pH-dependent 11° F₁F_O ATP synthase sub-steps reveal insight into the F_O torque generating mechanism.

Abstract

Most cellular ATP is made by rotary F₁F_O ATP synthases using proton translocation-generated clockwise torque on the F_O c-ring rotor, while F₁-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 F₁F_O structures. Here, single-molecule F₁F_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 <i>Escherichia coli</i> c₁₀-ring of F₁F_O against the torque of F₁-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 c₁₀-ring occur to sustain F₁F_O ATP synthesis.