Speaker:
Leah Bontreger
"At the Crossroads of Histidine and Copper-Catalyzed Oxidation: Investigation of Structures, Mechanisms, and Scope of Reactivity on Biologically-Relevant 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 Cu-catalyzed oxidation of histidine-containing peptides. Here, we use biologically-relevant model peptides that have histidine-containing Cu-binding sites to explore the reactions of histidine residues and Cu under reducing and oxidizing conditions. Characterization of the Histatin-5 family of peptides via LC-MS, MS/MS, UV-vis, and NMR revealed adjacent histidine residues of the bis-His site are the main target of Cu-catalyzed oxidation, with predominant modifications being 2-oxo-His and His-His crosslinks that give rise to distinctive electronic absorption features between 300-400 nm. Stopped-flow experiments revealed reaction intermediates that indicate the ATCUN and bis-His motifs present in Histatin-5 model peptides determine the mechanism by which the peptide will become oxidized. Reductive or oxidative conditions initiating Cu-catalyzed oxidation also determine the reaction rate and variety of oxidative modifications produced. The scope of Cu-catalyzed oxidation was expanded to biologically-relevant peptides TESHHK and Amyloid Beta, although some reactivity guidelines established by the Histatin family could not be replicated for these peptides, proving that the coordination requirements to enable reactivity are more nuanced than previously thought. Finally, progress was made towards the synthesis of a protected 2-oxo-His residue to be incorporated into peptides via solid phase peptide synthesis. Together, this work defined reactivity requirements, mechanistic paths, and resulting structures for histidine-rich peptides subjected to Cu-catalyzed oxidation, and provides a path to study the functional ramifications of oxidative modification.