Abstract
The two opposed rotary molecular motors of the F0F1-ATP synthase work together to provide the majority of ATP in biological organisms. Rotation occurs in 120° power strokes separated by dwells when F1 synthesizes or hydrolyzes ATP. F0 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 F0 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 F0F1 embedded in lipid bilayer nanodiscs, we observed that the ability of the F0 motor to form transient dwells increases with decreasing pH. Transient dwells can halt counterclockwise rotation powered by the F1-ATPase in steps equivalent to the rotation of single c-subunits in the c-ring of F0, and can push the common axle shared by the two motors clockwise by as much as one c-subunit. Because the F0 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.
Highlights
The two opposed rotary molecular motors of the F0F1-ATP synthase work together to provide the majority of ATP in biological organisms
The three catalytic sites for ATP synthesis/hydrolysis are located at the interfaces of ␣- and -subunit heterodimers that comprise the (␣)3-ring of the F1-ATPase, which serves as the stator [9]
These results show that even though both F0 half-channels were exposed to the same pH, the periplasmic half-channel that serves as the ATP synthase proton import channel is more protonated in a manner that halted ATPase-driven rotation by blocking ATPase-dependent proton pumping
Summary
The two opposed rotary molecular motors of the F0F1-ATP synthase work together to provide the majority of ATP in biological organisms. Some power strokes rotated CCW in continuous 120° events, many were periodically interrupted by “transient dwells.” These dwells occurred at an average interval of ϳ36°, consistent with single c-subunit stepping of the c10-ring of E. coli F0F1. Transient dwells formed at low pH at intervals of ϳ36° lasted an average of 118 –158 s and were able to push the c-ring CW against the force of ATPase-dependent CCW rotation by as much as one c-subunit These results show that even though both F0 half-channels were exposed to the same pH, the periplasmic half-channel that serves as the ATP synthase proton import channel is more protonated in a manner that halted ATPase-driven rotation by blocking ATPase-dependent proton pumping
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