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

December 2nd, 2013 by Steed, P. R., Fillingame, R. H.

Rotary catalysis in F1F0 ATP synthase is powered by proton translocation through the membrane-embedded F0 sector. Proton binding and release occur in the middle of the membrane at Asp61 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, and revealed extensive aqueous access in the region around Asp61 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 F1 from F0, with release of F1 from the membrane. In contrast, substitutions in the C-terminal half of the loop retained membrane bound F1 after metal treatment. In several of these cases, inhibition was shown to be due to blockage of passive H+ translocation through F0, as assayed with F0 reconstituted into liposomes. The results suggest that the C-terminal domain of the cytoplasmic loop may function in gating H+ translocation to the cytoplasm.
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