Single-Molecule Physiology on the Molecular Mechanism of the Rotary Motor F1-ATPase Kazuhiko Kinosita, Jr. Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences Higashiyama 5-1, Myodaiji, Okazaki 444-8787, Japan http://www.k2.ims.ac.jp 　　　　　The protein F1-ATPase is a rotary motor made of a single molecule. In 1997, 　　　　　we videotaped its motion under an optical microscope [1], as the final proof that 　　　　　its central γ subunit indeed rotates inside a surrounding cylinder made of α3β3 　　　　　subunits. Since then we have shown, by single-molecule imaging, that (i) the rotary 　　　　　torque is nearly independent of the rotation angle [2], (ii) 80-90 pN nm of 　　　　　mechanical work can be done per ATP hydrolyzed [3], (iii) binding of ATP causes 　　　　　80-90°rotation [4,5], and (iv) release of a hydrolysis product causes further 　　　　　40-30°rotation [4,5]. Point ii implies that the efficiency of chemo-mechanical 　　　　　conversion may reach 〜100%, though this statement needs to be qualified [6]. 　　　　　Points i-iv allowed us to infer the angle-dependent potential energies for γ rotation 　　　　　for each of chemical intermediates that appear during rotation [4]. On the basis 　　　　　of these potential energies and using a toy model for illustration, we have 　　　　　suggested how the free energy of ATP hydrolysis may be converted to mechanical 　　　　　torque, and more importantly, how the reverse rotation of the motor by an 　　　　　external force may lead to ATP synthesis [6]. The most important aspect is that 　　　　　binding (and release) of a nucleotide, rather than hydrolysis per se, is the major 　　　　　source of mechanical output. An equally important corollary is that mechanical 　　　　　motion (rotation) changes the affinity for a nucleotide by orders of magnitude, 　　　　　the essential ingredient of Boyer’s binding-change model for ATP synthesis [7]. 　　　　　We have now shown, using magnetic tweezers, that reverse rotation of F1 indeed 　　　　　produces ATP [8]. Possibly for the first time, energetically uphill chemical 　　　　　synthesis has been accomplished by the action of mechanical force generated by 　　　　　human artifacts (with the aid of the nature’s nano machine). If time allows, I will 　　　　　also mention our recent attempts at clarifying precisely how the three catalytic 　　　　　sites in F1 cooperate to produce rotation. 　　　　　[1] Noji, H., Yasuda, R., Yoshida, M. & Kinosita, K. Jr. Direct observation of the 　　　　　　　rotation of F1-ATPase. Nature 386, 299-302 (1997). 　　　　　[2] Kinosita, K. Jr., Yasuda, R., Noji, H. & Adachi, K. A rotary molecular motor 　　　　　　　that can work at near 100% efficiency. Phil. Trans. R. Soc. Lond. B 355, 　　　　　　　473-489 (2000). 　　　　　[3] Yasuda, R., Noji, H., Kinosita, K. Jr. & Yoshida, M. F1-ATPase is a highly 　　　　　　　efficient molecular motor that rotates with discrete 120°steps. Cell 93, 　　　　　　　1117-1124 (1998). 　　　　　[4] Yasuda, R., Noji, H., Yoshida, M., Kinosita, K. Jr. & Itoh, H. Resolution of 　　　　　　　distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase. 　　　　　　　Nature 410, 898-904 (2001). 　　　　　[5] Nishizaka, T., Oiwa, K., Noji, H., Kimura, S., Muneyuki, E., Yoshida, M. & Kinosita, 　　　　　　　K., Jr. Chemo-mechanical coupling in F1-ATPase revealed by simultaneous 　　　　　　　observation of nucleotide kinetics and rotation. Nature Struct. Mol. Biol. 11, 　　　　　　　142-148 (2004). 　　　　　[6] Kinosita, R., Jr., Adachi, K. & Itoh, H. Rotation of F1-ATPase: How an 　　　　　　　ATP-driven molecular machine may work. Annu. Rev. Biophys. Biomol. Struct. 　　　　　　　33, 245-268 (2004). 　　　　　[7] Boyer, P. D. The binding change mechanism for ATP synthase - some 　　　　　　　probabilities and possibilities. Biochim. Biophys. Acta 1140, 215-250 (1993). 　　　　　[8] Itoh, H., Takahashi, A., Adachi, K., Noji, H., Yasuda, R., Yoshida, M. & Kinosita, K. 　　　　　　　Jr. Mechanically-driven ATP synthesis by F1-ATPase. Nature 427, 465-468 　　　　　　　(2004).