Radical-Mediated Peptide Tyrosine Nitration In Vacuo: Experimental Evidence and Theoretical Examination

Abstract

Protein tyrosine nitration (PTN)—a hallmark of post-translational modification of proteins under nitrative stress in vivo—modification is believed to occur regioselectively and site-specifically at diverse local sequences with no observed consensus modification motif.1 The mechanistic details governing the site-specificity of the ortho-tyrosine nitration are largely unknown. Herein, the mechanism of radical-mediated PTN has been elucidated in detail at the molecular level using an integrated approach combining gas phase synthesis of prototypical tyrosine-containing peptide radical cations, ion–molecule reactions with nitrogen dioxide, isotopic labelling MS experiments, and DFT calculations at the B3LYP/6-31++G** level of theory. First, π-centered molecular radical cationic tyrosine-containing peptides were generated in situ through collision-induced intramolecular one-electron transfer oxidation of copper(II)–peptide complexes In Vacuo.2,3 The representative radical cationic tyrosine-containing tetrapeptide was mass-selected, trapped, and reacted with •NO2. Recombination of •NO2 with radical cationic peptide led to a substantial yield of a stable closed-shell nitrated product. Our experimental and theoretical investigations into radical-mediated tyrosine nitration revealed the necessity for phenoxyl radical formation prior to the production of distinct 3-nitrotyrosine (3-NT) products, which are influenced by the location of the radical site and the tautomerization barriers for radical and proton mobility; 3,4 a two-step mechanism, involving the generation of phenoxyl radical intermediates with viable isomerization barriers (<10 kcal mol–1) and concerted proton rearrangement prior to the formation of the stable closed-shell ortho-nitration product of the tyrosyl residue, has been determined unambiguously. The spin state of the phenoxyl radical governs the regioselectivity of the ortho-tyrosine nitration (3-NT). To better understand the site-specific formation of endogenous PTNs, the fundamental factors governing the site-specificity of the 3-NT have been investigated using computationally tractable prototypical dityrosyl-containing peptides that mimic the local topological characteristics of peptides found in PTNs in vitro from a M. fascicularis model of cerebral ischemia; 5 these nitrated peptides have been validated and shortlisted as potential selectivity determinants for the model peptides in their subsequent gas phase PTN reactions; selective tyrosine nitrations have been verified in several other instances of prototypical dityrosyl-containing peptides. A crucial aspect of the formation of the site-specific nitrated product ions is that they are likely preceded by favorable interconversion barrier(s) to generate phenoxyl radical intermediate structures prior to the formation of distinct 3-NT products; the exact site of tyrosyl nitration also depends on the local sequence; the types and locations of the essential neighboring residues and their proximity to the tyrosyl residue appear to significantly influence the competition between the isomerization and the site-specific phenoxyl radical formation. References: 1. Radi, R. Acc. Chem. Res. 2013, 46, 550-559. 2. Turecek, F.; Julian, R. R. Chem. Rev. 2013, 113, 6691-6733. 3. Laskin J. ; Yang Z.; Chu I. K. J. Am. Chem. Soc. 2008, 130, 3218-3230. 4. Chu I. K.; Zhao J.; Xu M.; Siu S. O.; Hopkinson A. C.; Siu K. W. M. J. Am. Chem. Soc. 2008, 130, 7862-7872. 5. Quan Q., Szeto S. S. W.; Law C. H.; Zhang Z.; Wang Y.; Chu I. K. Anal. Chem. 2015, 87, 10015- 10024