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J. Biol. Chem., Vol. 279, Issue 50, 52376-52381, December 10, 2004

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Catalase Reaction by Myoglobin Mutants and Native Catalase

MECHANISTIC INVESTIGATION BY KINETIC ISOTOPE EFFECT^*

Shigeru Kato, Takafumi Ueno, Shunichi Fukuzumi¶, and Yoshihito Watanabe||**

From the Department of Structural Molecule Science, The Graduate University for Advanced Studies, Okazaki 444-8585, Japan, Research Center for Materials Science, Nagoya University, Nagoya 464-8602, Japan, ^¶Department of Material and Life Science, Graduate School of Engineering, CREST, JST, Osaka University, Suita, Osaka 565-0871, Japan, and ^||Department of Chemistry, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan

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 H₂¹⁸O₂/H₂¹⁶O₂ 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.

Received for publication, March 31, 2004 , and in revised form, August 26, 2004.

^* This work was supported by Grants-in-aid for Scientific Research 11490036 and 1122828 (to Y. W.) and 13740384 (to T. U.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Appendices 1 and 2.

^** To whom correspondence should be addressed. Tel.: 81-52-789-3049; Fax: 81-52-789-2953; E-mail: yoshi{at}nucc.cc.nagoya-u.ac.jp.

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