Intramolecular Histidine Cross-Links Formed via Copper-Catalyzed Oxidation of Histatin Peptides

Histidine is a versatile amino acid with metal-binding, nucleophilic, and basic properties that endow many peptides and proteins with biological activity. However, histidine itself is susceptible to oxidative modifications via post-translational modifications, photo-oxidation, and metal-catalyzed oxidation. Despite multiple investigations into these different oxidation systems, the varied attributions and differential outcomes point to significant gaps in our understanding of the coordination requirements, spectral features, and reaction products that accompany the Cu-catalyzed oxidation of histidine-containing peptides. Here, we use model peptides of Histatin-5, a salivary peptide with Cu-potentiated antifungal activity that relies on its histidine residues, to characterize the complex mixture resulting from the reaction with Cu under physiologically relevant reducing and oxidizing conditions. Characterization via LC-MS, MS/MS, UV-vis, and NMR revealed that adjacent histidine residues of the <i>bis</i>-His site are the main target of Cu-catalyzed oxidation, with predominant modifications being 2-oxo-His and His-His cross-links that give rise to distinctive electronic absorption features between 300-400 nm. Doubly- and triply-oxygenated peptides, intramolecular His-His cross-links, and multimers in the case of a shorter model peptide were also observed. The configuration of the <i>bis</i>-His motif may enable Cu reactivity not available in systems where His residues are not adjacent in sequence or space. These results expand the possibilities of oxidative modifications available to other proteins and peptides containing multiple histidines.