ATP-driven stepwise rotation of F_oF₁-ATP synthase

F_oF₁-ATP synthase (F_oF₁) is a motor enzyme that couples ATP synthesis/hydrolysis with a transmembrane proton translocation. F₁, a water-soluble ATPase portion of F _oF₁, rotates by repeating ATP-waiting dwell, 80° substep rotation, catalytic dwell, and 40°-substep rotation. Compared with F₁, rotation of F_oF₁ has yet been poorly understood, and, here, we analyzed ATP-driven rotations of F_oF ₁. Rotation was probed with an 80-nm bead attached to the ring of c subunits in the immobilized F_oF₁ and recorded with a submillisecond fast camera. The rotation rates at various ATP concentrations obeyed the curve defined by a K_m of ≈30 μM and a V _max of ≈350 revolutions per second (at 37°C). At low ATP, ATP-waiting dwell was seen and the k_on-ATP was estimated to be 3.6 × 10⁷ M^-1·s^-1. At high ATP, fast, poorly defined stepwise motions were observed that probably reflect the catalytic dwells. When a slowly hydrolyzable substrate, adenosine 5′-[γ-thio]triphosphate, was used, the catalytic dwells consisting of two events were seen more clearly at the angular position of ≈80°. The rotational behavior of F_oF₁ resembles that of F ₁. This finding indicates that "friction" in F_o motor is negligible during the ATP-driven rotation. Tributyltin chloride, a specific inhibitor of proton translocation, slowed the rotation rate by 96%. However, dwells at clearly defined angular positions were not observed under these conditions, indicating that inhibition by tributyltin chloride is complex.