Kinetic and Spectroscopic Characterization of a Hydroperoxy Compound in the Reaction of Native Myoglobin with Hydrogen Peroxide

Tsuyoshi Egawa, Shiro Yoshioka, Satoshi Takahashi, Hiroshi Hori, Shingo Nagano, Hideo Shimada, Koichiro Ishimori, Isao Morishima, Makoto Suematsu, Yuzuru Ishimura

Research output: Contribution to journalArticle

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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-(OH]-) 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 (pKa app) from 6.95 to 7.27 depending on the metmyoglobins examined. This variation in pKa app paralleled that in pKa of the acid-alkaline transition (pKa AB) of metmyoglobins, suggesting that the accumulation of hydroperoxy compound is controlled by the distal histidine. We propose that the H2O 2 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.

Original languageEnglish (US)
Pages (from-to)41597-41606
Number of pages10
JournalJournal of Biological Chemistry
Volume278
Issue number43
DOIs
StatePublished - Oct 24 2003
Externally publishedYes

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Metmyoglobin
Myoglobin
Hydrogen Peroxide
Histidine
Kinetics
Paramagnetic resonance
Oxygen
Peroxidases
Protonation
Singular value decomposition
Heme
Freezing
Chemical activation
Acids
Experiments

ASJC Scopus subject areas

  • Biochemistry

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Kinetic and Spectroscopic Characterization of a Hydroperoxy Compound in the Reaction of Native Myoglobin with Hydrogen Peroxide. / Egawa, Tsuyoshi; Yoshioka, Shiro; Takahashi, Satoshi; Hori, Hiroshi; Nagano, Shingo; Shimada, Hideo; Ishimori, Koichiro; Morishima, Isao; Suematsu, Makoto; Ishimura, Yuzuru.

In: Journal of Biological Chemistry, Vol. 278, No. 43, 24.10.2003, p. 41597-41606.

Research output: Contribution to journalArticle

Egawa, T, Yoshioka, S, Takahashi, S, Hori, H, Nagano, S, Shimada, H, Ishimori, K, Morishima, I, Suematsu, M & Ishimura, Y 2003, 'Kinetic and Spectroscopic Characterization of a Hydroperoxy Compound in the Reaction of Native Myoglobin with Hydrogen Peroxide', Journal of Biological Chemistry, vol. 278, no. 43, pp. 41597-41606. https://doi.org/10.1074/jbc.M210383200
Egawa, Tsuyoshi ; Yoshioka, Shiro ; Takahashi, Satoshi ; Hori, Hiroshi ; Nagano, Shingo ; Shimada, Hideo ; Ishimori, Koichiro ; Morishima, Isao ; Suematsu, Makoto ; Ishimura, Yuzuru. / Kinetic and Spectroscopic Characterization of a Hydroperoxy Compound in the Reaction of Native Myoglobin with Hydrogen Peroxide. In: Journal of Biological Chemistry. 2003 ; Vol. 278, No. 43. pp. 41597-41606.
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T1 - Kinetic and Spectroscopic Characterization of a Hydroperoxy Compound in the Reaction of Native Myoglobin with Hydrogen Peroxide

AU - Egawa, Tsuyoshi

AU - Yoshioka, Shiro

AU - Takahashi, Satoshi

AU - Hori, Hiroshi

AU - Nagano, Shingo

AU - Shimada, Hideo

AU - Ishimori, Koichiro

AU - Morishima, Isao

AU - Suematsu, Makoto

AU - Ishimura, Yuzuru

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N2 - 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-(OH]-) 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 (pKa app) from 6.95 to 7.27 depending on the metmyoglobins examined. This variation in pKa app paralleled that in pKa of the acid-alkaline transition (pKa AB) of metmyoglobins, suggesting that the accumulation of hydroperoxy compound is controlled by the distal histidine. We propose that the H2O 2 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.

AB - 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-(OH]-) 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 (pKa app) from 6.95 to 7.27 depending on the metmyoglobins examined. This variation in pKa app paralleled that in pKa of the acid-alkaline transition (pKa AB) of metmyoglobins, suggesting that the accumulation of hydroperoxy compound is controlled by the distal histidine. We propose that the H2O 2 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.

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