Dioxygenation of tryptophan residues by superoxide and myeloperoxidase.

Abstract

When neutrophils ingest pathogens into phagosomes, they generate large amounts of the superoxide radical through the reduction of molecular oxygen. Superoxide is essential for effective antimicrobial defense, but the precise role it plays in bacterial killing is unknown. Within phagosomes, superoxide reacts with the heme enzyme myeloperoxidase (MPO) and is converted to hydrogen peroxide, then subsequently to the bactericidal oxidant hypochlorous acid. But other reactions of superoxide with MPO may also contribute to host defense. Here, we demonstrate that MPO uses superoxide to dioxygenate tryptophan residues within model peptides via two hypochlorous acid-independent pathways. Using mass spectrometry, we show that formation of N-formylkynurenine is the favored reaction. This reaction is consistent with a direct transfer of dioxygen from an intermediate of MPO, where superoxide is bound to the active site heme iron (compound III). In addition, hydroperoxides are formed when superoxide adds to tryptophan radicals, which are produced during the peroxidase cycle of MPO. Proteomic analysis revealed that tryptophan dioxygenation occurs on the abundant neutrophil protein calprotectin and lactoferrin during phagocytosis of Staphylococcus aureus, indicating that this is a physiologically relevant modification. Our study enhances the understanding of superoxide chemistry in the phagosome. It also suggests that tryptophan dioxygenation by MPO and superoxide may occur during infection and inflammation.