Identification of oxidative modifications of hemopexin and their predicted physiological relevance

Abstract Hemopexin protects against heme toxicity in hemolytic diseases and conditions, sepsis and sickle cell disease. This protection is sustained by heme-hemopexin complexes present in biological fluids that resist oxidative damage during heme-driven inflammation. However, apo-hemopexin is vulnerable to inactivation by reactive nitrogen and oxygen species that covalently modify amino acids. The consequent nitration of amino acids is considered a selective and specific effect reflecting biological events. Using liquid chromatography-tandem mass spectrometry, we discovered low endogenous levels of tyrosine nitration in the peptide YYCFQGNQFLR of human hemopexin, which was similarly nitrated in rabbit and rat hemopexins. Immuno-blotting and selective reaction monitoring were used to quantitate tyrosine nitration in these in vivo samples and when hemopexin was incubated in vitro with a nitrating system of nitrite/myeloperoxidase/glucose oxidase. Significantly, heme binding by hemopexin declined as tyrosine nitration proceeded in vitro. Three nitrated tyrosines reside in the heme binding site of heme-hemopexin and one, Tyr199, in YY199CFQGNQFLR interacts directly with the heme ring D propionate. We also investigated the oxidative modifications of amino acids in hemopexin after incubation with tert-butyl hydroperoxide and hypochlorous acid in vitro. We identified additional covalent oxidative modifications on four tyrosine residues and one tryptophan residue of hemopexin. Importantly, three of the four modified tyrosines, some of which having more than one modification, resided closely together in the heme-binding site, supporting a hierarchy of amino acids vulnerable to oxidative damage. Based on these in vivo and in vitro data, we propose that during inflammation nitration and oxidative modification of apo-hemopexin occurs in niches of the body where there are activated immune and endothelial cells that generate RNS and ROS, with the potential to impair the protective extracellular anti-oxidant function of hemopexin.