Residues in the polar loop of subunit c in Escherichia coli ATP synthase function in gating proton transport to the cytoplasm

J Biol Chem. 2014 Jan 24;289(4):2127-38. doi: 10.1074/jbc.M113.527879. Epub 2013 Dec 2.


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.

Keywords: ATP Synthase; Chemical Modifications Inhibiting Function; Cysteine Substitution Mutagenesis; F1F0-ATPase; Loops of Transmembrane Proteins; Membrane Energetics; Membrane Transport; Proton Transport; Subunit c.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Substitution
  • Cadmium / pharmacology
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism*
  • Ion Channel Gating / drug effects
  • Ion Channel Gating / physiology*
  • Ion Transport / drug effects
  • Ion Transport / physiology
  • Mutation, Missense
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Proton-Translocating ATPases / antagonists & inhibitors
  • Proton-Translocating ATPases / genetics
  • Proton-Translocating ATPases / metabolism*
  • Silver / pharmacology


  • Escherichia coli Proteins
  • Cadmium
  • Silver
  • Proton-Translocating ATPases