ATP hydrolysis by membrane-bound Escherichia coli F0F1 causes rotation of the γ subunit relative to the β subunits

We recently demonstrated that the gamma subunit in soluble F-1-ATPase from Escherichia coli rotates relative to surrounding beta subunits during catalytic turnover (Duncan et al. (1995) Proc. Natl. Acad. Sci. USA 92, 10964-10968). Here, we extend our studies to the more physiologically relevant membrane-bound F0F1 complex. It is shown that beta D380C-F-1, containing a beta-gamma intersubunit disulfide bond, can bind to F-1-depleted membranes and can restore coupled membrane activities upon reduction of the disulfide. Using a dissociation/reconstitution approach with crosslinked PD380C-F-1, beta subunits containing an N-terminal Flag epitope (beta(flag)) were incorporated into the two non-crosslinked beta positions and the hybrid F-1 was reconstituted with membrane-bound F-0. Following reduction and ATP hydrolysis, reoxidation resulted in a significant amount of crosslinking of beta(flag) to the gamma subunit. This demonstrates that gamma rotates within F-1 during catalytic turnover by membrane-bound F0F1. Furthermore, the rotation of gamma is functionally coupled to F-0, since preincubation with DCCD to modify F-0 blocked rotation.