Inactivation of Horseradish Peroxidase by Phenoxyl Radical Attack

To test the hypothesis that horseradish peroxidase (HRP) can be inactivated by phenoxyl radicals upon reaction with H2O2/phenol, we probed HRP-catalyzed phenol oxidation at various phenol/H2O2 concentrations. To this end the total protein, phenolic product, active protein, and iron concentrations in the aqueous phase were determined by protein assay, phenol-14C isotopic labeling, resonance Raman and atomic absorption spectroscopy, respectively. Additionally, resonance Raman and FTIR measurements were carried out to probe possible structural changes of the enzyme during the reaction. The data obtained provide the first experimental support for the hypothesis that HRP can be inactivated by a phenoxyl radical attack. The heme macrocycle destruction involving deprivation of the heme iron occurs as a result of the reaction. An intermediate type of the active protein was observed by Raman difference spectra at low concentrations which features a stabilization of the quantum mixed state of the heme iron and a significant amount of phenoxylphenol-type oligomers in solution and probably also in the heme pocket. This work provides a basis for evaluating the relative contributions of different HRP inactivation mechanisms and is thus critical for optimizing engineering applications involving HRP reactions.