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Catalytic generation of N2O3 by the concerted nitrite reductase and anhydrase activity of hemoglobin

Nature Chemical Biology volume 3, pages 785–794 (2007)Cite this article

Abstract

Nitrite reacts with deoxyhemoglobin to form nitric oxide (NO) and methemoglobin. Though this reaction is experimentally associated with NO generation and vasodilation, kinetic analysis suggests that NO should not be able to escape inactivation in the erythrocyte. We have discovered that products of the nitrite-hemoglobin reaction generate dinitrogen trioxide (N2O3) via a novel reaction of NO and nitrite-bound methemoglobin. The oxygen-bound form of nitrite-methemoglobin shows a degree of ferrous nitrogen dioxide (Fe(II)-NO2˙) character, so it may rapidly react with NO to form N2O3. N2O3 partitions in lipid, homolyzes to NO and readily nitrosates thiols, all of which are common pathways for NO escape from the erythrocyte. These results reveal a fundamental heme globin– and nitrite-catalyzed chemical reaction pathway to N2O3, NO and S-nitrosothiol that could form the basis of in vivo nitrite-dependent signaling. Because the reaction redox-cycles (that is, regenerates ferrous heme) and the nitrite-methemoglobin intermediate is not observable by electron paramagnetic resonance spectroscopy, this reaction has been 'invisible' to experimentalists over the last 100 years.

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Figure 1: HbFe(III)-NO2− (nitrite-MetHb) is the intermediate in the deoxyhemoglobin-nitrite reaction.
Figure 2: EPR silence of HbFe(III)-NO2−.
Figure 3: Electronic configuration of HbFe(III)-NO2−.
Figure 4: Reductive nitrosylation of HbFe(III)-NO2− catalyzed by NO.
Figure 5: Nitrite-mediated nitrosothiol formation.
Figure 6: NO-catalyzed reduction of HbFe(III)-NO2− generates gas-phase N2O3.
Figure 7: Model of nitrite- and Hb-mediated N2O3 export and nitrosation.

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Acknowledgements

We thank D.L.H. Williams for helpful discussion and for his seminal and pioneering work in nitrosation chemistry. This work was supported by US National Institutes of Health (NIH) grants HL58091 (D.B.K.-S.), GM55792 (N.H.) and HL62198 (S.B.K.). EPR spectrometry was facilitated by a grant from the North Carolina Biotechnology Center (2003-IDG-1013, D.B.K.-S.). D.B.K.-S. gratefully acknowledges further support from NIH grant K02 HL078706. A.G. thanks the Research Council of Norway for supercomputing time, and J.C. thanks the South African National Research Foundation for further support. M.T.G. is supported by the Division of Intramural Research of the US National Heart, Lung, and Blood Institute.

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Author notes

  1. Swati Basu and Rozalina Grubina: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Physics, Wake Forest University, 1834 Wake Forest Road, Winston-Salem, 27109, North Carolina, USA

    Swati Basu, Alice Jiang, Xiaojun He, Ivan Azarov, Ryan Seibert, Atul Mehta & Daniel B Kim-Shapiro

  2. Vascular Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, 10 Center Drive, MSC 1454, Bethesda, 20892, Maryland, USA

    Rozalina Grubina, Zhi Huang & Mark T Gladwin

  3. Howard Hughes Medical Institute–National Institutes of Health Research Scholars Program, 1 Cloister Court, Bethesda, 20814, Maryland, USA

    Rozalina Grubina

  4. Department of Chemistry, Wake Forest University, 1834 Wake Forest Road, Winston-Salem, 27109, North Carolina, USA

    Jinming Huang & Stephen Bruce King

  5. Department of Chemistry and Center for Theoretical and Computational Chemistry, University of Tromsø, N-9037, Tromsø, Norway

    Jeanet Conradie & Abhik Ghosh

  6. Chemistry Department, University of the Free State, Nelson Mandela Street, Bloemfontein, 9301, South Africa

    Jeanet Conradie

  7. Virginia Tech–Wake Forest University School of Biomedical Engineering and Sciences, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, 27157, North Carolina, USA

    Anne Jeffers & Daniel B Kim-Shapiro

  8. Department of Pathology, University of Alabama, 901 19th Street South, BMR-2, Room 302, Birmingham, 35294, Alabama, USA

    Rakesh Patel

  9. Department of Biophysics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, 53226, Wisconsin, USA

    Neil Hogg

  10. Critical Care Medicine Department, Clinical Center, National Institutes of Health, 10 Center Drive, MSC 1454, Bethesda, 20892, Maryland, USA

    Mark T Gladwin

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Contributions

The bulk of the experiments were performed by S.B. and R.G., who also helped in the design of these experiments and in writing the paper. A.G. and J.C. performed DFT calculations and wrote the text associated with these. D.B.K.-S. and M.T.G. directed the research, designed the experiments and wrote most of the paper. Significant other intellectual contributions (and some writing) were made by R.P., S.B.K and N.H. Other experiments were performed by J.H., A. Jeffers, A. Jiang, X.H., I.A., R.S., Z.H. and A.M.

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Correspondence to Mark T Gladwin or Daniel B Kim-Shapiro.

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Competing interests

R.P., M.T.G. and D.B.K.-S. are listed as coinventors on US National Institutes of Health government patent applications entitled “Use of Nitrite Salts for the Treatment of Cardiovascular Conditions” and “Nitrite-Methemoglobin therapy to detoxify stroma-free hemoglobin based blood substitutes.”

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Basu, S., Grubina, R., Huang, J. et al. Catalytic generation of N2O3 by the concerted nitrite reductase and anhydrase activity of hemoglobin. Nat Chem Biol 3, 785–794 (2007). https://doi.org/10.1038/nchembio.2007.46

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