The role of subunit a in promoting proton translocation and rotary motion in the Escherichia coli F 1 F o ATP synthase is poorly understood. In the membrane-bound F o sector of the enzyme, H + binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of subunit c . Protons are thought to reach Asp-61 at the center of the membrane via aqueous channels formed at least in part by one or more of the five TMHs of subunit a . Aqueous access pathways have previously been mapped to surfaces of a TMH4. Here we have substituted Cys into the second and fifth TMHs of subunit a and carried out chemical modification with Ag + and N -ethylmaleimide to define the aqueous accessibility of residues along these helices. Access to c Asp-61 at the center of the membrane may be mediated in part by Ag + -sensitive residues 248, 249, 251, and 252 in a TMH5. From the periplasmic surface, aqueous access to c Asp-61 may be mediated by silver-sensitive residues 115, 116, 119, 120, 122, and 126 in a TMH2. The Ag + -sensitive residues in TMH2, -4, and -5 form a continuum extending from the periplasmic to the cytoplasmic side of the membrane. In an arrangement of helices supported by second-site revertant and crosslinking analyses, these residues cluster at the interior of a four-helix bundle formed by TMH2–5. The aqueous access pathways at the interior of subunit a may be gated by a swiveling of helices in this bundle, alternately exposing cytoplasmic and periplasmic half channels to c Asp-61 during the H + transport cycle.

Angevine, C. M., Herold, K. A. G., & Fillingame, R. H. (2003). Aqueous access pathways in subunit a of rotary ATP synthase extend to both sides of the membrane. Proceedings of the National Academy of Sciences, 100(23), 13179–13183.