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 alpha 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). F-0 subunits b and c inserted normally into the membrane in the Delta uncB strain. This observation confirms a previous report (Hermolin, J., and Fillingame, R. H. (1995) J. Biol. Chem, 270, 2815-2817) that subunit alpha is not required for the insertion of subunits b and c, The Delta uncB strain has been used to characterize mutations in Arg-210 and Glu-219 of subunit alpha, residues previously postulated to be essential in proton translocation, The alpha E219G and alpha E219K 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 alpha Glu-219 residue cannot play a critical role in proton translocation. The alpha R210A mutant did not grow on succinate and membranes exhibited no ATPase-coupled proton translocation, However, on removal of F-1 from membrane, the alpha R210A mutant F-0 was active in passive proton translocation, i.e. in dissipating the Delta pH normally established by NADH oxidation with these membrane vesicles. alpha R210A membranes with F-1 bound were also proton permeable. Arg-210 of subunit a 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.