Chemical Reactivities of Cysteine Substitutions in Subunit a of ATP Synthase Define Residues Gating H+ Transport from Each Side of the Membrane

Subunit a plays a key role in coupling H+ transport to rotations of the subunit c-ring in F1Fo ATP synthase. In Escherichia coli, H+ binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of F-o subunit c. Based upon the Ag+ sensitivity of Cys substituted into subunit a, H+ are thought to reach Asp-61 via aqueous pathways mapping to surfaces of TMH 2-5. In this study we have extended characterization of the most Ag+-sensitive residues in subunit a with cysteine reactive methanethiosulfonate (MTS) reagents and Cd2+. The effect of these reagents on ATPase-coupled H+ transport was measured using inside-out membrane vesicles. Cd2+ inhibited the activity of all Ag+-sensitive Cys on the cytoplasmic side of the TMHs, and three of these substitutions were also sensitive to inhibition by MTS reagents. On the other hand, Cd2+ did not inhibit the activities of substitutions at residues 119 and 120 on the periplasmic side of TMH2, and residues 214 and 215 in TMH4 and 252 in TMH5 at the center of the membrane. When inside-out membrane vesicles from each of these substitutions were sonicated during Cd2+ treatment to expose the periplasmic surface, the ATPase-coupled H+ transport activity was strongly inhibited. The periplasmic access to N214C and Q252C, and their positioning in the protein at the a-c interface, is consistent with previous proposals that these residues may be involved in gating H+ access from the periplasmic half-channel to Asp-61 during the protonation step.