The proton pore in the Escherichia coli F0F1-ATPase: a requirement for arginine at position 210 of the a-subunit.
Site-directed mutagenesis was used to generate three mutations in the uncB gene encoding the a-subunit of the F0 portion of the F0F1-ATPase of Escherichia coli. These mutations directed the substitution of Arg-210 by Gln, or of His-245 by Leu, or of both Lys-167 and Lys-169 by Gln. The mutations were incorporated into plasmids carrying all the structural genes encoding the F0F1-ATPase complex and these plasmids were used to transform strain AN727 (uncB402). Strains carrying either the Arg-210 or His-245 substitutions were unable to grow on succinate as sole carbon source and had uncoupled growth yields. The substitution of Lys-167 and Lys-169 by Gln resulted in a strain with growth characteristics indistinguishable from a normal strain. The properties of the membranes from the Arg-210 or His-245 mutants were essentially identical, both being proton impermeable and both having ATPase activities resistant to the inhibitor DCCD. Furthermore, in both mutants, the F1-ATPase activities were inhibited by about 50% when bound to the membranes. The membrane activities of the mutant with the double lysine change were the same as for a normal strain. The results are discussed in relation to a previously proposed model for the F0 (Cox, G.B., Fimmel, A.L., Gibson, F. and Hatch, L. (1986) Biochim. Biophys. Acta 849, 62-69).
D000595:Amino Acid Sequence; D001120:Arginine; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D010086:Oxidative Phosphorylation Coupling Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011522:Protons; D013386:Succinates / Q000378:metabolism; D019802:Succinic Acid
D001426:Bacterial Proteins; D010086:Oxidative Phosphorylation Coupling Factors; D011522:Protons; D013386:Succinates; D004024:Dicyclohexylcarbodiimide; D001120:Arginine; D019802:Succinic Acid; D006180:Proton-Translocating ATPases