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, (αβ)3γδη, and is active as an ATPase1,2. 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 release6 at cooperative and rotating (or at least alternating) sites7. 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 γ) within (αβ)3, the catalytic hexagon of F1 as suggested by electron microscopy8, by X-ray crystal structure analysis9 and by the use of cleavable crosslinkers10. 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 γ. We observe the rotation of γ relative to immobilized (αβ)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 catalysis7,11, with γ acting as a crankshaft in (αβ)3. The rotation of γ is blocked when ATP is substituted with its non-hydrolysable analogue AMP-PNP.

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