INTERACTING HELICAL FACES OF SUBUNITS A AND C IN THE F1F0 ATP SYNTHASE OF ESCHERICHIA-COLI DEFINED BY DISULFIDE CROSS-LINKING

Subunits a and c of F-o are thought to cooperatively catalyze proton t ranslocation during ATP synthesis by the Escherichia call F1Fo ATP syn thase. Optimizing mutations in subunit a at residues A217, I221, and L 224 improves the partial function of the cA24D/cD61G double mutant and , on this basis, these three residues were proposed to lie on one face of a transmembrane helix of subunit a, which then interacted with the transmembrane helix of subunit c anchoring the essential aspartyl gro up. To test this model, in the present work Cys residues mere introduc ed into the second transmembrane helix of subunit c and the predicted fourth transmembrane helix of subunit a. After treating the membrane v esicles of these mutants with Cu(1,10-phenanthroline)(2)SO4 at 0 degre es, 10 degrees, or 20 degrees C, strong a-e dimer formation was observ ed at all three temperatures in membranes of 7 of the 65 double mutant s constructed, i.e., in the aS207C/cI55C, aN214C/cA62C, aN214C/cM65C, aI221C/cG69C, aI223C/cL72C, aL224C/cY73C, and aI225C/cY73C double muta nt proteins. The pattern of cross-linking aligns the helices in a para llel fashion over a span of 19 residues with the aN214C residue lying close to the cA62C and cM65C residues in the middle of the membrane. L esser a-c dimer formation was observed in nine of her double mutants a fter treatment at 20 degrees C in a pattern generally supporting that indicated by the seven landmark residues cited above. Cross-link forma tion was not observed between helix-1 of subunit c and helix-4 of subu nit a in 19 additional combinations of doubly Cys-substituted proteins . These results provide direct chemical evidence that helix-2 of subun it c and helix-4 of subunit a pack close enough to each other in the m embrane to interact during function. The proximity of helices supports the possibility of an interaction between Arg210 in helix-4 of subuni t a and Asp61 in helix-2 of subunit c during proton translocation, as has been suggested previously.