Proton-powered subunit rotation in single membrane-bound F0F1-ATP synthase

Abstract

Synthesis of ATP from ADP and phosphate, catalyzed by F(0)F(1)-ATP synthases, is the most abundant physiological reaction in almost any cell. F(0)F(1)-ATP synthases are membrane-bound enzymes that use the energy derived from an electrochemical proton gradient for ATP formation. We incorporated double-labeled F(0)F(1)-ATP synthases from Escherichia coli into liposomes and measured single-molecule fluorescence resonance energy transfer (FRET) during ATP synthesis and hydrolysis. The gamma subunit rotates stepwise during proton transport-powered ATP synthesis, showing three distinct distances to the b subunits in repeating sequences. The average durations of these steps correspond to catalytic turnover times upon ATP synthesis as well as ATP hydrolysis. The direction of rotation during ATP synthesis is opposite to that of ATP hydrolysis.

Bioenergetics and Biological Electron Transport

“…The 90 sub-step is driven by ATP binding and the 30 sub-step is postulated to correspond to product release. More recently, using FRET measurements, Diez et al [127] have shown that the g sub-unit rotates in the opposite direction during ATP synthesis, as expected. In addition, a further demonstration of the chemomechanical coupling has been provided by Itoh et al…”

Rotor/Stator Interactions of the ϵ Subunit in Escherichia coli ATP Synthase and Implications for Enzyme Regulation

“…Rotation of the ring of c subunits has also been documented (15)(16)(17)(18)(19)(20), as likely required for completion of the proton pathway through F 0 . Recent studies have confirmed that the rotor turns in opposite directions during ATP synthesis and hydrolysis (21,22).…”

Single-molecule FRET reveals sugar-induced conformational dynamics in LacY

“…Single-molecule f luorescence (Förster) resonance energy transfer (sm-FRET) (12)(13)(14) has been applied to the study of relatively few membrane proteins [e.g., F 1 F o -ATP synthase (15), epidermal growth factor (16), the antiporter OxlT (17), and SNARE proteins (18,19)]. Structural dynamics in these proteins has been probed by measuring sm-FRET either from freely diffusing molecules or by recording a time-trajectory from a single, immobilized protein.…”