Oxygenation is the main pathway for the reaction between superoxide anion and tryptophan radical

Oxygenation, the covalent addition of oxygen, is the most common chemical modification of proteins, leading to the loss of their structure and functionality. Tryptophan (TrpH) within peptides and proteins is considered one of the main sources of electrons for cellular oxidants, and corresponding tryptophan radicals may have long lifetimes. The reaction between superoxide anion (O₂^•—) and tryptophan radicals (Trp^•) is the fastest known bimolecular reaction of Trp^•, which may lead to the irreversible oxygenation of TrpH. This reaction may proceed via the addition, yielding oxygenated forms of TrpH, or the electron transfer with the restoration of the initial reagents. Currently, the mechanism of this reaction remains unclear due to contradicting conclusions reported previously. This work aims to elucidate the mechanism of this reaction using methods of time-resolved optical spectroscopy to visualise processes on the microsecond time scale and liquid chromatography-mass spectrometry to analyse the reagent degradation and product accumulation. The target radicals were generated using natural photosensitiser kynurenic acid irradiated by pulsed laser radiation. Our time-resolved data demonstrated that TrpH undergoes rapid oxygenation under aerobic conditions without significant restoration of its ground state. Hydroxyperoxides and N-formyl-kynurenine were found to be major photoproducts of TrpH degradation. Altogether, our data unambiguously indicated the addition as the major pathway for the reaction between superoxide anion and tryptophan radicals. Comparison of experimentally measured yields of reagent degradation with those calculated from the kinetic scheme provided an opportunity to estimate the ratio between addition/electron transfer reactions as 7/1 for N-acetyl-L-tryptophan.