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

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.