Mutational analysis of cytochrome b at the ubiquinol oxidation site of yeast complex III

Tina Wenz, Raul Covian, Petra Hellwig, Fraser MacMillan, Brigitte Meunier, Bernard L. Trumpower, Carola Hunte

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Abstract

The cytochrome bc(1) 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 bc(1) 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, Tyr(132) and Glu(272), and two residues proposed to be involved in docking ubiquinol into the center P pocket, Phe(129) and Tyr(279). Substitution of Phe(129) 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 Tyr(279) lowers the catalytic activity accompanied by a low level of bypass reactions. Pre-steady-state kinetics of the enzymes modified at Glu(272) and Tyr(132) 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.
Original languageEnglish
Pages (from-to)3977-3988
Number of pages12
JournalJournal of Biological Chemistry
Volume282
Issue number6
DOIs
Publication statusPublished - 9 Feb 2007

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