title: Kinetic and spectroscopic characterization of a hydroperoxy compound in the reaction of native myoglobin with hydrogen peroxide.
abstract: The reaction of metmyoglobin with H2O2 was investigated in a pH range between 8.5 and 6.0 with the aid of stopped flow-rapid scan and rapid freezing-EPR techniques. Singular value decomposition analyses of the stopped flow data at pH 8.5 revealed that a spectral species previously unknown accumulated during the reaction and exhibited a Soret absorption maximum at >/=423 nm. In the EPR experiments, the new species exhibited a set of g values at 2.32, 2.19, and 1.94, indicating that the species was assignable to a ferric hydroperoxy (Fe(III)[O-O-H]-) compound. In contrast, the hydroperoxy compound scarcely accumulated in the reaction at pH 6.0, and the dominant intermediate species accumulated was compound I, which was derived from the oxygen-oxygen bond cleavage of the hydroperoxy compound. The accumulated amount of the hydroperoxy compound relative to compound I showed a pH dependence with an apparent pKa (pKaapp) from 6.95 to 7.27 depending on the metmyoglobins examined. This variation in pKaapp paralleled that in pKa of the acid-alkaline transition (pKaAB) of metmyoglobins, suggesting that the accumulation of hydroperoxy compound is controlled by the distal histidine. We propose that the H2O2 activation by metmyoglobin is promoted at the acidic condition due to the imidazolium form of the distal histidine, and we further propose that the controlled protonation state of the distal histidine is important for the facile O-O bond cleavage in heme peroxidases.
authors.affiliation: Department of Biochemistry, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. egawa@sc.itc.keio.ac.jp
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Egawa
authors.forename: Shiro
authors.initials: S
authors.lastname: Yoshioka
authors.forename: Satoshi
authors.initials: S
authors.lastname: Takahashi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hori
authors.forename: Shingo
authors.initials: S
authors.lastname: Nagano
authors.forename: Hideo
authors.initials: H
authors.lastname: Shimada
authors.forename: Koichiro
authors.initials: K
authors.lastname: Ishimori
authors.forename: Isao
authors.initials: I
authors.lastname: Morishima
authors.forename: Makoto
authors.initials: M
authors.lastname: Suematsu
authors.forename: Yuzuru
authors.initials: Y
authors.lastname: Ishimura
mesh_terms: D000818:Animals; D004578:Electron Spin Resonance Spectroscopy; D005290:Ferric Compounds / Q000737:chemistry; D006639:Histidine; D006736:Horses; D006861:Hydrogen Peroxide / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008786:Metmyoglobin / Q000737:chemistry*; D008956:Models, Chemical; D010544:Peroxidases / Q000737:chemistry; D013057:Spectrum Analysis; D014907:Whales
chemical_list: D005290:Ferric Compounds; D008786:Metmyoglobin; D006639:Histidine; D006861:Hydrogen Peroxide; D010544:Peroxidases