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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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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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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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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DOI: https://doi.org/10.1038/nchembio.2007.46
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