Cooperation among c -subunits of F o F 1 -ATP synthase in rotation-coupled proton translocation.

In F o F 1 -ATP synthase, proton translocation through F o drives rotation of the c -subunit oligomeric ring relative to the a -subunit. Recent studies suggest that in each step of the rotation, key glutamic acid residues in different c -subunits contribute to proton release to and proton uptake from the a -subunit. However, no studies have demonstrated cooperativity among c -subunits toward F o F 1 -ATP synthase activity. Here, we addressed this using Bacillus PS3 ATP synthase harboring a c -ring with various combinations of wild-type and c E56D, enabled by genetically fused single-chain c -ring. ATP synthesis and proton pump activities were decreased by a single c E56D mutation and further decreased by double c E56D mutations. Moreover, activity further decreased as the two mutation sites were separated, indicating cooperation among c -subunits. Similar results were obtained for proton transfer-coupled molecular simulations. The simulations revealed that prolonged proton uptake in mutated c -subunits is shared between two c -subunits, explaining the cooperation observed in biochemical assays.