Cytoplasmic Loops of Subunits C and A in E. Coli F1Fo ATP Synthase Interact to Gate H+ Transport to the Cytoplasm

Abstract

Rotary catalysis in F1Fo ATP synthase is powered by proton transport through the membrane-embedded Fo sector, which functions like a proton-driven turbine. 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 transmembrane helices (TMHs). Previously, the aqueous accessibility of Cys substitutions in the TM regions of subunits c and a were probed by testing the inhibitory effects of Ag+ or Cd+2 on function, and defined two half-channels leading to the proton binding site at cAsp61. The half channel from the periplasm lies in the center of a four-helix bundle in subunit a. We show here that the gating of protons from the periplasmic half channel to the Asp61 binding site requires repositioning of helices at the aAsn214/aGln252 site of interaction between TMH4 and TMH5. In addition we show that Ag+ and Cd2+ sensitive Cys substitutions on the proton transporting pathway to the cytoplasm extend into the polar loop of subunit c. Further, Ag+ and Cd2+ sensitive Cys substitutions that are directly involved in proton transport through Fo are also found in two cytoplasmic loops of subunit a. We show here that Cys substitutions in the hairpin loop of subunit c and the two loops of subunit a, each of which are directly implicated in proton transport, can be cross-linked to each other. We suggest that the three loops pack as a single domain that serves to gate proton release to the cytoplasm.