Abstract
Most cellular ATP is made by rotary F(1)F(O) ATP synthases using proton translocation-generated clockwise torque on the F(O) c-ring rotor, while F(1)-ATP hydrolysis can force counterclockwise rotation and proton pumping. The F(O) torque-generating mechanism remains elusive even though the F(O) interface of stator subunit-a, which contains the transmembrane proton half-channels, and the c-ring is known from recent F(1)F(O) structures. Here, single-molecule F(1)F(O) rotation studies determined that the pKa values of the half-channels differ, show that mutations of residues in these channels change the pKa values of both half-channels, and reveal the ability of F(O) to undergo single c-subunit rotational stepping. These experiments provide evidence to support the hypothesis that proton translocation through F(O) operates via a Grotthuss mechanism involving a column of single water molecules in each half-channel linked by proton translocation-dependent c-ring rotation. We also observed pH-dependent 11° ATP synthase-direction sub-steps of the Escherichia coli c(10)-ring of F(1)F(O) against the torque of F(1)-ATPase-dependent rotation that result from H(+) transfer events from F(O) subunit-a groups with a low pKa to one c-subunit in the c-ring, and from an adjacent c-subunit to stator groups with a high pKa. These results support a mechanism in which alternating proton translocation-dependent 11° and 25° synthase-direction rotational sub-steps of the c(10)-ring occur to sustain F(1)F(O) ATP synthesis.