-108, after which disulfide bridges could be created by CuCl2 induced oxidation in high yield between a and e, b and e, a and g, b and g (endogenous Cys-87), and a and b. All of these cross-links lead to inhibition of ATP hydrolysis activity. In the two double mutants, containing a cysteine in eSer-108 along with either the DELSEED region of b (Glu-381) or the homologous region in a (Ser-411), there was a clear nucleotide dependence of the cross-link formation with the e subunit. In bE381C/eS108C the b-e cross-link was obtained preferentially when Mg and ADP 1 Pi (addition of MgCl2 1 ATP) was present, while the a-e cross-link product was strongly favored in the aS411C/eS108C mutant in the Mg ATP state (addition of MgCl2 1 5*-adenylyl-b,g-imidodiphosphate). In the triple mutant aS411C/bE381C/eS108C, the e subunit bound to the b subunit in Mg-ADP and to the a subunit in Mg-ATP, indicating a significant movement of this subunit. The g subunit cross-linked to the b subunit in higher yield in Mg-ATP than in Mg-ADP, and when possible, i.e. in the triple mutant, always preferred the interaction with the b over the a subunit.