Protonation-dependent stepped rotation of the F-type ATP synthase c-ring observed by single-molecule measurements [Molecular Biophysics]

The two opposed rotary molecular motors of the FOF1 ATP synthase work together to provide the majority of ATP in most biological organisms. Rotation occurs in 120° power strokes separated by dwells when F1 synthesizes or hydrolyzes ATP. FO and F1 complexes connect via a central rotor stalk and a peripheral stator stalk. A major unresolved question is the mechanism in which the interaction between subunit a and rotating subunit c-ring in the FO motor uses the flux of H+ across the membrane to induce clockwise rotation against the force of counterclockwise rotation driven by the F1-ATPase. In single-molecule measurements of FOF1 embedded in lipid bilayer nanodiscs, we observed that the ability of the FO motor to form transient dwells increases with decreasing pH. Transient dwells can halt CCW rotation powered by the F1-ATPase in steps equivalent to the rotation of single c-subunits in the c-ring of FO, and can push the common axle shared by the two motors clockwise by as much as one c-subunit. Since the FO proton half-channels that access the periplasm and the cytoplasm are exposed to the same pH, these data are consistent with the conclusion that the periplasmic half-channel is more easily protonated in a manner that halts ATPase-driven rotation by blocking ATPase-dependent proton pumping. The fit of transient dwell occurrence to the sum of three Gaussian curves suggests that the asymmetry of the three ATPase-dependent 120° power strokes imposed by the relative positions of the central and peripheral stalks affects c-subunit stepping efficiency.

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