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, (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.

The vacuolar H+-ATPase: a universal proton pump of eukaryotes

“…The crucial linking subunit is the γ polypeptide, which forms a spindle of extended α-helices at the centre of the pseudo-hexameric arrangement of alternating α and β subunits in the F " sector [6]. There is now experimental evidence to show that, during the catalytic cycle, the γ subunit rotates about its long axis with respect to each α\β dimer in the F " sector, in a socalled ' entropic motor ' [173,[214][215][216]. The γ subunit also interfaces with the ε subunit [217], and both of these subunits in turn contact both the β subunit in F " [218] and subunit c in the F o sector [143,219,220].…”

A rotary molecular motor that can work at near 100% efficiency

“…In experiments where catalysis was not allowed between the two cross-linking treatments, the second target was the same as the ¢rst one. In another set of experiments (Sabbert et al 1996;HÌsler et al 1998), a £uorescent dye was attached to the g-subunit. Time-resolved polarization Figure 2.…”

Intragenic and intergenic suppression of the Escherichia coli ATP synthase subunit a mutation of Gly-213 to Asn: functional interactions between residues in the proton transport site

“…The catalytic domain, whose structure was solved by X-ray crystallography [5], consists of α $ β $ γ with one active site in each β subunit with some contribution from the neighbouring α subunit. Rotation of the γ subunit relative to the α $ β $ hexamer has been shown to have an integral role in steady-state turnover [6][7][8][9].…”