Nitric oxide generation from heme/copper assembly mediated nitrite reductase activity

  • Shabnam Hematian
  • Maxime A. Siegler
  • Kenneth D. KarlinEmail author
Original Paper
Part of the following topical collections:
  1. Topical Issue in honor of Ivano Bertini


Nitric oxide (NO) as a cellular signaling molecule and vasodilator regulates a range of physiological and pathological processes. Nitrite (NO2 ) is recycled in vivo to generate nitric oxide, particularly in physiologic hypoxia and ischemia. The cytochrome c oxidase binuclear heme a 3/CuB active site is one entity known to be responsible for conversion of cellular nitrite to nitric oxide. We recently reported that a partially reduced heme/copper assembly reduces nitrite ion, producing nitric oxide; the heme serves as the reductant and the cupric ion provides a Lewis acid interaction with nitrite, facilitating nitrite (N–O) bond cleavage (Hematian et al., J. Am. Chem. Soc. 134:18912–18915, 2012). To further investigate this nitrite reductase chemistry, copper(II)–nitrito complexes with tridentate and tetradentate ligands were used in this study, where either O,O′-bidentate or O-unidentate modes of nitrite binding to the cupric center are present. To study the role of the reducing ability of the ferrous heme center, two different tetraarylporphyrinate–iron(II) complexes, one with electron-donating para-methoxy peripheral substituents and the other with electron-withdrawing 2,6-difluorophenyl substituents, were used. The results show that differing modes of nitrite coordination to the copper(II) ion lead to differing kinetic behavior. Here, also, the ferrous heme is in all cases the source of the reducing equivalent required to convert nitrite to nitric oxide, but the reduction ability of the heme center does not play a key role in the observed overall reaction rate. On the basis of our observations, reaction mechanisms are proposed and discussed in terms of heme/copper heterobinuclear structures.


Nitric oxide Model compounds Nitrite reductase Nitrite coordination modes Cytochrome c oxidase 



We are grateful to the US National Institutes of Health for support of this research (GM60353).

Supplementary material

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Supplementary material 1 (PDF 1135 kb)


