Residues in the Polar Loop of Subunit c in Escherichia coli ATP Synthase Function in Gating Proton Transport to the Cytoplasm

Rotary catalysis in F1F0 ATP synthase is powered by proton translocation through the membrane-embedded F-0 sector. Proton binding and release occur in the middle of the membrane at Asp-61 on the second transmembrane helix (TMH) of subunit c, which folds in a hairpin-like structure with two TMHs. Previously, the aqueous accessibility of Cys substitutions in the transmembrane regions of subunit c was probed by testing the inhibitory effects of Ag+ or Cd2+ on function, which revealed extensive aqueous access in the region around Asp-61 and on the half of TMH2 extending to the cytoplasm. In the current study, we surveyed the Ag+ and Cd2+ sensitivity of Cys substitutions in the loop of the helical hairpin and used a variety of assays to categorize the mechanisms by which Ag+ or Cd2+ chelation with the Cys thiolates caused inhibition. We identified two distinct metal-sensitive regions in the cytoplasmic loop where function was inhibited by different mechanisms. Metal binding to Cys substitutions in the N-terminal half of the loop resulted in an uncoupling of F-1 from F-0 with release of F-1 from the membrane. In contrast, substitutions in the C-terminal half of the loop retained membrane-bound F-1 after metal treatment. In several of these cases, inhibition was shown to be due to blockage of passive H+ translocation through F-0 as assayed with F-0 reconstituted into liposomes. The results suggest that the C-terminal domain of the cytoplasmic loop may function in gating H+ translocation to the cytoplasm.