title: Catalytic mechanism of heme oxygenase through EPR and ENDOR of cryoreduced oxy-heme oxygenase and its Asp 140 mutants.
abstract: Heme oxygenase (HO) catalyzes the O(2)- and NADPH-cytochrome P450 reductase-dependent conversion of heme to biliverdin, Fe, and CO through a process in which the heme participates both as a prosthetic group and as a substrate. In the present study, we have generated a detailed reaction cycle for the first monooxygenation step of HO catalysis, conversion of the heme to alpha-meso-hydroxyheme. We employed EPR (using both (16)O(2) and (17)O(2)) and (1)H, (14)N ENDOR spectroscopies to characterize the intermediates generated by 77 K radiolytic cryoreduction and subsequent annealing of wild-type oxy-HO and D140A, F mutants. One-electron cryoreduction of oxy-HO yields a hydroperoxoferri-HO with g-tensor, g = [2.37, 2.187, 1.924]. Annealing of this species to 200 K is accompanied by spectroscopic changes that include the appearance of a new (1)H ENDOR signal, reflecting rearrangements in the active site. Kinetic measurements at 214 K reveal that the annealed hydroperoxoferri-HO species, denoted R, generates the ferri-alpha-meso-hydroxyheme product in a first-order reaction. Disruption of the H-bonding network within the distal pocket of HO by the alanine and phenylalanine mutations of residue D140 prevents product formation. The hydroperoxoferri-HO (D140A) instead undergoes heterolytic cleavage of the O-O bond, ultimately yielding an EPR-silent compound II-like species that does not form product. These results, which agree with earlier suggestions, establish that hydroperoxoferri-HO is indeed the reactive species, directly forming the alpha-meso-hydroxyheme product by attack of the distal OH of the hydroperoxo moiety at the heme alpha-carbon.
authors.affiliation: Department of Chemistry, Northwestern University, Evanston, Illinois 60208, USA.
authors.forename: Roman
authors.initials: R
authors.lastname: Davydov
authors.forename: Viktoria
authors.initials: V
authors.lastname: Kofman
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Fujii
authors.forename: Tadashi
authors.initials: T
authors.lastname: Yoshida
authors.forename: Masao
authors.initials: M
authors.lastname: Ikeda-Saito
authors.forename: Brian M
authors.initials: BM
authors.lastname: Hoffman
mesh_terms: D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics; D002384:Catalysis; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006419:Heme Oxygenase (Decyclizing) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction
chemical_list: D001224:Aspartic Acid; D006418:Heme; D006419:Heme Oxygenase (Decyclizing)