THE ATP SYNTHASE GAMMA-SUBUNIT - SUPPRESSOR MUTAGENESIS REVEALS 3 HELICAL REGIONS INVOLVED IN ENERGY COUPLING

A role in coupling proton transport to catalysis of ATP synthesis has been demonstrated for the Escherichia coli F0F1 ATP synthase gamma sub unit. Previously, functional interactions between the terminal regions that were important for coupling were shown by finding several mutati ons in the carboxyl-terminal region of the gamma subunit (involving re sidues at positions 242 and 269-280) that restored efficient coupling to the mutation, gamma Met-23 --> Lys (Nakamoto, R. K., Maeda, M., and Futai, M. (1993) J. Biol. Chem. 268, 867-872). In this study, we used suppressor mutagenesis to establish that the terminal regions can be separated into three interacting segments. Second-site mutations that cause pseudo reversion of the primary mutations, gamma GLn-269 --> Glu or gamma Thr-273 --> Val, map to an amino-terminal segment with chang es at residues 18, 34, and 35, and to a segment near the carboxyl term inus with changes at residues 236, 238, 242, and 246. Each second-site mutation suppressed the effects of both gamma Gln-269 --> Glu and gam ma Thr-273 --> Val, and restored efficient coupling to enzyme complexe s containing either of the primary mutations. Mapping of these residue s in the recently reported x-ray crystallographic structure of the F-1 complex (Abrahams, J. P., Leslie, A. G., Lutter, R., and Walker, J. E . (1994) Nature 370, 621-628), reveals that the second-site mutations do not directly interact with gamma Gln-269 and gamma Thr-273 and that the effect of suppression occurs at a distance. We propose that the t hree gamma subunit segments defined by suppressor mutagenesis, residue s gamma 18-35, gamma 236-246, and gamma 269-280, constitute a domain t hat is critical for both catalytic function and energy coupling.