CHARACTERIZATION OF MUTATIONS IN THE B-SUBUNIT OF F(1)F(0) ATP SYNTHASE IN ESCHERICHIA-COLI

Abstract

Site-directed mutagenesis was used to investigate the restrictions on Ala-79 of the b subunit in F1F0 adenosine triphosphate synthase. This amino acid had been previously identified as particularly sensitive to mutation (McCormick, K. A., and Cain, B. D. (1991) J. Bacteriol. 173, 7240-7248). Mutant uncF (b) genes were placed under control of the lac promoter and monitored for F1F0 ATP synthase function in an uncF(b) deletion strain. Three deleterious b(Ala-79) mutations were moved to the unc operon in the chromosome by homologous recombination. Decreases in enzymatic activity in the uncF (b) mutant strains resulted from reduced amounts of enzyme. With the exception of the b(Ala-79-->Pro) mutation, high expression of mutant uncF (b) genes resulted in increases in F1F0 ATP synthase activity which were sufficient to overcome the defects. In addition to the decrease in the amount of enzyme, the b(Ala-79-->Lys) mutation affected ATP synthesis to a much greater extent than ATP-driven proton translocation. The evidence supports our earlier hypothesis, in which b(Ala-79) was proposed to play an important, but not essential, structural role in F1F0 ATP synthase assembly or stability.