ATP-driven stepwise rotation of FOF1,-ATP synthase

FoF1-ATP synthase (FoF1) is a motor enzyme that couples ATP synthesis/hydrolysis with a transmembrane proton translocation. F-1, a water-soluble ATPase portion of FoF1, rotates by repeating ATP-waiting dwell, 80degrees substep rotation, catalytic dwell, and 40degrees-substep rotation. Compared with F-1, rotation of FoF1 has yet been poorly understood, and, here, we analyzed ATIP-driven rotations of FoF1. Rotation was probed with an 80-nm bead attached to the ring of c subunits in the immobilized FoF1 and recorded with a submillisecond fast camera. The rotation rates at various ATP concentrations obeyed the curve defined by a K-m of approximate to30 muM and a V-max of 350 revolutions per second (at 37degreesC). At low ATP, ATP-waiting dwell was seen and the k(on-ATP) was estimated to be 3.6 x 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'+[gamma-thio]triphosphate, was used, the catalytic dwells consisting of two events were seen more clearly at the angular position of approximate to80degrees. The rotational behavior of FoF1 resembles that of F-1. This finding indicates that friction in F-o motor is negligible during the ATIP-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.