pH-dependent 11° F1FO ATP synthase sub-steps reveal insight into the FO torque generating mechanism

Most cellular ATP is made by rotary F1FO 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 F1FO structures. Here, single-molecule F1FO 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 degrees ATP synthase-direction sub-steps of the Escherichia coli c(10)-ring of F1FO 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 degrees and 25 degrees synthase-direction rotational sub-steps of the c(10)-ring occur to sustain F1FO ATP synthesis.

Automated summary

This study investigates the torque-generating mechanism in the F1FO ATP synthase, showing that mutations in the half-channels can change their pKa values and ultimately affect proton translocation. The experiments also reveal the ability of F-O to undergo single c-subunit rotational stepping, supporting a Grotthuss mechanism involving single water molecules in each half-channel linked by c-ring rotation. Furthermore, the pH-dependent 11-degree ATP synthase-direction sub-steps of the c(10)-ring were observed, providing evidence for a mechanism involving alternating proton translocation-dependent rotational sub-steps to sustain F1FO ATP synthesis.