Obstruction of transmembrane helical movements in subunit a blocks proton pumping by F1Fo ATP synthase.

Subunit a plays a key role in promoting H+ transport-coupled rotary motion of the subunit c ring in F1Fo ATP synthase. H+ binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of Fo subunit c. H+ are thought to reach cAsp61 via aqueous half-channels formed by TMHs 2–5 of subunit a. Movements of TMH4 and TMH5 have been proposed to facilitate protonation of cAsp61 from a half channel centered in a four helix bundle at the periplasmic side of subunit a. The possible necessity of these proposed TMH movements was investigated by assaying ATP driven H+ pumping function before and after cross-linking paired Cys substitutions at the center of TMHs within subunit a. The cross-linking of the Cys pairs aG218C/I248C in TMH4 and TMH5, and aL120C/H245C in TMH2 and TMH5, inhibited H+ pumping by 85–90%. H+ pumping function was largely unaffected by modification of the same Cys residues in the absence of cross-link formation. The inhibition is consistent with the proposed requirement for TMH movements during the gating of periplasmic H+ access to cAsp61. The cytoplasmic loops of subunit a have been implicated in gating H+ release to the cytoplasm, and previous cross-linking experiments suggest that the chemically reactive regions of the loops may pack as a single domain. Here we show that Cys substitutions in these domains can be cross-linked with retention of function and conclude that these domains need not undergo large conformational changes during enzyme function.