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.

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.

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