title: Coupled oxidation vs heme oxygenation: insights from axial ligand mutants of mitochondrial cytochrome b5.
abstract: Mutation of His-39, one of the axial ligands in rat outer mitochondrial membrane cytochrome b(5) (OM cyt b(5)), to Val produces a mutant (H39V) capable of carrying out the oxidation of heme to biliverdin when incubated with hydrazine and O(2). The reaction proceeds via the formation of an oxyferrous complex (Fe(II)(-)O(2)) that is reduced by hydrazine to a ferric hydroperoxide (Fe(III)(-)OOH) species. The latter adds a hydroxyl group to the porphyrin to form meso-hydroxyheme. The observation that catalase does not inhibit the oxidation of the heme in the H39V mutant is consistent with the formation of a coordinated hydroperoxide (Fe(III)(-)OOH), which in heme oxygenase is the precursor of meso-hydroxyheme. By comparison, mutation of His-63, the other axial ligand in OM cyt b(5), to Val results in a mutant (H63V) capable of oxidizing heme to verdoheme in the absence of catalase. However, the oxidation of heme by H63V is completely inhibited by catalase. Furthermore, whereas the incubation of Fe(III)(-)H63V with H(2)O(2) leads to the nonspecific degradation of heme, the incubation of Fe(II)(-)H63V with H(2)O(2) results in the formation of meso-hydroxyheme, which upon exposure to O(2) is rapidly converted to verdoheme. These findings revealed that although meso-hydroxyheme is formed during the degradation of heme by the enzyme heme oxygenase or by the process of coupled oxidation of model hemes and hemoproteins not involved in heme catabolism, the corresponding mechanisms by which meso-hydroxyheme is generated are different. In the coupled oxidation process O(2) is reduced to noncoordinated H(2)O(2), which reacts with Fe(II)-heme to form meso-hydroxyheme. In the heme oxygenation reaction a coordinated O(2) molecule (Fe(II)(-)O(2)) is reduced to a coordinated peroxide molecule (Fe(III)(-)OOH), which oxidizes heme to meso-hydroxyheme.
authors.affiliation: Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74078-3071, USA.
authors.forename: Ludivina
authors.initials: L
authors.lastname: Avila
authors.forename: Hong-wei
authors.initials: HW
authors.lastname: Huang
authors.forename: Christopher O
authors.initials: CO
authors.lastname: Damaso
authors.forename: Shen
authors.initials: S
authors.lastname: Lu
authors.forename: Pierre
authors.initials: P
authors.lastname: Moënne-Loccoz
authors.forename: Mario
authors.initials: M
authors.lastname: Rivera
mesh_terms: D000818:Animals; D001664:Biliverdine / Q000737:chemistry / Q000378:metabolism; D002374:Catalase / Q000737:chemistry / Q000494:pharmacology; D015786:Cytochromes b5 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D051381:Rats
chemical_list: C099547:verdoheme; D006418:Heme; D015786:Cytochromes b5; D006861:Hydrogen Peroxide; D002374:Catalase; D001664:Biliverdine