ARRANGEMENT OF THE MULTICOPY H-TRANSLOCATING SUBUNIT-C IN THE MEMBRANE SECTOR OF THE ESCHERICHIA-COLI F1F0 ATP SYNTHASE()

The multicopy subunit c of the H+-transporting F1F0 ATP synthase of Es cherichia coli is thought to fold across the membrane as a hairpin of two hydrophobic cu-helices. The conserved Asp(61), centered in the sec ond transmembrane helix, is essential for H+ transport. In this study, we have made sequential Cys substitutions across both transmembrane h elices and used disulfide cross-link formation to determine the oligom eric arrangement of the c subunits. Cross link formation between singl e Cys substitutions in helix 1 provided initial limitations on how the subunits could be arranged. Double Cys substitutions at positions 14/ 16, 16/18, and 21/23 in helix 1 and 70/72 in helix 2 led to the format ion of cross-linked multimers upon oxidation. Double Cys substitutions in helix 1 and helix 2, at residues 14/72, 21/65, and 20/66, respecti vely, also formed cross-linked multimers. These results indicate that at least 10 and probably 12 subunits c interact in a front-to-back fas hion to form a ring-like arrangement in F-0. Helix 1 packs at the inte rior and helix 2 at the periphery of the ring. The model indicates tha t the Asp(61) carboxylate is centered between the helical faces of adj acent subunit c at the center of a four-helix bundle.