  1. 1.
    Schopfer MP, Wang J, Karlin KD (2010) Inorg Chem 49:6267–6282PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Tavares P, Pereira AS, Moura JJG, Moura I (2006) J Inorg Biochem 100:2087–2100PubMedCrossRefGoogle Scholar
  3. 3.
    Zumft WG (1997) Microbiol Mol Biol Rev 61:533–616PubMedCentralPubMedGoogle Scholar
  4. 4.
    Poole RK (2005) Biochem Soc Trans 33:176–180PubMedCrossRefGoogle Scholar
  5. 5.
    Samouilov A, Kuppusamy P, Zweier JL (1998) Arch Biochem Biophys 357:1–7PubMedCrossRefGoogle Scholar
  6. 6.
    Dezfulian C, Raat N, Shiva S, Gladwin MT (2007) Cardiovasc Res 75:327–338PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Lundberg JO, Gladwin MT, Ahluwalia A, Benjamin N, Bryan NS, Butler A, Cabrales P, Fago A, Feelisch M, Ford PC, Freeman BA, Frenneaux M, Friedman J, Kelm M, Kevil CG, Kim-Shapiro DB, Kozlov AV, Lancaster JR, Lefer DJ, McColl K, McCurry K, Patel RP, Petersson J, Rassaf T, Reutov VP, Richter-Addo GB, Schechter A, Shiva S, Tsuchiya K, van Faassen EE, Webb AJ, Zuckerbraun BS, Zweier JL, Weitzberg E (2009) Nat Chem Biol 5:865–869PubMedCrossRefGoogle Scholar
  8. 8.
    van Faassen EE, Bahrami S, Feelisch M, Hogg N, Kelm M, Kim-Shapiro DB, Kozlov AV, Li H, Lundberg JO, Mason R, Nohl H, Rassaf T, Samouilov A, Slama-Schwok A, Shiva S, Vanin AF, Weitzberg E, Zweier J, Gladwin MT (2009) Med Res Rev 29:683–741PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Fukuto JM, Carrington SJ, Tantillo DJ, Harrison JG, Ignarro LJ, Freeman BA, Chen A, Wink DA (2012) Chem Res Toxicol 25:769–793PubMedCrossRefGoogle Scholar
  10. 10.
    Kozlov AV, Staniek K, Nohl H (1999) FEBS Lett 454:127–130PubMedCrossRefGoogle Scholar
  11. 11.
    Crane BR (2008) Biochem Soc Trans 036:1149–1154CrossRefGoogle Scholar
  12. 12.
    Zhu Y, Silverman RB (2008) Biochemistry 47:2231–2243PubMedCrossRefGoogle Scholar
  13. 13.
    Gladwin MT, Schechter AN, Kim-Shapiro DB, Patel RP, Hogg N, Shiva S, Cannon RO, Kelm M, Wink DA, Espey MG, Oldfield EH, Pluta RM, Freeman BA, Lancaster JR, Feelisch M, Lundberg JO (2005) Nat Chem Biol 1:308–314PubMedCrossRefGoogle Scholar
  14. 14.
    Gladwin MT, Shiva S (2009) Circ Res 104:1136–1138PubMedCrossRefGoogle Scholar
  15. 15.
    Zweier JL, Samouilov A, Kuppusamy P (1999) Biochim Biophys Acta 1411:250–262PubMedCrossRefGoogle Scholar
  16. 16.
    Cosby K, Partovi KS, Crawford JH, Patel RP, Reiter CD, Martyr S, Yang BK, Waclawiw MA, Zalos G, Xu XL, Huang KT, Shields H, Kim-Shapiro DB, Schechter AN, Cannon RO, Gladwin MT (2003) Nat Med 9:1498–1505PubMedCrossRefGoogle Scholar
  17. 17.
    Shiva S, Huang Z, Grubina R, Sun JH, Ringwood LA, MacArthur PH, Xu XL, Murphy E, Darley-Usmar VM, Gladwin MT (2007) Circ Res 100:654–661PubMedCrossRefGoogle Scholar
  18. 18.
    Shiva S, Rassaf T, Patel RP, Gladwin MT (2011) Cardiovasc Res 89:566–573PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Rassaf T, Flogel U, Drexhage C, Hendgen-Cotta U, Kelm M, Schrader J (2007) Circ Res 100:1749–1754PubMedCrossRefGoogle Scholar
  20. 20.
