Oxidative phosphorylation by mutant Escherichia coli membranes with impaired proton permeability

Abstract

The effect on the function of the E. coli F1F0-ATPase of the substitution of Leu-31 by phenylalanine in the c-subunit of the enzyme was examined. The assembly of the mutant c-subunit requires an increased gene dosage and this was achieved by incorporation of the uncE408 or uncE463 alleles on to F-plasmids or multicopy plasmids. Membranes from strains carrying the uncE463 or uncE408 alleles on F-plasmids or multicopy plasmids were capable of carrying out oxidative phosphorylation. Membranes from strain AN1928 (pAN162, uncE463) gave phosphorylation rates and P/O ratios equal to or greater than those obtained for the control strain AN1460 (pAN45, unc+). The mutant membranes, on removal of the F1-ATPase, appeared to be proton-impermeable. The ATPase activity of the mutant membranes was also resistant to the inhibitor dicyclohexylcarbodi-imide.