Characterization of the temperature-sensitive reaction of F(1)-ATPase by using single-molecule manipulation

F(1)-ATPase (F(1)) is a rotary motor protein that couples ATP hydrolysis to mechanical rotation with high efficiency. In our recent study, we observed a highly temperature-sensitive (TS) step in the reaction catalyzed by a thermophilic F(1) that was characterized by a rate constant remarkably sensitive to temperature and had a Q(10) factor of 6–19. Since reactions with high Q(10) values are considered to involve large conformational changes, we speculated that the TS reaction plays a key role in the rotation of F(1). To clarify the role of the TS reaction, in this study, we conducted a stall and release experiment using magnetic tweezers, and assessed the torque generated during the TS reaction. The results indicate that the TS reaction generates the same amount of rotational torque as does ATP binding, but more than that generated during ATP hydrolysis. Thus, we confirmed that the TS reaction contributes significantly to the rotation of F(1).