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J. Biol. Chem., Vol. 282, Issue 6, 3977-3988, February 9, 2007

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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, and Carola Hunte¹

From the Department Molecular Membrane Biology, Max Planck Institute of Biophysics, D-60438 Frankfurt am Main, Germany, the Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, the ^¶Institut de Chimie, UMR 7177 LC3, Université Louis Pasteur, 4 Rue Blaise Pascal, F-67000 Strasbourg, France, the ^||Institute for Physical and Theoretical Chemistry and Center for Biomolecular Magnetic Resonance, Johann Wolfgang Goethe University, D-60439 Frankfurt am Main, Germany, and the ^**Centre de Génétique Moléculaire, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France

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.

Received for publication, July 7, 2006 , and in revised form, December 4, 2006.

^* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 472 (to C. H., P. H., and F. M.), Boehringer Ingelheim Fonds (to T. W.), the Centre for Membrane Proteomics (to F. M.), and National Institutes of Health Grant GM 20379 (to B. L. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Max-Planck-Institute of Biophysics, Dept. Molecular Membrane Biology, Max-von-Laue-Str. 3, 60438 Frankfurt am Main, Germany. Tel.: 49-69-6303-1062; Fax: 49-69-6303-1002; E-mail: Carola.Hunte{at}mpibp-frankfurt.mpg.de.

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