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Convergence of biological nitration and nitrosation via symmetrical nitrous anhydride

Nature Chemical Biology volume 11, pages 504–510 (2015)Cite this article

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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.

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Figure 1: 15NO2− incorporation into NO2-CLA is dependent on •NO production by activated RAW264.7 cells.
Figure 2: 15NO2− participates in nitration of CLA in the absence of cellular components.
Figure 3: 15NO2− mediates glutathione nitrosation in the presence of •NO.
Figure 4: NO2− incorporation into NO2-CLA and GSNO is associated with symN2O3 formation.
Figure 5: NO2− incorporation into nitrating and nitrosating equivalents requires reaction with •NO-derived species.
Figure 6: Inflammatory conditions promote NO2−-dependent generation of symN2O3 in vivo.

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Acknowledgements

This study was supported by US National Institutes of Health grants R01-HL058115, R01-HL64937, P01-HL103455 (B.A.F.), R01-AT006822 (F.J.S.) and AHA #14GRNT20170024 (F.J.S.). We thank M.T. Gladwin, J. Tejero, A.M. Ferreira and B. Alvarez for helpful discussions and T. Sparwasser for expert assistance with flow cytometry experiments.

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Authors and Affiliations

  1. Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Dario A Vitturi, Sonia R Salvatore, Marco Fazzari, Jack R Lancaster Jr, Bruce A Freeman & Francisco J Schopfer

  2. Cátedra de Inmunología, Universidad de la República, Montevideo, Uruguay

    Lucia Minarrieta

  3. Department of Environmental Health Sciences, University of Alabama at Birmingham, Birmingham, Alabama, USA

    Edward M Postlethwait

  4. Fondazione Ri.MED, Palermo, Italy

    Marco Fazzari

  5. Laboratorio de Fisicoquímica Biológica, Universidad de la República, Montevideo, Uruguay

    Gerardo Ferrer-Sueta

  6. Center for Free Radical and Biomedical Research, Universidad de la República, Montevideo, Uruguay

    Gerardo Ferrer-Sueta

  7. Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Jack R Lancaster Jr

  8. Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Jack R Lancaster Jr

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Contributions

D.A.V. designed, performed and analyzed experiments, and wrote the manuscript. L.M. designed, performed and analyzed cell-based experiments. S.R.S. performed high-resolution LC-MS/MS experiments and contributed to overall LC-MS/MS method development. E.M.P. designed and performed •NO2 gas experiments. M.F. developed extraction methods for in vivo experiments. G.F.-S. contributed to data interpretation. J.R.L. designed experiments, contributed to data interpretation and provided critical insight into manuscript content. B.A.F. contributed to the overall concept, experimental design and manuscript preparation. F.J.S. designed experiments and contributed to data analysis and interpretation as well as manuscript writing.

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Correspondence to Bruce A Freeman or Francisco J Schopfer.

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Competing interests

B.A.F. and F.J.S. acknowledge financial interest in Complexa, Inc.

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Vitturi, D., Minarrieta, L., Salvatore, S. et al. Convergence of biological nitration and nitrosation via symmetrical nitrous anhydride. Nat Chem Biol 11, 504–510 (2015). https://doi.org/10.1038/nchembio.1814

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