Mutational analysis of cytochrome b at the ubiquinol oxidation site of yeast complex III.
The cytochrome bc1 complex is a dimeric enzyme of the inner mitochondrial membrane that links electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which ubiquinol is oxidized at one center in the enzyme, referred to as center P, and ubiquinone is rereduced at a second center, referred to as center N. To better understand the mechanism of ubiquinol oxidation, we have examined catalytic activities and pre-steady-state reduction kinetics of yeast cytochrome bc1 complexes with mutations in cytochrome b that we expected would affect oxidation of ubiquinol. We mutated two residues thought to be involved in proton conduction linked to ubiquinol oxidation, Tyr132 and Glu272, and two residues proposed to be involved in docking ubiquinol into the center P pocket, Phe129 and Tyr279. Substitution of Phe129 by lysine or arginine yielded a respiration-deficient phenotype and lipid-dependent catalytic activity. Increased bypass reactions were detectable for both variants, with F129K showing the more severe effects. Substitution with lysine leads to a disturbed coordination of a b heme as deduced from changes in the midpoint potential and the EPR signature. Removal of the aromatic side chain in position Tyr279 lowers the catalytic activity accompanied by a low level of bypass reactions. Pre-steady-state kinetics of the enzymes modified at Glu272 and Tyr132 confirmed the importance of their functional groups for electron transfer. Altered center N kinetics and activation of ubiquinol oxidation by binding of cytochrome c in the Y132F and E272D enzymes indicate long range effects of these mutations.
Wenz
Tina
T
Department Molecular Membrane Biology, Max Planck Institute of Biophysics, D-60438 Frankfurt am Main, Germany.
Covian
Raul
R
Hellwig
Petra
P
Macmillan
Fraser
F
Meunier
Brigitte
B
Trumpower
Bernard L
BL
Hunte
Carola
C
D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D003460:Crystallization; D045303:Cytochromes b / Q000235:genetics* / Q000378:metabolism*; D045304:Cytochromes c / Q000235:genetics; D004252:DNA Mutational Analysis; D014450:Electron Transport Complex III / Q000378:metabolism*; D004789:Enzyme Activation / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D014443:Tyrosine / Q000235:genetics; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000378:metabolism
D014451:Ubiquinone; D001224:Aspartic Acid; D018698:Glutamic Acid; D014443:Tyrosine; D010649:Phenylalanine; D045304:Cytochromes c; D045303:Cytochromes b; D014450:Electron Transport Complex III; C003741:ubiquinol