A novel method for detecting F
1-ATPase rotation in a manner sufficiently sensitive to achieveacquisition rates with a time resolution of 2.5
s (equivalent to 400 000 fps) is reported. This is sufficientfor resolving the rate at which the
-subunit travels from one dwell state to another (transition time).Rotation is detected via a gold nanorod attached to the rotating
-subunit of an immobilized F
1-ATPase.Variations in scattered light intensity allow precise measurement of changes in the angular position of therod below the diffraction limit of light. Using this approach, the transition time of
Escherichia coli F
1-ATPase
-subunit rotation was determined to be 7.62 ± 0.15 (st
andard deviation) rad/ms. The averagerate-limiting dwell time between rotation events observed at the saturating substrate concentration was8.03 ms, comparable to the observed Mg
2+-ATPase
kcat of 130 s
-1 (7.7 ms). Histograms of scattered lightintensity from ATP-dependent nanorod rotation as a function of polarization angle allowed the determinationof the nanorod orientation with respect to the axis of rotation
and plane of polarization. This informationallowed the drag coefficient to be determined, which implied that the instantaneous torque generated byF
1 was 63.3 ± 2.9 pN nm. The high temporal resolution of rotation allowed the measurement of theinstantaneous torque of F
1, resulting in direct implications for its rotational mechanism.