The cytoplasmic loops of subunit a of Escherichia coli ATP synthase may participate in the proton translocating mechanism

Subunit a plays a key role in promoting H+ transport and the coupled rotary motion of the subunit c ring in F1F0-ATP synthase. H+ binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of F-0 subunit c. H+ are thought to reach Asp-61 via aqueous pathways mapping to the surfaces of TMHs 2 - 5 of subunit a based upon the chemical reactivity of Cys substituted into these helices. Here we substituted Cys into loops connecting TMHs 1 and 2 ( loop 1 - 2) and TMHs 3 and 4 ( loop 3 - 4). A large segment of loop 3 - 4 extending from loop residue 192 loop to residue 203 in TMH4 at the lipid bilayer surface proved to be very sensitive to inhibition by Ag+. Cys-161 and - 165 at the other end of the loop bordering TMH3 were also sensitive to inhibition by Ag+. Further Cys substitutions in residues 86 and 93 in the middle of the 1 - 2 loop proved to be Ag+-sensitive. We next asked whether the regions of Ag+-sensitive residues clustered together near the surface of the membrane by combining Cys substitutions from two domains and testing for cross-linking. Cys-161 and - 165 in loop 3 - 4 were found to cross-link with Cys-202, - 203, or - 205, which extend into TMH4 from the cytoplasm. Further Cys at residues 86 and 93 in loop 1 - 2 were found to cross-link with Cys-195 in loop 3 - 4. We conclude that the Ag+-sensitive regions of loops 1 - 2 and 3 - 4 may pack in a single domain that packs at the ends of TMHs 3 and 4. We suggest that the Ag+- sensitive domain may be involved in gating H+ release at the cytoplasmic side of the aqueous access channel extending through F-0.