The stalk region of the Escherichia coli ATP synthase - Tyrosine 205 of the gamma subunit is in the interface between the F-1 and F-0 parts and can interact with both the epsilon and c oligomer

The soluble portion of the Escherichia coli F1F0 ATP synthase (ECF(1)) and E. coli F1F0 ATP synthase (ECF(1)F(0)) have been isolated hom a novel mutant gamma Y205C. ECF(1) isolated from this mutant had an ATPase activity 3.5-fold higher than that of wild-type enzyme and could be activated further by maleimide modification of the introduced cysteine. This effect was not seen in ECF(1)F(0). The mutation partly disrupts the F-1 to F-0 interaction, as indicated by a reduced efficiency of proton pumping, ECF(1) containing the mutation gamma Y205C was bound to the membrane-bound portion of the E. coli F1F0 ATP synthase (ECF(0)) isolated from mutants cA39C, cQ42C, cP43C, and cD44C to reconstitute hybrid enzymes, Cu2+ treatment or reaction with 5,5'-dithio-bis(2-nitro-benzoic acid) induced disulfide bond formation between the Cys at gamma position 205 and a Cys residue at positions 42, 43, or 44 in the c subunit but not at position 39, Using Cu2+ treatment, this covalent cross-linking was obtained in yields as high as 95% in the hybrid ECF(1) gamma Y205C/cQ42C and in ECF(1)F(0) isolated from the double mutant of the same composition, The covalent linkage of the gamma to a c subunit had little effect on ATPase activity, However, ATP hydrolysis linked proton translocation was lost, by modification of both gamma Cys-205 and c Cys-42 by bulky reagents such as 5,5'-dithio-bis (2 nitro-benzoic acid) or benzophenone-4-maleimide. In both ECF(1) and ECF(1)F(0) containing a Cys at gamma 205 and a Cys in the epsilon subunit (at position 38 or 43), cross linking of the gamma to the epsilon subunit was induced in high yield by Cu2+, NO cross-linking was observed in hybrid enzymes in which the Cys was at position 10, 65, or 108 of the epsilon subunit, Cross-linking of gamma to epsilon had only a minimal effect on ATP hydrolysis, The reactivity of the Cys at gamma 205 showed a nucleotide dependence of reactivity to maleimides in both ECF(1) and ECF(1)F(0), which was lost in ECF(1) when the epsilon subunit was removed, Our results show that there is close interaction of the gamma and a subunits for the full-length of the stalk region in ECF(1)F(0). We argue that this interaction controls the coupling between nucleotide binding sites and the proton channel in ECF(1)F(0).