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A motif for reversible nitric oxide interactions in metalloenzymes


Nitric oxide (NO) participates in numerous biological processes, such as signalling in the respiratory system and vasodilation in the cardiovascular system. Many metal-mediated processes involve direct reaction of NO to form a metal–nitrosyl (M–NO), as occurs at the Fe2+ centres of soluble guanylate cyclase or cytochrome c oxidase. However, some copper electron-transfer proteins that bear a type 1 Cu site (His2Cu–Cys) reversibly bind NO by an unknown motif. Here, we use model complexes of type 1 Cu sites based on tris(pyrazolyl)borate copper thiolates [CuII]-SR to unravel the factors involved in NO reactivity. Addition of NO provides the fully characterized S-nitrosothiol adduct [CuI](κ1-N(O)SR), which reversibly loses NO on purging with an inert gas. Computational analysis outlines a low-barrier pathway for the capture and release of NO. These findings suggest a new motif for reversible binding of NO at bioinorganic metal centres that can interconvert NO and RSNO molecular signals at copper sites.

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Figure 1: T1Cu electron-transfer site and synthetic model complexes.
Figure 2: Capture of NO by iPr2TpCu-SCPh3 (1a) to form unstable S-nitrosothiol adduct 1b.
Figure 3: Synthesis of MesTpCu(κ1-N(O)SCPh3) (2b) and MesTpCu-SCPh3 (1b).
Figure 4: X-ray crystal structures of MesTpCu-SCPh3 (1b) and MesTpCu(κ1-N(O)SCPh3) (2b).
Figure 5: Orbital interactions and pathway for reversible capture of NO by MesTpCu-SCPh3 (2b).
Figure 6: Interconnections between NO and S-nitrosothiols promoted by copper.

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  1. 1

    Ignarro, L. J. Nitric Oxide, Biology and Pathobiology 2nd edn (Academic, 2010).

    Google Scholar 

  2. 2

    Cary, S. P. L., Winger, J. A., Derbyshire, E. R. & Marletta, M. A. Nitric oxide signaling: no longer simply on or off. Trends Biochem. Sci. 31, 231–239 (2006).

    CAS  Article  Google Scholar 

  3. 3

    Francis, S. H., Busch, J. L. & Corbin, J. D. cGMP-dependent protein kinases and cGMP phosphodiesterases in nitric oxide and cGMP action. Pharmacol. Rev. 62, 525–563 (2010).

    CAS  Article  Google Scholar 

  4. 4

    Hematian, S. et al. Nitrogen oxide atom-transfer redox chemistry; mechanism of NO(g) to nitrite conversion utilizing μ-oxo heme-FeIII–O–CuII(L) constructs. J. Am. Chem. Soc. 137, 6602–6615 (2015).

    CAS  Article  Google Scholar 

  5. 5

    Mason, M. G., Nicholls, P., Wilson, M. T. & Cooper, C. E. Nitric oxide inhibition of respiration involves both competitive (heme) and noncompetitive (copper) binding to cytochrome c oxidase. Proc. Natl Acad. Sci. USA 103, 708–713 (2006).

    CAS  Article  Google Scholar 

  6. 6

    Brown, G. C. Nitric oxide regulates mitochondrial respiration and cell functions by inhibiting cytochrome oxidase. FEBS Lett. 369, 136–139 (1995).

    CAS  Article  Google Scholar 

  7. 7

    Ohta, K. et al. X-ray structure of the NO-bound CuB in bovine cytochrome c oxidase. Acta Crystallogr. F 66, 251–253 (2010).

    CAS  Article  Google Scholar 

  8. 8

    Tocheva, E. I., Rosell, F. I., Mauk, A. G. & Murphy, M. E. P. Side-on copper nitrosyl coordination by nitrite reductase. Science 304, 867–870 (2004).

    CAS  Article  Google Scholar 

  9. 9

    Merkle, A. C. & Lehnert, N. Binding and activation of nitrite and nitric oxide by copper nitrite reductase and corresponding model complexes. Dalton Trans. 41, 3355–3368 (2012).