    Totzeck M, Hendgen-Cotta UB, Luedike P, Berenbrink M, Klare JP, Steinhoff HJ, Semmler D, Shiva S, Williams D, Kipar A, Gladwin MT, Schrader J, Kelm M, Cossins AR, Rassaf T (2012) Circulation 126:325–334PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Tiso M, Js Tejero, Basu S, Azarov I, Wang X, Simplaceanu V, Frizzell S, Jayaraman T, Geary L, Shapiro C, Ho C, Shiva S, Kim-Shapiro DB, Gladwin MT (2011) J Biol Chem 286:18277–18289PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Basu S, Azarova NA, Font MD, King SB, Hogg N, Gladwin MT, Shiva S, Kim-Shapiro DB (2008) J Biol Chem 283:32590–32597PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Gladwin MT (2005) Nat Chem Biol 1:245–246PubMedCrossRefGoogle Scholar
  24. 24.
    Lundberg JO, Weitzberg E, Gladwin MT (2008) Nat Rev Drug Discov 7:156–167PubMedCrossRefGoogle Scholar
  25. 25.
    de Mel A, Murad F, Seifalian AM (2011) Chem Rev 111:5742–5767PubMedCrossRefGoogle Scholar
  26. 26.
    Babcock GT, Wikström M (1992) Nature 356:301–309PubMedCrossRefGoogle Scholar
  27. 27.
    Ferguson-Miller S, Babcock GT (1996) Chem Rev 96:2889–2908PubMedCrossRefGoogle Scholar
  28. 28.
    Kim E, Chufán EE, Kamaraj K, Karlin KD (2004) Chem Rev 104:1077–1133PubMedCrossRefGoogle Scholar
  29. 29.
    Poyton RO, Ball KA (2011) Discov Med 57:154–159Google Scholar
  30. 30.
    Shiva S (2013) Redox Biol 1:40–44PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Castello PR, David PS, McClure T, Crook Z, Poyton RO (2006) Cell Metab 3:277–287PubMedCrossRefGoogle Scholar
  32. 32.
    Gupta KJ, Stoimenova M, Kaiser WM (2005) J Exp Bot 56:2601–2609PubMedCrossRefGoogle Scholar
  33. 33.
    Castello PR, Woo DK, Ball K, Wojcik J, Liu L, Poyton RO (2008) Proc Natl Acad Sci USA 105:8203–8208PubMedCentralPubMedCrossRefGoogle Scholar
  34. 34.
    Poyton RO, Castello PR, Ball KA, Woo DK, Pan N (2009) Ann N Y Acad Sci 1177:48–56PubMedCrossRefGoogle Scholar
  35. 35.
    Gupta KJ, Igamberdiev AU (2011) Mitochondrion 11:537–543PubMedCrossRefGoogle Scholar
  36. 36.
    Hematian S, Siegler MA, Karlin KD (2012) J Am Chem Soc 134:18912–18915PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Kopf M-A, Neuhold Y-M, Zuberbühler AD, Karlin KD (1999) Inorg Chem 38:3093–3102CrossRefGoogle Scholar
  38. 38.
    Ghiladi RA, Kretzer RM, Guzei I, Rheingold AL, Neuhold Y-M, Hatwell KR, Zuberbühler AD, Karlin KD (2001) Inorg Chem 40:5754–5767PubMedCrossRefGoogle Scholar
  39. 39.
    Torres J, Sharpe MA, Rosquist A, Cooper CE, Wilson MT (2000) FEBS Lett 475:263–266PubMedCrossRefGoogle Scholar
  40. 40.
    Ford PC, Lorkovic IM (2002) Chem Rev 102:993–1017PubMedCrossRefGoogle Scholar
  41. 41.
    Wang J, Schopfer MP, Sarjeant AAN, Karlin KD (2009) J Am Chem Soc 131:450–451PubMedCentralPubMedCrossRefGoogle Scholar
  42. 42.
    Schopfer MP, Mondal B, Lee D-H, Sarjeant AAN, Karlin KD (2009) J Am Chem Soc 131:11304–11305PubMedCentralPubMedCrossRefGoogle Scholar
  43. 43.
    Liang H-C, Zhang CX, Henson MJ, Sommer RD, Hatwell KR, Kaderli S, Zuberbuehler AD, Rheingold AL, Solomon EI, Karlin KD (2002) J Am Chem Soc 124:4170–4171PubMedCrossRefGoogle Scholar
  44. 44.
    Park GY, Deepalatha S, Puiu SC, Lee D-H, Mondal B, Narducci Sarjeant AA, del Rio D, Pau MYM, Solomon EI, Karlin KD (2009) J Biol Inorg Chem 14:1301–1311PubMedCentralPubMedCrossRefGoogle Scholar
  45. 45.
    Vogel KM, Kozlowski PM, Zgierski MZ, Spiro TG (1999) J Am Chem Soc 121:9915–9921CrossRefGoogle Scholar
  46. 46.
    Sheldrick GM (2008) Acta Crystallogr Sect A 64:112–122CrossRefGoogle Scholar
