Suppressor mutations in F1 subunit epsilon recouple ATP-driven H+ translocation in uncoupled Q42E subunit c mutant of Escherichia coli F1F0 ATP synthase.
The Q42E mutation in the polar loop of subunit c of the Escherichia coli F1F0 ATP synthase leads to an uncoupling of H+ translocation through F0 and ATP synthesis/hydrolysis in F1. We have isolated four second-site suppressor mutants in which the coupling defect is corrected. Substitutions for Glu31 in F1 subunit epsilon were found in each suppressor mutant, where the substitutions were E31G, E31V, and E31K (the last being found twice). The different substitutions vary in effectiveness in restoring wild type growth properties in the order epsilon E31G > epsilon E31V > epsilon E31K. Biochemical properties of epsilon E31G/cQ42E and epsilon E31K/cQ42E membranes were compared. In epsilon E31G/cQ42E mutant membranes, ATP-driven H+ translocation by F1F0 and the binding and coupling of F1 to F0 showed a striking pH dependence. Near normal function was observed at pH 7.0, but function was lost at pH 7.8. The function of epsilon E31K/cQ42E membranes was much less affected by changes in pH. Relative to epsilon E31G/cQ42E membranes, the ATP-driven H+ transport function of epsilon E31K/cQ42E membranes was approximately the same at pH 7.5, greater at pH 7.8, and less at pH 7.0. The differences between mutants could be explained if cGlu42 ionized at pH 7.8 with loss of function in epsilon E31G/cQ42E membrane and a similar ionization were compensated for by the positively charged Lys in the epsilon E31K/cQ42E membrane.
Zhang
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Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison 53706.
Oldenburg
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Fillingame
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D000255:Adenosine Triphosphate / Q000378:metabolism*; D001692:Biological Transport; D002462:Cell Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D016153:Genes, Suppressor; D005971:Glutamates / Q000235:genetics; D018698:Glutamic Acid; D006859:Hydrogen / Q000378:metabolism*; D009154:Mutation; D009876:Operon; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*
D005971:Glutamates; D018698:Glutamic Acid; D006859:Hydrogen; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases