, 
*Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259, Yokohama 226-8503, Japan; ATP System Project, Exploratory Research for Advanced Technology, Japan Science and Technology Agency, Nagatsuta 5800-3, Yokohama 226-0026, Japan; and Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Higashiyama 5-1 Myodaiji, Okazaki 444-8787, Japan
Edited by Paul D. Boyer, University of California, Los Angeles, CA and approved December 13, 2004 (received for review October 21, 2004)
FoF1-ATP synthase (FoF1) is a motor enzyme that couples ATP synthesis/hydrolysis with a transmembrane proton translocation. F1, a water-soluble ATPase portion of FoF1, rotates by repeating ATP-waiting dwell, 80° substep rotation, catalytic dwell, and 40°-substep rotation. Compared with F1, rotation of FoF1 has yet been poorly understood, and, here, we analyzed ATP-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 Km of 
30 µM and a Vmax of 350 revolutions per second (at 37°C). At low ATP, ATP-waiting dwell was seen and the kon-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'-[
-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 FoF1 resembles that of F1. This finding indicates that "friction" in Fo 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.
ATP hydrolysis | binding change mechanism | membrane protein | single-molecule imaging
Freely available online through the PNAS open access option.
Abbreviations: ATPS, adenosine 5'-[-thio]triphosphate; DCCD, N,N'-dicyclohexylcarbodiimide; LPC, lysophosphatidylcholine; Ni2+-NTA, Ni2+-nitrilotriacetic acid; rps, revolutions per second; TBT-Cl, tributyltin chloride.
To whom correspondence should be addressed. E-mail: myoshida{at}res.titech.ac.jp.
© 2005 by The National Academy of Sciences of the USA
