Mechanism of horseradish peroxidase catalyzed epinephrine oxidation: obligatory role of endogenous O₂^- and H₂O₂

Horseradish peroxidase (HRP) catalyzes cyanide sensitive oxidation of epinephrine to adrenochrome at physiological pH in the absence of added H₂O₂ with concurrent consumption of O₂. Both adrenochrome formation and O₂ consumption are significantly inhibited by catalase, indicating a peroxidative mechanism as a major part of oxidation due to intermediate formation of H₂O₂. Sensitivity to superoxide dismutase (SOD) also indicates involvement of O₂^- in the oxidation. Although SOD-mediated H₂O₂ formation should continue epinephrine oxidation through a peroxidative mechanism, low catalytic turnover, on the contrary, indicates that O₂^- takes part in a vital reaction to form an intermediate for adrenochrome formation and O₂ consumption. Generation of O₂^- is evidenced by ferricytochrome c reduction sensitive to SOD. On addition of H₂O₂, both adrenochrome formation and O₂ consumption are further increased due to reaction of molecular oxygen with some intermediate oxidation product. Peroxidative oxidation proceeds by one-electron transfer generating o-semiquinone and similar free radicals which when stabilized with Zn²⁺ or spin-trap, α-phenyl-tert-butylnitrone (PBN), inhibit adrenochrome formation and O₂ consumption. The free radicals thus favor reduction of O₂ rather than the disproportionation reaction. Spectral studies indicate that, during epinephrine oxidation in the presence of catalase, HRP remains in the ferric state absorbing at 403 nm. This suggests that HRP catalyzes epinephrine oxidation by its oxidase activity through Fe³⁺/Fe²⁺ shuttle consuming O₂, where the rate of reduction of ferric HRP with epinephrine is slower than subsequent oxidation of ferrous HRP by O₂ to form compound III. Compound III was not detected spectrally because of its quick reduction to the ferric state by epinephrine or its subsequent oxidation product. In the absence of catalase, peroxidative cycles predominate when HRP still remains in the ferric state through the transient formation of compounds I and II not detectable spectrally. Among various mono- and dihydroxyl aromatic donors tested, only epinephrine shows the oxidase reaction. Binding studies indicate that epinephrine interferes with the binding of CN^-, SCN^-, and guaiacol indicating that HRP preferentially binds epinephrine near the heme iron close to the anion or aromatic donor binding site to catalyze electron transfer for oxidation. HRP thus initiates epinephrine oxidation by its oxidase activity generating O₂^- and H₂O₂. Once H₂O₂ is generated, the peroxidative cycle continues with the consumption of O₂, through the intermediate formation of O₂^- and H₂O₂ which play an obligatory role in subsequent cycles of peroxidation