title: Residues in the polar loop of subunit c in Escherichia coli ATP synthase function in gating proton transport to the cytoplasm.
abstract: 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 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 Cd(2+) 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 Cd(2+) 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 Cd(2+) 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.
authors.affiliation: From the Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin 53706.
authors.forename: P Ryan
authors.initials: PR
authors.lastname: Steed
authors.forename: Robert H
authors.initials: RH
authors.lastname: Fillingame
mesh_terms: D019943:Amino Acid Substitution; D002104:Cadmium / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D017136:Ion Transport / Q000187:drug effects / Q000502:physiology; D020125:Mutation, Missense; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012834:Silver / Q000494:pharmacology
chemical_list: D029968:Escherichia coli Proteins; D002104:Cadmium; D012834:Silver; D006180:Proton-Translocating ATPases
keywords: ATP Synthase; Chemical Modifications Inhibiting Function; Cysteine Substitution Mutagenesis; F1F0-ATPase; Loops of Transmembrane Proteins; Membrane Energetics; Membrane Transport; Proton Transport; Subunit c