Arrangement of the epsilon subunit in the Escherichia coli ATP synthase from the reactivity of cysteine residues introduced at different positions in this subunit.

ECF1F0 has been purified from three mutants in which a Cys has been incorporated by site-directed mutagenesis in the epsilon subunit: these mutants are epsilon S10C, epsilon H38C and epsilon S108C, respectively. ECF1F0 from the mutant epsilon S10C had a 2-fold higher activity than wild-type enzyme, due to altered association of the epsilon subunit with the rest of the complex, and yet showed normal proton pumping function. The other two mutants had ATPase activities similar to wild-type enzyme. The introduced Cys was exposed for reaction with maleimides in epsilon S10C and epsilon S108C. In epsilon H38C, the introduced Cys reacted readily with N-ethylmaleimide in isolated ECF1, but was unavailable for reaction with this or other maleimides in ECF1F0. When this Cys at position 38 in the epsilon subunit was reacted with various maleimides in isolated ECF1 and then the ECF1 bound back to F0, the interaction between the two parts was perturbed. While ECF1F0 reconstituted with unmodified ECF1 functioned normally, enzyme with maleimide-reacted Cys-38 showed much reduced proton pumping, had only around 50% of the DCCD inhibition of unmodified or wild-type enzyme, and had a much higher LDAO activation (as much as 8.3-fold, c.f. 4-fold for wild type). Nucleotide-dependent conformational changes have been observed previously, in studies of ECF1 from the mutants epsilon S10C and epsilon S108C. Identical nucleotide-dependent structural changes were observed in cross-linking experiments with tetrafluorophenylazide maleimides when the intact ECF1F0 from these mutants was examined. Taken together, the Cys reactivity data and cross-linking results provide the orientation of the epsilon subunit in the enzyme complex.