ON THE ROLE OF ARG-210 AND GLU-219 OF SUBUNIT-ALPHA IN PROTON TRANSLOCATION BY THE ESCHERICHIA-COLI F0F1-ATP SYNTHASE

A strain of Escherichia coli was constructed which had a complete dele tion of the chromosomal uncB gene encoding subunit alpha of the F0F1-A TP 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 membra ne in the Delta uncB strain. This observation confirms a previous repo rt (Hermolin, J., and Fillingame, R. H. (1995) J. Biol. Chem, 270, 281 5-2817) that subunit alpha is not required for the insertion of subuni ts b and c, The Delta uncB strain has been used to characterize mutati ons in Arg-210 and Glu-219 of subunit alpha, residues previously postu lated to be essential in proton translocation, The alpha E219G and alp ha E219K mutants grew on a succinate carbon source via oxidative phosp horylation and membranes from these mutants exhibited ATPase-coupled p roton translocation (i.e. ATP driven 9-amino-6-chloromethoxyacridine q uenching responses that were 60-80% of wild type membranes), We conclu de that the alpha Glu-219 residue cannot play a critical role in proto n translocation. The alpha R210A mutant did not grow on succinate and membranes exhibited no ATPase-coupled proton translocation, However, o n 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 norm ally 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 cou pled to ATP synthesis or hydrolysis, but is not essential for the tran slocation of protons across the membranes.