    CAS  Article  Google Scholar 

  10. 10

    Usov, O. M., Sun, Y., Grigoryants, V. M., Shapleigh, J. P. & Scholes, C. P. EPR–ENDOR of the Cu(I)NO complex of nitrite reductase. J. Am. Chem. Soc. 128, 13102–13111 (2006).

    CAS  Article  Google Scholar 

  11. 11

    Fujisawa, K. et al. Structural and spectroscopic characterization of mononuclear copper(I) nitrosyl complexes: end-on versus side-on coordination of NO to copper(I). J. Am. Chem. Soc. 130, 1205–1213 (2008).

    CAS  Article  Google Scholar 

  12. 12

    Wever, R., Van Leeuwen, F. X. R. & Van Gelder, B. F. The reaction of nitric oxide with ceruloplasmin. Biochim. Biophys. Acta 302, 236–239 (1973).

    CAS  Article  Google Scholar 

  13. 13

    Gorren, A. C. F., De Boer, E. & Wever, R. The reaction of nitric oxide with copper proteins and the photodissociation of copper–nitric oxide complexes. Biochim. Biophys. Acta 916, 38–47 (1987).

    CAS  Article  Google Scholar 

  14. 14

    Van Leeuwen, F. X. R., Wever, R., Van Gelder, B. F., Avigliano, L. & Mondovi, B. The interaction of nitric oxide with ascorbate oxidase. Biochim. Biophys. Acta 403, 285–291 (1975).

    CAS  Article  Google Scholar 

  15. 15

    Ehrenstein, D., Filiaci, M., Scharf, B., Engelhard, M. & Nienhaus, G. U. Ligand binding and protein dynamics in cupredoxins. Biochemistry 34, 12170–12177 (1995).

    CAS  Article  Google Scholar 

  16. 16

    Solomon, E. I. et al. Copper active sites in biology. Chem. Rev. 114, 3659–3853 (2014).

    CAS  Article  Google Scholar 

  17. 17

    Liu, J. et al. Metalloproteins containing cytochrome, iron–sulfur, or copper redox centers. Chem. Rev. 114, 4366–4469 (2014).

    CAS  Article  Google Scholar 

  18. 18

    Solomon, E. I., Szilagyi, R. K., DeBeer George, S. & Basumallick, L. Electronic structures of metal sites in proteins and models: contributions to function in blue copper proteins. Chem. Rev. 104, 419–458 (2004).

    CAS  Article  Google Scholar 

  19. 19

    Inoue, K. et al. Nitrosothiol formation catalyzed by ceruloplasmin. J. Biol. Chem. 274, 27069–27075 (1999).

    CAS  Article  Google Scholar 

  20. 20

    Shiva, S. et al. Ceruloplasmin is a NO oxidase and nitrite synthase that determines endocrine NO homeostasis. Nature Chem. Biol. 2, 486–493 (2006).

    CAS  Article  Google Scholar 

  21. 21

    Rassaf, T. et al. Plasma nitrosothiols contribute to the systemic vasodilator effects of intravenously applied NO. Experimental and clinical study on the fate of NO in human blood. Circ. Res. 91, 470–477 (2002).

    CAS  Article  Google Scholar 

  22. 22

    Stamler, J. S. & Toone, E. J. The decomposition of thionitrites. Curr. Opin. Chem. Biol. 6, 779–785 (2002).

    CAS  Article  Google Scholar 

  23. 23

    Zhang, S., Çelebi-Ölçüm, N., Melzer, M. M., Houk, K. N. & Warren, T. H. Copper(I) nitrosyls from reaction of copper(II) thiolates with S-nitrosothiols: mechanism of NO release from RSNOs at Cu. J. Am. Chem. Soc. 135, 16746–16749 (2013).

    CAS  Article  Google Scholar 

  24. 24

    Hess, D. T., Matsumoto, A., Kim, S.-O., Marshall, H. E. & Stamler, J. S. Protein S-nitrosylation: purview and parameters. Nature Rev. Mol. Cell Biol. 6, 150–166 (2005).

    CAS  Article  Google Scholar 

  25. 25

    Foster, M. W., Hess, D. T. & Stamler, J. S. Protein S-nitrosylation in health and disease: a current perspective. Trends Mol. Med. 15, 391–404 (2009).

