Site-Selective Tyrosine Modification and Cross-Linking of the Grass Pollen Allergen Phl p 5a by Peroxynitrite.

Reactive oxygen and nitrogen species can modify proteins and alter their chemical structure and biological function, including their inflammatory and allergenic potential. Peroxynitrite is a particularly relevant reactant associated with oxidative stress and inflammatory diseases. Hydroxyl radicals, carbonate radicals, and nitrogen dioxide released upon decomposition of peroxynitrite can react with tyrosine residues, resulting in protein nitration, hydroxylation, and covalent cross-linking through dityrosine. These radical-mediated processes are increasingly recognized as molecular pathways linking environmental oxidative stress to adverse health effects. Yet, the determinants governing site-specific tyrosine modification within protein macromolecules are not well understood. Here, we investigate tyrosine modifications in the major grass pollen allergen Phl p 5a exposed to varying doses of peroxynitrite. Site-specific nitration, hydroxylation, and cross-linking were characterized by LC-Orbitrap-MS/MS combined with SEC-HPLC and interpreted using atom-level solvent accessibilities derived from a structural model. All 12 tyrosines were nitrated, but degrees varied strongly between sites, from ∼ 8% at Y236 to ∼ 95% at Y65 at the highest peroxynitrite-to-tyrosine ratio (5:1). Hydroxylation was detected to a lower extent ( ∼ 0.7% at Y127 to ∼ 5% at Y17). Nitration increased at all residues, and hydroxylation at 8 out of 12 residues, with increasing molar ratio. A total of 14 covalent cross-links were detected between 8 tyrosine residues, with the highest number of cross-link peptide spectrum matches observed at an intermediate peroxynitrite-to-tyrosine molar ratio of 3:1. Our findings provide mechanistic insights into how oxidative stress can induce post-translational modifications of protein macromolecules. This may be relevant for human health and inflammatory or allergic diseases.