Intersubunit rotation in active F-ATPase.
The enzyme ATP synthase, or F-ATPase, is present in the membranes of bacteria, chloroplasts and mitochondria. Its structure is bipartite, with a proton-conducting, integral membrane portion, F0, and a peripheral portion, F1. Solubilized F1 is composed of five different subunits, (alpha beta)3 gamma delta epsilon, and is active as an ATPase. The function of F-ATPase is to couple proton translocation through F0 with ATP synthesis in F1 (ref.3). Several lines of evidence support the spontaneous formation of ATP on F1 (refs 4,5) and its endergonic release at cooperative and rotating (or at least alternating) sites. The release of ATP at the expense of protonmotive force might involve mechanical energy transduction from F0 into F1 by rotation of the smaller subunits (mainly gamma) within (alpha beta)3, the catalytic hexagon of F1 as suggested by electron microscopy, by X-ray crystal structure analysis and by the use of cleavable crosslinkers. Here we record an intersubunit rotation in real time in the functional enzyme by applying polarized absorption relaxation after photobleaching to immobilized F1 with eosin-labelled gamma. We observe the rotation of gamma relative to immobilized (alpha beta)3 in a timespan of 100 ms, compatible with the rate of ATP hydrolysis by immobilized F1. Its angular range, which is of at least 200 degrees, favours a triple-site mechanism of catalysis, with gamma acting as a crankshaft in (alpha beta)3. The rotation of gamma is blocked when ATP is substituted with its non-hydrolysable analogue AMP-PNP.
Sabbert
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D
Abteilung Biophysik, Fachbereich Biologie/Chemie, Universität Osnabrück, Germany.
Engelbrecht
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S
Junge
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W
D000255:Adenosine Triphosphate / Q000378:metabolism; D016880:Anisotropy; D004789:Enzyme Activation; D004800:Enzymes, Immobilized; D006868:Hydrolysis; D008958:Models, Molecular; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism*; D018724:Spinacia oleracea
D004800:Enzymes, Immobilized; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases