Interacting cytoplasmic loops of subunits a and c of Escherichia coli F1F0 ATP synthase gate H+ transport to the cytoplasm

Abstract H+-transporting F1F0 ATP synthase catalyzes the synthesis of ATP via coupled rotary motors within F0 and F1. H+ transport at the subunit a–c interface in transmembranous F0 drives rotation of a cylindrical c10 oligomer within the membrane, which is coupled to rotation of subunit γ within the α3β3 sector of F1 to mechanically drive ATP synthesis. F1F0 functions in a reversible manner, with ATP hydrolysis driving H+ transport. ATP-driven H+ transport in a select group of cysteine mutants in subunits a and c is inhibited after chelation of Ag+ and/or Cd+2 with the substituted sulfhydryl groups. The H+ transport pathway mapped via these Ag+(Cd+2)-sensitive Cys extends from the transmembrane helices (TMHs) of subunits a and c into cytoplasmic loops connecting the TMHs, suggesting these loop regions could be involved in gating H+ release to the cytoplasm. Here, using select loop-region Cys from the single cytoplasmic loop of subunit c and multiple cytoplasmic loops of subunit a, we show that Cd+2 directly inhibits passive H+ transport mediated by F0 reconstituted in liposomes. Further, in extensions of previous studies, we show that the regions mediating passive H+ transport can be cross-linked to each other. We conclude that the loop-regions in subunits a and c that are implicated in H+ transport likely interact in a single structural domain, which then functions in gating H+ release to the cytoplasm.