Stochastic high‐speed rotation of Escherichia coli ATP synthase F 1 sector: the ε subunit‐sensitive rotation

Abstract

The γ subunit of the ATP synthase F1 sector rotates at the center of the α 3β 3 hexamer during ATP hydrolysis. A gold bead (40 ~ 200 nm diameter) was attached to the γ subunit of E. 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 nm and 60 nm beads were essentially similar: 721 and 671 rps (revolutions/second), 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 bulk ATPase activity, and that ~ 10 % of F1 molecules are active on the m sec time scale. Furthermore, the real ATP turnover number (number of ATP molecules converted to ADP and phosphate per sec), 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 significantly increased the pausing time during the rotation. Stochastic fluctuation of the catalysis may be a general property of an enzymes, although its understanding requires combining studies of steady-state kinetics and single molecule observation.