title: Catalase reaction by myoglobin mutants and native catalase: mechanistic investigation by kinetic isotope effect.
abstract: The catalase reaction has been studied in detail by using myoglobin (Mb) mutants. Compound I of Mb mutants (Mb-I), a ferryl species (Fe(IV)=O) paired with a porphyrin radical cation, is readily prepared by the reaction with a nearly stoichiometric amount of m-chloroperbenzoic acid. Upon the addition of H2O2 to an Mb-I solution, Mb-I is reduced back to the ferric state without forming any intermediates. This indicates that Mb-I is capable of performing two-electron oxidation of H2O2 (catalatic reaction). Gas chromatography-mass spectroscopy analysis of the evolved O2 from a 50:50 mixture of H2(18)O2/H2(16)O2 solution containing H64D or F43H/H64L Mb showed the formation of 18O2 (m/e = 36) and 16O2 (m/e = 32) but not 16O18O (m/e = 34). This implies that O2 is formed by two-electron oxidation of H2O2 without breaking the O-O bond. Deuterium isotope effects on the catalatic reactions of Mb mutants and catalase suggest that the catalatic reactions of Micrococcus lysodeikticus catalase and F43H/H64L Mb proceed via an ionic mechanism with a small isotope effect of less than 4.0, since the distal histidine residue is located at a proper position to act as a general acid-base catalyst for the ionic reaction. In contrast, other Mb mutants such as H64X (X is Ala, Ser, and Asp) and L29H/H64L Mb oxidize H2O2 via a radical mechanism in which a hydrogen atom is abstracted by Mb-I with a large isotope effect in a range of 10-29, due to a lack of the general acid-base catalyst.
authors.affiliation: Department of Structural Molecule Science, The Graduate University for Advanced Studies, Okazaki 444-8585, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Kato
authors.forename: Takafumi
authors.initials: T
authors.lastname: Ueno
authors.forename: Shunichi
authors.initials: S
authors.lastname: Fukuzumi
authors.forename: Yoshihito
authors.initials: Y
authors.lastname: Watanabe
mesh_terms: D000818:Animals; D001665:Binding Sites; D002374:Catalase / Q000378:metabolism*; D002417:Cattle; D017666:Deuterium Oxide; D066298:In Vitro Techniques; D007700:Kinetics; D008837:Micrococcus / Q000201:enzymology; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation; D009211:Myoglobin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010103:Oxygen Isotopes; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013053:Spectrophotometry; D013816:Thermodynamics; D014907:Whales
chemical_list: D009211:Myoglobin; D010103:Oxygen Isotopes; D011994:Recombinant Proteins; D002374:Catalase; D017666:Deuterium Oxide