Stochastic High-speed Rotation of Escherichia coli ATP Synthase F1 Sector

The γ subunit of the ATP synthase F1 sector rotates at the center of the α3β3 hexamer during ATP hydrolysis. A gold bead (40–200 nm diameter) was attached to the γ subunit of Escherichia coli F1, and then its ATP hydrolysis-dependent rotation was studied. The rotation speeds were variable, showing stochastic fluctuation. The high-speed rates of 40- and 60-nm beads were essentially similar: 721 and 671 rps (revolutions/s), respectively. The average rate of 60-nm beads was 381 rps, which is ∼13-fold faster than that expected from the steady-state ATPase turnover number. These results indicate that the F1 sector rotates much faster than expected from the bulk of ATPase activity, and that ∼10% of the F1 molecules are active on the millisecond time scale. Furthermore, the real ATP turnover number (number of ATP molecules converted to ADP and phosphate/s), as a single molecule, is variable during a short period. The ϵ subunit inhibited rotation and ATPase, whereas ϵ fused through its carboxyl terminus to cytochrome b562 showed no effect. The ϵ subunit significantly increased the pausing time during rotation. Stochastic fluctuation of catalysis may be a general property of an enzyme, although its understanding requires combining studies of steady-state kinetics and single molecule observation.