# Peroxidase-Catalyzed and Photo-Oxidation of Tryptophan Results in Distinct Isomeric Tryptophan Dimers

## TL;DR

This study shows that heme peroxidases like HRP can produce distinct isomeric tryptophan dimers through oxidation, with different results depending on the method and pH.

## Contribution

The study reveals the formation of multiple tryptophan dimer isomers via HRP/H2O2 oxidation and compares it to photo-oxidation.

## Key findings

- HRP/H2O2 oxidation of tryptophan produces multiple di-Trp isomers, with higher yields at pH 9.2.
- Photo-oxidation with riboflavin results in a single dominant di-Trp dimer.
- In silico studies suggest di-Trp forms within the HRP catalytic pocket.

## 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/H2O2 has 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 H2O2 with 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.