Probing the role of E272 in quinol oxidation of mitochondrial complex III.
Bifurcated electron transfer during ubiquinol oxidation is the key reaction of complex III catalysis, but the molecular basis of this process is still not clear. E272 of the conserved cytochrome b PEWY motif has been suggested as a ligand and proton acceptor for ubiquinol oxidation at center P. We introduced the two replacement mutations, E272D and E272Q, into the mitochondrially encoded cytochrome b gene by biolistic transformation to study their effects on substrate binding and catalysis. Both substitutions resulted in a lower ubiquinol cytochrome c reductase activity and affect the KM for ubiquinol. The E272 carboxylate stabilizes stigmatellin binding, and in accordance, both variants are resistant to stigmatellin. Large structural changes in the cofactor environment as well as in the binding pocket can be excluded. The mutations do not perturb the midpoint potentials of the heme groups. The sensitivity toward the respective distal and proximal niche inhibitors HDBT and myxothiazol is retained. However, both mutations provoke subtle structural alterations detected by redox FTIR. They affect binding and oxidation of ubiquinol, and they promote electron short-circuit reactions resulting in production of reactive oxygen species. The aspartate substitution modifies the environment of the reduced Rieske protein as monitored by EPR. Both variants alter the pH dependence of the enzyme activity. Diminished activity at low pH coincides with the loss of one protonatable group with a pKa of approximately 6.2 compared to three pKa values in the wild type, supporting the role of E272 in proton transfer. The conserved glutamate appears to influence the accurate formation of the enzyme-substrate complex and to govern the efficiency of catalysis.
Wenz
Tina
T
Department Molecular Membrane Biology, Max-Planck-Institute of Biophysics, 60438 Frankfurt am Main, Germany.
Hellwig
Petra
P
MacMillan
Fraser
F
Meunier
Brigitte
B
Hunte
Carola
C
D019943:Amino Acid Substitution / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D014450:Electron Transport Complex III / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics; D005973:Glutamine / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011199:Potentiometry; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D017550:Spectroscopy, Fourier Transform Infrared; D013481:Superoxides / Q000378:metabolism; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D029701:Saccharomyces cerevisiae Proteins; D005973:Glutamine; D013481:Superoxides; D014451:Ubiquinone; D001224:Aspartic Acid; D018698:Glutamic Acid; D014450:Electron Transport Complex III; C003741:ubiquinol