Cooperation amongc-subunits of F_oF₁-ATP synthase in rotation-coupled proton translocation

In F_oF₁-ATP synthase, proton translocation through F_odrives rotation of thec-subunit oligomeric ring relative to thea-subunit. Recent studies suggest that in each step of the rotation, key glutamic acid residues in differentc-subunits contribute to proton release to and proton uptake from thea-subunit. However, no studies have demonstrated cooperativity amongc-subunits toward F_oF₁-ATP synthase activity. Here, we addressed this usingBacillusPS3 ATP synthase harboringc-ring with various combinations of wild-type andcE56D, enabled by genetically fused single-chainc-ring. ATP synthesis and proton pump activities were decreased by a singlecE56D mutation and further decreased by doublecE56D mutations. Moreover, activity further decreased as the two mutation sites were separated, indicating cooperation amongc-subunits. Similar results were obtained for proton transfer-coupled molecular simulations. Simulations revealed that prolonged proton uptake in mutatedc-subunits is shared between twoc-subunits, explaining the cooperation observed in biochemical assays.