Formation of porphyrin .pi. cation radical in zinc-substituted horseradish peroxidase

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Abstract
Zn-substituted horseradish peroxidase is oxidized by K2IrCl6 to a characteristic state which retains 1 oxidizing equivalent more than the Zn peroxidase. The oxidized enzyme gives an optical absorption spectrum similar to that of compound I of peroxidase and catalase and a g = 2 EPR signal which has an intensity corresponding to the porphyrin content. It is reduced to Zn peroxidase by a stoichiometric amount of ferrocyanide or by an excess of K2IrCl6. From the equilibrium data, the value of E0'' for the Zn peroxidase couple is 0.74 V at pH 6. The oxidized Zn peroxidase is also formed by the addition of H2O2 or upon illumination with white light. The rate constants for the oxidation by K2IrCl6 and H2O2 at pH 8.0 are 8 .times. 105 and 8 .times. 102 M-1 s-1, respectively. No essential spectral change can be observed when K2IrCl6 is added to the metal-free peroxidase (protoporphyrin-apoperoxidase complex) or to Zn-substituted sperm whale myoglobin.