title: Identification of the heme axial ligands in the cytochrome b562 of the Saccharomyces cerevisiae succinate dehydrogenase.
abstract: Succinate dehydrogenase (SDH) plays a key role in energy generation by coupling the oxidation of succinate to the reduction of ubiquinone in the mitochondrial electron transport chain. The Saccharomyces cerevisiae SDH is composed of a catalytic dimer of the Sdh1p and Sdh2p subunits containing flavin adenine dinucleotide (FAD) and iron-sulfur clusters and a heme b-containing membrane-anchoring domain comprised of the Sdh3p and Sdh4p subunits. We systematically mutated all the histidine and cysteine residues in Sdh3p and Sdh4p to identify the residues involved in axial heme ligation. The mutants were characterized for growth on a non-fermentable carbon source, for enzyme assembly, for succinate-dependent quinone reduction, for heme b content, and for heme spectral properties. Mutation of Sdh3p His-46 or His-113 leads to a marked reduction in the catalytic efficiency of the enzyme for quinone reduction, suggesting that these residues form part of a quinone-binding site. We identified Sdh3p His-106 and Sdh4p Cys-78 as the most probable axial ligands for cytochrome b(562). Replacement of His-106 or Cys-78 with an alanine residue leads to a marked reduction in cytochrome b(562) content and to altered heme spectral characteristics that are consistent with a direct perturbation of heme b environment. This is the first identification of a cysteine residue serving as an axial ligand for heme b in the SDH family of enzymes. Loss of cytochrome b(562) has no effect on enzyme assembly and quinone reduction; the role of the heme in enzyme structure and function is discussed.
authors.affiliation: Canadian Institutes of Health Research Group in Membrane Protein Research, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Kayode S
authors.initials: KS
authors.lastname: Oyedotun
authors.forename: Paul F
authors.initials: PF
authors.lastname: Yau
authors.forename: Bernard D
authors.initials: BD
authors.lastname: Lemire
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003573:Cytochrome b Group / Q000737:chemistry / Q000235:genetics*; D006418:Heme / Q000737:chemistry; D008024:Ligands; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics*; D013385:Succinate Dehydrogenase / Q000737:chemistry / Q000235:genetics*
chemical_list: D003573:Cytochrome b Group; D008024:Ligands; D029701:Saccharomyces cerevisiae Proteins; D006418:Heme; D013385:Succinate Dehydrogenase