| Research Abstract |
We demonstrate that ATP synthase is the enzyme which has a rotating subunit. ATP synthase is composed of two separable parts, a membraneous portion, Fo, which mediates proton translocation, and a peripheral portion, F_1-ATPase, which catalyzes ATP hydrolysis. Active sites of both reactions are separated from each other by a narrow, long (-45) stalk. Hypothesis of rotational catalysis of ATP synthase gained a structural support from the crystal structure of mitochondrial F_1-ATPase in which the coiled-coil alpha helices of the gamma subunit extend from the central cavity of the hexagonal alpha_3beta_3 subassembly into the stalk region. Then, large motion of the gamma subunit was suggested from biochemical and optical studies. We show direct, real time observation of the rotation of the gamma subunit in a single molecule of alpha_3beta_3gamma subcomplex of thermophilic F_1-ATPase. We fixed the molecules on a glass plate, attached fluorescently labeled actin filament on top of the gamma subunit, and observed its rotation. The rotation was absolutely dependent on ATP,continued for more than 100 revolutions, and was inhibited by NaN_3, an inhibitor of F_1-ATPase. The gamma subunit rotated anti-clockwise when viewed from the membrane side. The rotary torque amounted to as much as -40 pN・nm.
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