F0F1-ATPase gamma subunit mutations perturb the coupling between catalysis and transport.
We introduced mutations to test the function of the conserved amino-terminal region of the gamma subunit from the Escherichia coli ATP synthase (F0F1-ATPase). Plasmid-borne mutant genes were expressed in an uncG strain which is deficient for the gamma subunit (gamma Gln-14-->end). Most of the changes, which were between gamma Ile-19 and gamma Lys-33, gamma Asp-83 and gamma Cys-87, or at gamma Asp-165, had little effect on growth by oxidative phosphorylation, membrane ATPase activity, or H+ pumping. Notable exceptions were gamma Met-23-->Arg or Lys mutations. Strains carrying these mutations grew only very slowly by oxidative phosphorylation. Membranes prepared from the strains had substantial levels of ATPase activity, 100% compared with wild type for gamma Arg-23 and 65% for gamma Lys-23, but formed only 32 and 17%, respectively, of the electrochemical gradient of protons. In contrast, other mutant enzymes with similar ATPase activities (including gamma Met-23-->Asp or Glu) formed H+ gradients like the wild type. Membranes from the gamma Arg-23 and gamma Lys-23 mutants were not passively leaky to protons and had functional F0 sectors. These results suggested that substitution by positively charged side chains at position 23 perturbed the energy coupling. The catalytic sites of the mutant enzymes were still regulated by the electrochemical H+ gradient but were inefficiently coupled to H+ translocation in both ATP-dependent H+ pumping and delta mu H+ driven ATP synthesis.
Shin
K
K
Department of Organic Chemistry and Biochemistry, Osaka University, Japan.
Nakamoto
R K
RK
Maeda
M
M
Futai
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004024:Dicyclohexylcarbodiimide; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D006180:Proton-Translocating ATPases