ARRANGEMENT OF THE EPSILON-SUBUNIT IN THE ESCHERICHIA-COLI ATP SYNTHASE FROM THE REACTIVITY OF CYSTEINE RESIDUES INTRODUCED AT DIFFERENT POSITIONS IN THIS SUBUNIT

ECF(1)F(0) 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. ECF(1)F(0) 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 ES108C. In epsilon H38C, the introduced Cys reacted readily with N-ethylmaleimide in isolated ECF,, but was unavailable for reaction with this or other maleimides in ECF(1)F(0). When this Cys at position 38 in the epsilon subunit was reacted with various maleimides in isolated ECF(1) and then the ECF(1) bound back to F-0, the interaction between the two parts was perturbed. While ECF(1)F(0) reconstituted with unmodified ECF(1) functioned normally, enzyme with maleimide-nacted 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 ECF, 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 ECF(1)F(0) 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.