Cooperation among <i>c</i>-subunits of F_oF₁-ATP synthase in rotation-coupled proton translocation.

Abstract

In F_oF₁-ATP synthase, proton translocation through F_o drives rotation of the <i>c</i>-subunit oligomeric ring relative to the <i>a</i>-subunit. Recent studies suggest that in each step of the rotation, key glutamic acid residues in different <i>c</i>-subunits contribute to proton release to and proton uptake from the <i>a</i>-subunit. However, no studies have demonstrated cooperativity among <i>c</i>-subunits toward F_oF₁-ATP synthase activity. Here, we addressed this using <i>Bacillus</i> PS3 ATP synthase harboring a <i>c</i>-ring with various combinations of wild-type and <i>c</i>E56D, enabled by genetically fused single-chain <i>c</i>-ring. ATP synthesis and proton pump activities were decreased by a single <i>c</i>E56D mutation and further decreased by double <i>c</i>E56D mutations. Moreover, activity further decreased as the two mutation sites were separated, indicating cooperation among <i>c</i>-subunits. Similar results were obtained for proton transfer-coupled molecular simulations. The simulations revealed that prolonged proton uptake in mutated <i>c</i>-subunits is shared between two <i>c</i>-subunits, explaining the cooperation observed in biochemical assays.