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

Abstract

The two opposed rotary molecular motors of the FF-ATP synthase work together to provide the majority of ATP in biological organisms. Rotation occurs in 120° power strokes separated by dwells when F synthesizes or hydrolyzes ATP. F and F 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 F motor uses the flux of H across the membrane to induce clockwise rotation against the force of counterclockwise rotation driven by the F-ATPase. In single-molecule measurements of FF embedded in lipid bilayer nanodiscs, we observed that the ability of the F motor to form transient dwells increases with decreasing pH. Transient dwells can halt counterclockwise rotation powered by the F-ATPase in steps equivalent to the rotation of single c-subunits in the c-ring of F, and can push the common axle shared by the two motors clockwise by as much as one c-subunit. Because the F 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.

Rotary substates of mitochondrial ATP synthase reveal the basis of flexible F1-Fo coupling

“…A recent single-molecule study of F 1 F o reported forward and backward stepping of the rotor by ~30°, i.e. up to one c-subunit (18). However, with F 1 anchored to the support by subunit b and a probe attached to c, flexibility at OSCP would not be detected.…”

“…Studies aiming to characterize flexible coupling have focused on the central stalk (17), often working with an F 1 and rotor subcomplex. When the whole F 1 F o complex was examined, it was nonetheless normally attached to substrate and reporter molecules at subunits b and c to probe for flexibility along the F 1 -F o axis (9,18,19). A recent single-molecule study of F 1 F o reported forward and backward stepping of the rotor by ~30°, i.e.…”

Rotary substates of mitochondrial ATP synthase reveal the basis of flexible F 1 -F o coupling

“…Studies aiming to characterize flexible coupling have focused on the central stalk (21), often working with an F 1 and rotor subcomplex. When the whole F 1 F o complex was examined, it was typically attached to substrate and reporter molecules at subunits b and c to probe for flexibility along the F 1 -F o axis (9,22,23). A recent singlemolecule study of F 1 F o reported forward and backward stepping of the rotor by~30°(i.e., up to one c subunit) (22).…”

“…When the whole F 1 F o complex was examined, it was typically attached to substrate and reporter molecules at subunits b and c to probe for flexibility along the F 1 -F o axis (9,22,23). A recent singlemolecule study of F 1 F o reported forward and backward stepping of the rotor by~30°(i.e., up to one c subunit) (22). However, with F 1 anchored to the support by subunit b and a probe attached 4 of 10 to c, flexibility at OSCP would not be detected.…”

Structural Asymmetry and Kinetic Limping of Single Rotary F-ATP Synthases

“…When viewed from the membrane side, the two half-channels are asymmetrically arranged: the right semi-channel turns towards the P-side of the membrane, while the left semi-channel faces the N-side, to ensure obliged proton translocation into one direction only that is linked to the rotation of the central stalk and the catalysis reaction in F 1 [106,107]. Recent experiments have shown that access to the two half-channels is not the same from both sides of the membrane [108].…”