TY - JOUR
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
PY - 2003/10/24
Y1 - 2003/10/24
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 (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 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 (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 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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U2 - 10.1074/jbc.M210383200
DO - 10.1074/jbc.M210383200
M3 - Article
C2 - 12902339
AN - SCOPUS:0142149195
SN - 0021-9258
VL - 278
SP - 41597
EP - 41606
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 43
ER -