    CAS  Article  Google Scholar 

  26. 26

    Kim, S. F., Huri, D. A. & Snyder, S. H. Inducible nitric oxide synthase binds, S-nitrosylates, and activates cyclooxygenase-2. Science 310, 1966–1970 (2005).

    CAS  Article  Google Scholar 

  27. 27

    Johnson, M. A., Macdonald, T. L., Mannick, J. B., Conaway, M. R. & Gaston, B. Accelerated S-nitrosothiol breakdown by amyotrophic lateral sclerosis mutant copper, zinc-superoxide dismutase. J. Biol. Chem. 276, 39872–39878 (2001).

    CAS  Article  Google Scholar 

  28. 28

    Schonhoff, C. M. et al. S-nitrosothiol depletion in amyotrophic lateral sclerosis. Proc. Natl Acad. Sci. USA 103, 2404–2409 (2006).

    CAS  Article  Google Scholar 

  29. 29

    Romeo, A. A., Filosa, A., Capobianco, J. A. & English, A. M. Metal chelators inhibit S-nitrosation of Cysβ93 in oxyhemoglobin. J. Am. Chem. Soc. 123, 1782–1783 (2001).

    CAS  Article  Google Scholar 

  30. 30

    Pawloski, J. R., Hess, D. T. & Stamler, J. S. Export by red blood cells of nitric oxide bioactivity. Nature 409, 622–626 (2001).

    CAS  Article  Google Scholar 

  31. 31

    McMahon, T. J. et al. A nitric oxide processing defect of red blood cells created by hypoxia: deficiency of S-nitrosohemoglobin in pulmonary hypertension. Proc. Natl Acad. Sci. USA 102, 14801–14806 (2005).

    CAS  Article  Google Scholar 

  32. 32

    Ford, P. C. & Lorkovic, I. M. Mechanistic aspects of the reactions of nitric oxide with transition-metal complexes. Chem. Rev. 102, 993–1018 (2002).

    CAS  Article  Google Scholar 

  33. 33

    McCleverty, J. A. Chemistry of nitric oxide relevant to biology. Chem. Rev. 104, 403–418 (2004).

    CAS  Article  Google Scholar 

  34. 34

    Ford, P. C., Fernandez, B. O. & Lim, M. D. Mechanisms of reductive nitrosylation in iron and copper models relevant to biological systems. Chem. Rev. 105, 2439–2455 (2005).

    CAS  Article  Google Scholar 

  35. 35

    Kitajima, N., Fujisawa, K., Tanaka, M. & Morooka, Y. X-ray structure of thiolatocopper(II) complexes bearing close spectroscopic similarities to blue copper proteins. J. Am. Chem. Soc. 114, 9232–9233 (1992).

    CAS  Article  Google Scholar 

  36. 36

    Qiu, D., Kilpatrick, L., Kitajima, N. & Spiro, T. G. Modeling blue copper protein resonance Raman spectra with thiolate–CuII complexes of a sterically hindered tris(pyrazolyl)borate. J. Am. Chem. Soc. 116, 2585–2590 (1994).

    CAS  Article  Google Scholar 

  37. 37

    Randall, D. W. et al. Spectroscopic and electronic structural studies of blue copper model complexes. Part 1. Perturbation of the thiolate–Cu bond. J. Am. Chem. Soc. 122, 11620–11631 (2000).

    CAS  Article  Google Scholar 

  38. 38

    Arulsamy, N. et al. Interrelationships between conformational dynamics and the redox chemistry of S-nitrosothiols. J. Am. Chem. Soc. 121, 7115–7123 (1999).

    CAS  Article  Google Scholar 

  39. 39

    Fujisawa, K. et al. Structural and electronic differences of copper(I) complexes with tris(pyrazolyl)methane and hydrotris(pyrazolyl)borate ligands. Inorg. Chem. 45, 1698–1713 (2006).

    CAS  Article  Google Scholar 

  40. 40

    Rheingold, A. L., White, C. B. & Trofimenko, S. Hydrotris(3-mesitylpyrazol-1-yl)borate and hydrobis(3-mesitylpyrazol-1-yl)(5-mesitylpyrazol-1-yl)borate: symmetric and asymmetric ligands with rotationally restricted aryl substituents. Inorg. Chem. 32, 3471–3477 (1993).

