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 THEINTACT 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 inclu des mutations that allow disulfide bond formation between the Cys at a lpha 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 gener ated between the Cys at alpha 2 and Cys-140 of the delta subunit, whic h 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 cros s-linked products. These are alpha-gamma-delta, alpha-gamma, alpha-del ta-epsilon, alpha-delta, and alpha-epsilon. The ratio of alpha-gamma-d elta 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 subu nit at which the delta subunit is bound. Also, 20% of the epsilon subu nit was present as a alpha-delta-epsilon product. With regard to the d elta 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 w as done after incubation in ATP + MgCl2. The amounts of these three pr oducts were 40, 22, and 38%, respectively, in experiments where Cu2+ w as added after preincubation in ATP + Mg2+ + azide. The delta subunit is fixed to, and therefore identifies, one specific alpha subunit (alp ha(delta)). A distribution of the gamma and epsilon subunits, which is essentially random with respect to the alpha subunits, can only be ex plained by rotation of gamma-epsilon relative to the alpha(3) beta(3) domain in ECF1F0.