ROTATION OF SUBUNITS DURING CATALYSIS BY ESCHERICHIA-COLI F1-ATPASE

During oxidative and photo-phosphorylation, F0F1-ATP synthases couple the movement of protons down an electrochemical gradient to the synthe sis of ATP, One proposed mechanistic feature that has remained specula tive is that this coupling process requires the rotation of subunits w ithin F0F1. Guided by a recent, high-resolution structure for bovine F -1 [Abrahams, J. P., Leslie, A, G., Lutter, R. & Walker, J, E, (1994) Nature (London) 370, 621-628], we have developed a critical test for r otation of the central gamma subunit relative to the three catalytic b eta subunits in soluble F-1 from Escherichia coli. In the bovine F-1 s tructure, a specific point of contact between the gamma subunit and on e of the three catalytic beta subunits includes positioning of the hom olog of E. coli gamma-subunit C-87 (gamma C87) close to the beta-subun it (380)DELSEED(386) sequence. A beta D380C mutation allowed us to ind uce formation of a specific disulfide bond between beta and gamma C87 in soluble E. coli F-1. Formation of the crosslink inactivated beta D3 80C-F-1, and reduction restored full activity. Using a dissociation/re assembly approach with cross-linked beta D380C-F-1, we incorporated ra diolabeled beta subunits into the two noncrosslinked beta-subunit posi tions of F-1, After reduction of the initial nonradioactive beta-gamma crosslink, only exposure to conditions for catalytic turnover results in similar reactivities of unlabeled and radiolabeled beta subunits w ith gamma C87 upon reoxidation. The results demonstrate that gamma sub unit rotates relative to the beta subunits during catalysis.