Intersubunit rotation in active F-ATPase

Abstract

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 ATPase^1,2. 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 catalysis^7,11, 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.