Chemo-Mechanical Coupling in F₁-ATPase Revealed by Catalytic Site Occupancy during Catalysis

Rieko Shimo-Kon^†, Eiro Muneyuki^‡, Hiroshi Sakai^§, Kengo Adachi^†, Masasuke Yoshida^¶ and Kazuhiko Kinosita^†*

^† Department of Physics, Faculty of Science and Engineering, Waseda University, Okubo, Shinjuku-ku, Tokyo, Japan
^‡ Department of Physics, Faculty of Science and Engineering, Chuo University, Kasuga, Bunkyo-ku, Tokyo, Japan
^§ Department of Food and Nutritional Sciences, Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, Yada, Shizuoka, Japan
^¶ Bio-Resources Division, Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama, Japan

^*Corresponding author

Abstract

F₁-ATPase is a rotary molecular motor in which the central γ subunit rotates inside a cylinder made of α₃β₃ subunits. To clarify how ATP hydrolysis in three catalytic sites cooperate to drive rotation, we measured the site occupancy, the number of catalytic sites occupied by a nucleotide, while assessing the hydrolysis activity under identical conditions. The results show hitherto unsettled timings of ADP and phosphate releases: starting with ATP binding to a catalytic site at an ATP-waiting γ angle defined as 0°, phosphate is released at ∼200°, and ADP is released during quick rotation between 240° and 320° that is initiated by binding of a third ATP. The site occupancy remains two except for a brief moment after the ATP binding, but the third vacant site can bind a medium nucleotide weakly.