Abstract

Heme peroxidases, including horseradish peroxidase (HRP), catalyze the oxidation of a wide variety of substrates by hydrogen peroxide (H2O2) via the peroxidase cycle of these enzymes. Oxidation of free tryptophan (Trp) by HRP/H2O2has been previously reported, but the formation of tryptophan dimers (di-Trp), which are biologically relevant, has not been studied. Here, we report on di-Trp production arising from oxidation of free Trp, at pH 5.5 and 9.2, by HRP/H2O2, as determined by liquid chromatography–mass spectrometry (LC-MS/MS) and selected reaction monitoring (SRM). These data were compared with those from riboflavin-sensitized photo-oxidation, and the products were rationalized by in silico studies. Incubation of varying concentrations of Trp and H2O2with HRP, irrespective of the pH, resulted in the consumption of ?2 mol of Trp per mole H2O2. Formation of multiple di-Trp isomers was detected, using m/z 407 ? 203 and m/z 407 ? 390 transitions, with greater yields detected at pH 9.2 than 5.5. These results contrast with riboflavin-mediated photo-oxidation where one di-Trp dimer predominated as detected by the m/z 407 ? 203 transition. In silico docking studies suggest di-Trp formation within the catalytic pocket of HRP, and subsequent release is a probable mechanism, although other alternative scenarios are also possible. © 2025 The Authors. Published by American Chemical Society