    CAS  Article  Google Scholar 

  41. 41

    Schneider, J. L., Carrier, S. M., Ruggiero, C. E., Young, V. G. & Tolman, W. B. Influences of ligand environment on the spectroscopic properties and disproportionation reactivity of copper–nitrosyl complexes. J. Am. Chem. Soc. 120, 11408–11418 (1998).

    CAS  Article  Google Scholar 

  42. 42

    Perissinotti, L. L., Estrin, D. A., Leitus, G. & Doctorovich, F. A surprisingly stable S-nitrosothiol complex. J. Am. Chem. Soc. 128, 2512–2513 (2006).

    CAS  Article  Google Scholar 

  43. 43

    Grimme, S., Antony, J., Ehrlich, S. & Krieg, H. A consistent and accurate ab initio parametrization of density functional dispersion correction (DFT-D) for the 94 elements H–Pu. J. Chem. Phys. 132, 154104 (2010).

    Article  Google Scholar 

  44. 44

    Frisch, M. J. Gaussian 09, Revision D.01 (Gaussian, 2009).

  45. 45

    Baciu, C., Cho, K.-B. & Gauld, J. W. Influence of Cu+ on the RS–NO bond dissociation energy of S-nitrosothiols. J. Phys. Chem. B 109, 1334–1336 (2005).

    CAS  Article  Google Scholar 

  46. 46

    Wright, A. M., Wu, G. & Hayton, T. W. Structural characterization of a copper nitrosyl complex with a {CuNO}10 configuration. J. Am. Chem. Soc. 132, 14336–14337 (2010).

    CAS  Article  Google Scholar 

  47. 47

    Williams, D. L. H. The chemistry of S-nitrosothiols. Acc. Chem. Res. 32, 869–876 (1999).

    CAS  Article  Google Scholar 

  48. 48

    Schwane, J. D. & Ashby, M. T. FTIR investigation of the intermediates formed in the reaction of nitroprusside and thiolates. J. Am. Chem. Soc. 124, 6822–6823 (2002).

    CAS  Article  Google Scholar 

  49. 49

    Perissinotti, L. L., Turjanski, A. G., Estrin, D. A. & Doctorovich, F. Transnitrosation of nitrosothiols: characterization of an elusive intermediate. J. Am. Chem. Soc. 127, 486–487 (2005).

    CAS  Article  Google Scholar 

  50. 50

    Melzer, M. M., Li, E. & Warren, T. H. Reversible RS–NO bond cleavage and formation at copper(I) thiolates. Chem. Commun. 5847–5849 (2009).

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This material is based on work supported by the US National Science Foundation (award no. CHE-1459090, to T.H.W.). The authors thank the Purdue University Department of Chemistry and the Vorisek Endowment at Georgetown University for additional financial support (to T.H.W.), as well as the TUBITAK ULAKBIM High Performance and Grid Computing Center (TRUBA, Turkey) for computer time (N.Ç.-O.).

Author information




S.Z., N.Ç.-Ö. and T.H.W. conceived and designed the research. S.Z., M.M.M., S.N.S. and N.Ç.-Ö. collected data and performed calculations. S.Z., M.M.M., S.N.S., N.Ç.-Ö. and T.H.W. analysed the data. S.Z., N.Ç.-Ö. and T.H.W. co-wrote the paper.

Corresponding authors

Correspondence to Nihan Çelebi-Ölçüm or Timothy H. Warren.

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The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 5913 kb)

Supplementary information

Crystallographic data for compound MesTpCu-SCPh3 (1b) (CIF 1080 kb)

Supplementary information

Crystallographic data for compound MesTpCu(κ1-N(O)SCPh3) (2b) (CIF 1541 kb)

Supplementary information

Crystallographic data for compound iPr2TpCuI(CNAr2,6-Me2) (3a) (CIF 1461 kb)

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Zhang, S., Melzer, M., Sen, S. et al. A motif for reversible nitric oxide interactions in metalloenzymes. Nature Chem 8, 663–669 (2016).

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