On the role of Arg-210 and Glu-219 of subunit α in proton translocation by the Escherichia coli F₀F₁-ATP synthase

A strain of Escherichia coli was constructed which had a complete deletion of the chromosomal uncB gene encoding subunit a of the F₀F₁-ATP synthase. Gene replacement was facilitated by a selection protocol that utilized the sacB gene of Bacillus subtilis cloned in a kanamycin resistance cartridge (Ried, J. L., and Collmer, A. (1987) Gene (Amst.) 57, 239-246). F₀ subunits b and c inserted normally into the membrane in the ΔuncB strain. This observation confirms a previous report (Hermolin, J., and Fillingame, R. H. (1995) J. Biol. Chem. 270, 2815-2817) that subunit a is not required for the insertion of subunits b and c. The ΔuncB strain has been used to characterize mutations in Arg-210 and Glu-219 of subunit α, residues previously postulated to be essential in proton translocation. The αE219G and αE219K mutants grew on a succinate carbon source via oxidative phosphorylation and membranes from these mutants exhibited ATPase-coupled proton translocation (i.e. ATP driven 9-amino-6-chloromethoxyacridine quenching responses that were 60-80% of wild type membranes). We conclude that the αGlu-219 residue cannot play a critical role in proton translocation. The αR210A mutant did not grow on succinate and membranes exhibited no ATPase-coupled proton translocation. However, on removal of F₁ from membrane, the αR210A mutant F₀ was active in passive proton translocation, i.e. in dissipating the ΔpH normally established by NADH oxidation with these membrane vesicles. αR210A membranes with F₁ bound were also proton permeable. Arg-210 of subunit α may play a critical role in active H⁺ transport that is coupled to ATP synthesis or hydrolysis, but is not essential for the translocation of protons across the membranes.