Nitrosation

Reactive-nitrogen species (RNS) such as peroxynitrite (ONOO−), that is, the reaction product of nitric oxide (•NO) and superoxide (O2−•), nitryl chloride (NO2Cl) and •NO2 react with the activated aromatic ring of tyrosine to form 3-nitrotyrosine. This modification, which has been known for more than a century, occurs to both the free form of the amino acid (i.e., soluble/free tyrosine) and to tyrosine residues covalently bound within the backbone of peptides and proteins. Nitration of tyrosine is thought to be of biological significance and has been linked to health and disease, but determining its role has proved challenging. Several key questions have been the focus of much of the research activity: (a) to what extent is free/soluble tyrosine nitrated in biological tissues and fluids, and (b) are there specific site(s) of nitration within peptides/proteins and to what extent (i.e., stoichiometry) does this modification occur? These issues have been addressed in a wide range of sample types (e.g., blood, urine, CSF, exhaled breath condensate and various tissues) and a diverse array of physiological/pathophysiological scenarios. The accurate determination of nitrated tyrosine is, however, a stumbling block. Despite extensive study, the extent to which nitration occurs in vivo, the specificity of the nitration reaction, and its importance in health and disease, remain unclear. In this review, we highlight the analytical challenges and discuss the approaches adopted to address them. Mass spectrometry, in combination with either gas chromatography (GC-MS, GC-MS/MS) or liquid chromatography (LC-MS/MS), has played the central role in the analysis of 3-nitrotyrosine and tyrosine-nitrated biological macromolecules. We discuss its unique attributes and highlight the role of stable-isotope labeled 3-nitrotyrosine analogs in both accurate quantification, and in helping to define the biological relevance of tyrosine nitration. We show that the application of sophisticated mass spectrometric techniques is advantageous if not essential, but that this alone is by no means a guarantee of accurate findings. We discuss the important analytical challenges in quantifying 3-nitrotyrosine, possible workarounds, and we attempt to make sense of the disparate findings that have been reported so far. © 2013 Wiley Periodicals, Inc. Mass Spec RevReactive-nitrogen species (RNS) such as peroxynitrite (ONOO−), that is, the reaction product of nitric oxide (•NO) and superoxide (O2−•), nitryl chloride (NO2Cl) and •NO2 react with the activated aromatic ring of tyrosine to form 3-nitrotyrosine. This modification, which has been known for more than a century, occurs to both the free form of the amino acid (i.e., soluble/free tyrosine) and to tyrosine residues covalently bound within the backbone of peptides and proteins. Nitration of tyrosine is thought to be of biological significance and has been linked to health and disease, but determining its role has proved challenging. Several key questions have been the focus of much of the research activity: (a) to what extent is free/soluble tyrosine nitrated in biological tissues and fluids, and (b) are there specific site(s) of nitration within peptides/proteins and to what extent (i.e., stoichiometry) does this modification occur? These issues have been addressed in a wide range of sample types (e.g., blood, urine, CSF, exhaled breath condensate and various tissues) and a diverse array of physiological/pathophysiological scenarios. The accurate determination of nitrated tyrosine is, however, a stumbling block. Despite extensive study, the extent to which nitration occurs in vivo, the specificity of the nitration reaction, and its importance in health and disease, remain unclear. In this review, we highlight the analytical challenges and discuss the approaches adopted to address them. Mass spectrometry, in combination with either gas chromatography (GC-MS, GC-MS/MS) or liquid chromatography (LC-MS/MS), has played the central role in the analysis of 3-nitrotyrosine and tyrosine-nitrated biological macromolecules. We discuss its unique attributes and highlight the role of stable-isotope labeled 3-nitrotyrosine analogs in both accurate quantification, and in helping to define the biological relevance of tyrosine nitration. We show that the application of sophisticated mass spectrometric techniques is advantageous if not essential, but that this alone is by no means a guarantee of accurate findings. We discuss the important analytical challenges in quantifying 3-nitrotyrosine, possible workarounds, and we attempt to make sense of the disparate findings that have been reported so far. © 2013 Wiley Periodicals, Inc. Mass Spec Rev

Human carbonyl reductases 1 and 3 (CBR1 and CBR3) are monomeric NADPH-dependent enzymes of the short-chain dehydrogenase/reductase superfamily. Despite 72% identity in primary structure they exhibit substantial differences in substrate specificity. Recently, the endogenous low molecular weight S-nitrosothiol S-nitrosoglutathione (GSNO) has been added to the broad substrate spectrum of CBR1. The current study initially addressed whether CBR3 could equally reduce GSNO which was not the case. Neither the introduction of residues which contribute to glutathione binding in CBR1, i.e. K106Q and S97V/D98A, nor the exchange C143S, which prevents a theoretical disulfide bond with C227 in CBR3, could engender activity towards GSNO. However, exchanging amino acids 236-244 in CBR3 to correspond to CBR1 was sufficient to engender catalytic activity towards GSNO. Catalytic efficiency was further improved by the exchanges Q142M, C143S, P230W and H270S. Hence, the same residues previously reported as important for reduction of carbonyl compounds appear to be key to CBR1-mediated reduction of GSNO. Furthermore, for CBR1-mediated reduction of GSNO, considerable substrate inhibition at concentrations >5 K(m) was observed. Treatment of CBR1 with GSNO followed by removal of low molecular weight compounds decreased the GSNO reducing activity, suggesting a covalent modification. Treatment with dithiothreitol, but not with ascorbic acid, could rescue the activity, indicating S-glutathionylation rather than S-nitrosation as the underlying mechanism. As C227 has previously been identified as the reactive cysteine in CBR1, the variant CBR1 C227S was generated, which, in comparison to the wild-type protein, displayed a similar k(cat), but a 30-fold higher K(m), and did not show substrate inhibition. Collectively, the results clearly argue for a physiological role of CBR1, but not for CBR3, in GSNO reduction and thus ultimately in regulation of NO signaling. Furthermore, at higher concentrations, GSNO appears to work as a suicide inhibitor for CBR1, probably through glutathionylation of C227.