F(1)-ATPase is a rotary molecular motor that proceeds in 120 degrees steps, each driven by ATP hydrolysis. How the chemical reactions that occur in three catalytic sites are coupled to mechanical rotation is the central question. Here, we show by high-speed imaging of rotation in single molecules of F(1) that phosphate release drives the last 40 degrees of the 120 degrees step, and that the 40 degrees rotation accompanies reduction of the affinity for phosphate. We also show, by single-molecule imaging of a fluorescent ATP analog Cy3-ATP while F(1) is forced to rotate slowly, that release of Cy3-ADP occurs at approximately 240 degrees after it is bound as Cy3-ATP at 0 degrees . This and other results suggest that the affinity for ADP also decreases with rotation, and thus ADP release contributes part of energy for rotation. Together with previous results, the coupling scheme is now basically complete.

F(1)-ATPase is a rotary molecular motor in which unidirectional rotation of the central gamma subunit is powered by ATP hydrolysis in three catalytic sites arranged 120 degrees apart around gamma. To study how hydrolysis reactions produce mechanical rotation, we observed rotation under an optical microscope to see which of the three sites bound and released a fluorescent ATP analog. Assuming that the analog mimics authentic ATP, the following scheme emerges: (i) in the ATP-waiting state, one site, dictated by the orientation of gamma, is empty, whereas the other two bind a nucleotide; (ii) ATP binding to the empty site drives an approximately 80 degrees rotation of gamma; (iii) this triggers a reaction(s), hydrolysis and/or phosphate release, but not ADP release in the site that bound ATP one step earlier; (iv) completion of this reaction induces further approximately 40 degrees rotation.

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The unbinding and rebinding of motor proteins and their substrate filaments are the main components of sliding movement. We have measured the unbinding force between an actin filament and a single motor molecule of muscle, myosin, in the absence of ATP, by pulling the filament with optical tweezers. The unbinding force could be measured repeatedly on the same molecule, and was independent of the number of measurements and the direction of the imposed loads within a range of +/- 90 degrees. The average unbinding force was 9.2 +/- 4.4 pN, only a few times larger than the sliding force but an order of magnitude smaller than other intermolecular forces. From its kinetics we suggest that unbinding occurs sequentially at the molecular interface, which is an inherent property of motor molecules.