Convergence of biological nitration and nitrosation via symmetrical nitrous anhydride

Abstract

The current perspective holds that the generation of secondary signaling mediators from nitrite (NO2(-)) requires acidification to nitrous acid (HNO2) or metal catalysis. Herein, the use of stable isotope-labeled NO2(-) and LC-MS/MS analysis of products reveals that NO2(-) also participates in fatty acid nitration and thiol S-nitrosation at neutral pH. These reactions occur in the absence of metal centers and are stimulated by autoxidation of nitric oxide ((•)NO) via the formation of symmetrical dinitrogen trioxide (nitrous anhydride, symN2O3). Although theoretical models have predicted physiological symN2O3 formation, its generation is now demonstrated in aqueous reaction systems, cell models and in vivo, with the concerted reactions of (•)NO and NO2(-) shown to be critical for symN2O3 formation. These results reveal new mechanisms underlying the NO2(-) propagation of (•)NO signaling and the regulation of both biomolecule function and signaling network activity via NO2(-)-dependent nitrosation and nitration reactions.

Figure 1. ¹⁵NO₂⁻ incorporation into NO₂-CLA is dependent on ^•NO production by activated RAW264.7 cells

a, CLA (50 μM) nitration by RAW264.7 cells activated with LPS and IFNγ for 24 h in the presence or absence of ¹⁵NO₂⁻. Under these conditions, all ^14•NO₂ and ¹⁴NO₂⁻ is derived from endogenous ^14•NO. *, # p<0.0001 versus 0 μM ¹⁵NO₂⁻ for ¹⁴NO₂-CLA and ¹⁵NO₂-CLA respectively. b, NO₂-CLA formation by activated RAW264.7 cells in the presence of 1400W (100 μM). *, # p<0.0001 versus corresponding non-1400W treatments (in panel a) for ¹⁴NO₂-CLA and ¹⁵NO₂-CLA respectively. c, CLA nitration by 1400W-treated activated cells in the presence of deta-NONOate (200 μM). *,# p < 0.0001 versus corresponding 1400W alone treatment (in panel b). For all panels, data are mean ± SD (n=4) and two-way ANOVA plus Bonferroni's multiple comparison were used to test statistical significance.

Figure 2. ¹⁵NO₂⁻ participates in CLA nitration in the absence of cellular components

Representative LCMS/MS traces for ¹⁴NO₂-CLA (a) and ¹⁵NO₂-CLA (b) detection. c-f, Kinetic traces of ¹⁴NO₂-CLA and ¹⁵NO₂-CLA formation from 25 μM MNO and 20 μM CLA in the absence (c) or presence of 20 μM (d), 200 μM (e) and 2 mM (f) ¹⁵NO₂⁻. Data are representative traces generated by combining time-staggered replicate reactions (n=4). g, Total yields of NO -CLA formation versus ¹⁵NO₂⁻ concentration. Data are mean ± SD (n=4), * p < 0.05 versus 0 mM ¹⁵NO₂⁻ as determined by one way ANOVA and Bonferroni's multiple comparison test. No NO -CLA formation was detected from 2 mM ¹⁵NO₂⁻ in the absence of MNO.

Figure 3. ¹⁵NO₂⁻ mediates glutathione nitrosation in the presence of ^•NO

a-b, Representative LCMS/MS traces for GS¹⁴NO (a) and GS¹⁵NO (b) formation. c-f, Traces showing GS¹⁴NO and GS¹⁵NO formation from 2.5 μM MNO and 20 μM GSH in the absence (c) or presence of 20 μM (d), 200 μM (e) and 2 mM (f) ¹⁵NO₂⁻. Traces are representative and reflect time-staggered replicate reactions (n=4). g, Total GSNO yields versus ¹⁵NO₂⁻ concentration. Data are mean ± SD (n=4), no statistical differences were found as determined by one way ANOVA. No GSNO was formed from 2 mM ¹⁵NO₂⁻ in the absence of MNO.

Figure 4. NO₂⁻ incorporation into NO₂-CLA and GSNO is associated with symN₂O₃ formation

a, Scheme illustrating the asymmetrical (1) and symmetrical (2) conformations of N₂O₃. Arrows indicate alternative bond cleavage patterns. Whereas asymN₂O₃ homolysis produces a unique set of products (1a, 1b), alternative cleavage of the O-N-O bonds in symN₂O₃ can be evidenced by isotopic labeling (blue and red represent ¹⁴N and ¹⁶O respectively, dark blue and green are ¹⁵N and ¹⁸O). b, Distribution of NO₂-CLA isotopologues versus ¹⁵N¹⁸O₂⁻ concentration in the presence of 25 μM MNO and 20 μM CLA. c, Isotopic GSNO distribution versus ¹⁵N¹⁸O₂⁻ in the presence of 2.5 μM MNO and 20 μM GSH. Data for panels b and c are mean ± SD (n=4). Error bars are not distinguishable as they overlap with data points.

Figure 5. NO₂⁻ incorporation into nitrating and nitrosating equivalents requires reaction with ^• NO-derived species

a, LC-MS/MS trace showing the formation of ¹⁴NO₂-CLA and ¹⁵N¹⁸O₂-CLA from CLA (20 μM) nitration by pure ^•NO gas in the presence of 2 mM ¹⁵N¹⁸O₂⁻. b, NO₂-CLA yields obtained from CLA nitration by ^•NO gas and ¹⁵N¹⁸O₂⁻. Data are means ± SD (n=3). c-d, NO₂-CLA formation from the reaction between 50 μM NOBF and either ¹⁴NO₂⁻ (c) or ¹⁵N¹⁸O₂⁻ (d) in acetonitrile. Data are mean ± SD (n=4), with no NO₂-CLA formation observed in the absence of nitrite. e-f, NTA formation from the reaction between 20 μM DAN and 50 μM NOBF₄ in the absence (e) or presence (f) of 20 μM ¹⁵NO₂⁻ ₂in acetonitrile. Data are means ± SD (n=4). g, Proposed mechanisms for NO₂⁻ incorporation into nitrating and nitrosating species via symN₂O₃ formation.

Figure 6. Inflammatory conditions promote NO₂⁻-dependent symN₂O₃ generation in vivo

a-c, Total concentrations and relative isotopic distributions of NO₂-CLA generated during peritoneal inflammation. Points represent measurements from individual animals with mean ± SD indicated by the lines. Mice were injected i.p. with 20 μg LPS and 18 h later received a second injection containing 2.5 mg CLA plus 0 (a), 20 (b) or 200 (c) nmol ¹⁵N¹⁸O₂⁻. d-f, Representative LC-MS/MS traces of NO₂-CLA isotopologue formation after administration of 0 (d), 20 (e) and 200 (f) nmol ¹⁵N¹⁸O₂⁻. ¹⁴N¹⁸O¹⁶O-containing NO₂-CLA levels were <0.5% of total and were not included in the isotopic distributions (a-c), ¹⁴N¹⁸O¹⁸O-containing NO₂-CLA was not detected.