Rotation of the ε subunit during catalysis by Escherichia coli F0F1-ATP synthase

We report evidence for catalysis-dependent rotation of the single epsilon subunit relative to the three catalytic beta subunits of functionally coupled, membrane-bound FOF1-ATP synthase, Cysteines substituted at beta 380 and epsilon 108 allowed rapid formation of a specific beta-epsilon disulfide cross-link upon oxidation, Consistent with a need for epsilon to rotate during catalysis, tethering epsilon to one of the beta subunits resulted in the inhibition of both ATP synthesis and hydrolysis, These activities were fully restored upon reduction of the beta-epsilon cross-link. As a more critical test for rotation, a subunit dissociation/reassociation procedure was used to prepare a beta-epsilon cross-linked hybrid F-1, having epitope-tagged beta D380C subunits (beta(flag)) exclusively in the two noncross-linked positions. This allowed the beta subunit originally aligned with epsilon to form the crosslink to be distinguished from the other two beta s. The cross-linked hybrid was reconstituted with F-O in F-1-depleted membranes. After reduction of the beta-epsilon cross-link and a brief period of catalytic turnover, reoxidation resulted in a significant amount of beta(flag) in the beta-epsilon cross-linked product. In contrast, exposure to ligands that bind to the catalytic site but do not allow catalysis resulted in the subsequent cross-linking of epsilon to the original untagged beta, Furthermore, catalysis dependent rotation of epsilon was prevented by prior treatment of membranes with N,N'-dicyclohexylcarbodiimide to block. proton translocation through F-O, From these results, we conclude that epsilon is part of the rotor that couples proton transport to ATP synthesis.