  47. 47.
    Yang L, Powell DR, Houser RP (2007) Dalton Trans 955–964Google Scholar
  48. 48.
    Mukhopadhyay U, Bernal I, Massoud SS, Mautner FA (2004) Inorg Chim Acta 357:3673–3682CrossRefGoogle Scholar
  49. 49.
    Nakamoto K (1997) Infrared and Raman spectra of inorganic and coordination compounds, 5th edn. Wiley Interscience, New York, pp 48–53Google Scholar
  50. 50.
    Quant Hatcher L, Karlin KD (2004) J Biol Inorg Chem 9:669–683Google Scholar
  51. 51.
    Hatcher LQ, Karlin KD (2006) Adv Inorg Chem 58:131–184CrossRefGoogle Scholar
  52. 52.
    Chufan EE, Mondal B, Gandhi T, Kim E, Rubie ND, Moenne-Loccoz P, Karlin KD (2007) Inorg Chem 46:6382–6394PubMedCrossRefGoogle Scholar
  53. 53.
    Kakuda S, Peterson RL, Ohkubo K, Karlin KD, Fukuzumi S (2013) J Am Chem Soc 135:6513–6522PubMedCentralPubMedCrossRefGoogle Scholar
  54. 54.
    Das D, Lee Y-M, Ohkubo K, Nam W, Karlin KD, Fukuzumi S (2013) J Am Chem Soc 135:2825–2834PubMedCentralPubMedCrossRefGoogle Scholar
  55. 55.
    Itoh S (2011) In: Itoh S, Karlin KD (eds) Copper-oxygen chemistry. Wiley, Hoboken, pp 225–282Google Scholar
  56. 56.
    Halime Z, Kieber-Emmons MT, Qayyum MF, Mondal B, Gandhi T, Puiu SC, Chufán EE, Sarjeant AAN, Hodgson KO, Hedman B, Solomon EI, Karlin KD (2010) Inorg Chem 49:3629–3645PubMedCentralPubMedCrossRefGoogle Scholar
  57. 57.
    Fox S, Nanthakumar A, Wikström M, Karlin KD, Blackburn NJ (1996) J Am Chem Soc 118:24–34CrossRefGoogle Scholar
  58. 58.
    Obias HV, van Strijdonck GPF, Lee D-H, Ralle M, Blackburn NJ, Karlin KD (1998) J Am Chem Soc 120:9696–9697CrossRefGoogle Scholar
  59. 59.
    Wang J, Schopfer MP, Puiu SC, Sarjeant AAN, Karlin KD (2010) Inorg Chem 49:1404–1419PubMedCentralPubMedCrossRefGoogle Scholar
  60. 60.
    Karlin KD, Nanthakumar A, Fox S, Murthy NN, Ravi N, Huynh BH, Orosz RD, Day EP (1994) J Am Chem Soc 116:4753–4763CrossRefGoogle Scholar
  61. 61.
    Helms JH, Terhaar LW, Hatfield WE, Harris DL, Jayaraj K, Toney GE, Gold A, Mewborn TD, Pemberton JR (1986) Inorg Chem 25:2334–2337CrossRefGoogle Scholar
  62. 62.
    Yi J, Heinecke J, Tan H, Ford PC, Richter-Addo GB (2009) J Am Chem Soc 131:18119–18128PubMedCentralPubMedCrossRefGoogle Scholar
  63. 63.
    Silaghi-Dumitrescu R (2004) Inorg Chem 43:3715–3718PubMedCrossRefGoogle Scholar
  64. 64.
    Williams PA, Fulop V, Garman EF, Saunders NFW, Ferguson SJ, Hajdu J (1997) Nature 389:406–412PubMedCrossRefGoogle Scholar
  65. 65.
    Perissinotti LL, Marti MA, Doctorovich F, Luque FJ, Estrin DA (2008) Biochemistry 47:9793–9802PubMedCrossRefGoogle Scholar
  66. 66.
    Blomberg LM, Blomberg MRA, Siegbahn PEM (2006) Biochim Biophys Acta 1757:240–252PubMedCrossRefGoogle Scholar
  67. 67.
    Blomberg LM, Blomberg MRA, Siegbahn PEM (2007) J Biol Inorg Chem 12:79–89PubMedCrossRefGoogle Scholar
  68. 68.
    Halime Z, Kotani H, Li Y, Fukuzumi S, Karlin KD (2011) Proc Natl Acad Sci USA 108:13990–13994PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© SBIC 2014

Authors and Affiliations

  • Shabnam Hematian
    • 1
  • Maxime A. Siegler
    • 1
  • Kenneth D. Karlin
    • 1
    Email author
  1. 1.Department of ChemistryJohns Hopkins UniversityBaltimoreUSA

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