Mg-substituted horseradish peroxidases were oxidized by K2IrCl6 or K3Fe(CN)6 to their porphyrin radical form and the 1:1 stoichiometric relationship was confirmed by spectrophotometric, fluorophotometric, and ESR titration methods. The values of E'0 for oxidations of Mg peroxidases A and C were both 0.63 V at pH 6 and depended on pH in the same way as postulated for the Compound I/Compound II couples of the corresponding enzymes. Unlike Zn peroxidase C, Mg peroxidase C was not directly oxidized by H2O2. The oxidation was catalyzed by the native peroxidase. Mg peroxidase C was photooxidized to the radical form faster than Zn peroxidase C, but its oxidation was accomplished by irreversible changes in the porphyrin in the early stage of reaction. The rate of reduction of the oxidized Mg peroxidases in the presence of various electron donors was measured at varying pH values and compared with the rate of Compound I reduction. A role of porphyrin as a site of electron transfer in the peroxidase catalysis was suggested.
Electron Spin Resonance Spectroscopy Horseradish Peroxidase