Abstract
Peroxynitrite (ONOO−, PN) has long been considered a potent nitrating agent implicated in numerous inflammation-mediated diseases. The current work highlights an unexplored oxidation chemistry initiated under conditions of sustained PN exposure. Impetus for this investigation developed from mass spectral results that suggested dimerization of a model peptide with a single tyrosine residue that was first nitrated following extended exposure to PN generated in situ. In attempts to substantiate this dimerization event and divulge the possible mode of linkage between the tyrosine derivatives of the peptide monomers, 3-nitrotyrosine (3-NT) was exposed to sustained fluxes of PN in a two-component PN-generating platform developed in this laboratory. Such exposure afforded products with tandem mass spectrometry and fluorescence spectroscopy profiles indicative of C–O coupling between 3-NT moieties. Synthesis and comparative analysis of the C–C coupled 3-NT isomer corroborated these findings. Most notably, the mass spectral data of the C–C coupled 3-NT dimer displayed a 226.80 m/z peak following exposure to high collision energy, corresponding to symmetric cleavage of the parent dimer peak (m/z = 453) along with a fragmentation product at m/z = 180.04 (–NO2 species). This fragmentation profile was distinct from the C–O coupled 3-NT dimer that exhibited a predominant 209.14 m/z peak with a small secondary 226.15 m/z peak indicative of asymmetric cleavage of the parent dimer. Results of this study indicate that formation of C–O coupled 3-NT dimer is promoted by elevated levels of 3-NT formed under high and sustained flux of PN.
Highlights
Tyrosine nitration has remained tightly associated to the reactive nitrogen species (RNS) peroxynitrite (PN) since the discovery of PN in the early 1990’s
Results of the present study reveal an alternative pathway of peptide crosslinking through C–O coupling between nitrated tyrosine residues upon prolonged exposure to high PN flux
Nitration of tyrosine in protein has been considered as the predominant consequence of PN exposure, it is apparent that 3-NT could be an intermediate in the formation of an array of unsuspected and unexplored PN-mediated products
Summary
Tyrosine nitration has remained tightly associated to the reactive nitrogen species (RNS) peroxynitrite (PN) since the discovery of PN in the early 1990’s. Work exploited the nitration of tyrosine by PN as a strategy to define and support the physiological existence of PN. The fleeting nature of PN has remained a technical hurdle impeding investigation of PN-mediated nitration, where limitations in PN-production necessitate the use of chemical PN sources [5]. Under these conditions, target tyrosine-containing proteins or peptides are exposed to a burst of PN, affording a snapshot of PN-mediated nitration [6,7]. One question that has not been posed in the field of PN is, what happens after tyrosine nitration?
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