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

Abstract

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.