Stochastic high-speed rotation of Escherichia coli ATP synthase F1 sector -: The ε subunit-sensitive rotation

The gamma subunit of the ATP synthase F-1 sector rotates at the center of the alpha(3)beta(3) hexamer during ATP hydrolysis. A gold bead (40-200 nm diameter) was attached to the gamma 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 similar to 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 similar to 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 epsilon subunit inhibited rotation and ATPase, whereas epsilon fused through its carboxyl terminus to cytochrome b(562) showed no effect. The epsilon 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.