The molecular mechanism of the photocatalytic oxidation reactions by horseradish peroxidase in the presence of histidine

Abstract

Oxidation reactions catalyzed by horseradish peroxidase (HRP) involve the conversion of ferric heme to the ferryl state by hydrogen peroxide (H₂O₂). As a result, HRP is typically considered unreactive without the presence of H₂O₂. Herein, we report the first use of histidine (His) and HRP in the presence of daylight and oxygen to initiate photocatalytic oxidation reactions without the addition of H₂O₂. We demonstrate that the oxidation reactions of norbixin (NBX) molecules proceed 2-times faster using this HRP/His system in comparison with HRP/H₂O₂. Most importantly, we also demonstrate that new species of NBX oxidized products are formed using HRP/His. In this work, we investigate the key steps of the HRP mechanism in the presence of His using electron paramagnetic resonance spectroscopy, liquid chromatography-mass spectrometry, and ultraviolet-visible spectrophotometry. Bearing in mind that H₂O₂ is normally thought indispensable for initiating the oxidation reactions of HRP, this work suggests a potential paradigm shift in our understanding of possible HRP mechanisms in the catalysis of biochemical reactions. The use of His, a natural essential amino acid, to mediate the formation of ferryl heme intermediates suggests the HRP/His system could serve as a natural catalyst for applications in photochemistry and bioelectrocatalysis.