Formation of a Porphyrin π-Cation Radical in the Fluoride Complex of Horseradish Peroxidase

Horseradish peroxidase (HRP) was oxidized by IrCl₆^2- to a mixture of compounds I and II, the rate of oxidation and the ratio of the mixture being greatly affected by pH (Hayashi & Yamazaki, 1979). Oxidation of HRP by IrCl₆^2- in the presence of fluoride was significantly accelerated. This resulted in the formation of a new compound which is a ferric fluoride complex containing a porphyrin π-cation radical. The spectrum of the new compound showed a decreased absorption band in the Soret region and a broad band at 570 nm; which was converted to that of the original ferric fluoride complex by addition of ascorbate or hydroquinone. Addition of cyanide slowed down the oxidation of HRP by IrCl₆^2-, and the oxidation product was the same as that obtained in the absence of cyanide. Compound I was formed when H₂O₂ was added to HRP in the presence of fluoride or cyanide. The one-electron reduction potential (E₀‘) of the oxidized HRP-fluoride complex was measured at several pH values, the E₀‘ value at pH 7 being 861 ± 4 mV. The ratio of ∆E₀‘ to ∆pH was 49 mV/pH unit.