Rotation of a gamma-epsilon subunit domain in the Escherichia coli F1F0-ATP synthase complex - The gamma-epsilon subunits are essentially randomly distributed relative to the alpha(3)beta(3)delta domain in the intact complex

A triple mutant of Escherichia coli F1F0-ATP synthase, alpha Q2C/alpha S411C/epsilon S108C, has been generated for studying movements of the gamma and epsilon subunits during functioning of the enzyme, It includes mutations that allow disulfide bond formation between the Cys at alpha 411 and both Cys-87 of gamma and Cys-108 of epsilon, two covalent cross-links that block enzyme function (Aggeler, R., and Capaldi, R. A. (1996) J. Biol. Chem. 271, 13888-13891). A cross-link is also generated between the Cys at alpha 2 and Cys-140 of the delta subunit, which has no effect on functioning (Ogilvie, I., Aggeler, R., and Capaldi, R. A. (1997) J. Biol. Chem. 272, 16652-16656), CuCl2 treatment of the mutant alpha Q2C/ alpha S411C/epsilon S108C generated five major cross-linked products. These are alpha-gamma-delta, alpha-gamma, alpha-delta-epsilon, alpha-delta, and alpha-epsilon. The ratio of alpha-gamma-delta to the alpha-gamma product was close to 1:2, i.e. in one-third of the ECF1F0 molecules the gamma subunit was attached to the alpha subunit at which the delta subunit is bound. Also, 20% of the epsilon subunit was present as a alpha-delta-epsilon product. With regard to the delta subunit, 30% was in the alpha-gamma-delta, 20% in the alpha-delta-epsilon, and 50% in the alpha-delta products when the cross-linking was done after incubation in ATP + MgCl2. The amounts of these three products were 40, 22, and 38%, respectively, in experiments where Cu2+ was added after preincubation in ATP + Mg2+ + azide. The delta subunit is fixed to, and therefore identifies, one specific alpha subunit (alpha(delta)). A distribution of the gamma and epsilon subunits, which is essentially random with respect to the alpha subunits, can only be explained by rotation of gamma-epsilon relative to the alpha(3) beta(3) domain in ECF1F0.