Catalase reaction by myoglobin mutants and native catalase: Mechanistic investigation by kinetic isotope effect

Shigeru Kato, Takafumi Ueno, Shunichi Fukuzumi, Yoshihito Watanabe

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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 H218O 2/H216O2 solution containing H64B 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 (Y is Ala, Ser, and Asp) and L29B/H64L Mb oxidize H 2O2 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.

Original languageEnglish
Pages (from-to)52376-52381
Number of pages6
JournalJournal of Biological Chemistry
Issue number50
StatePublished - 10 Dec 2004


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