Reductive nitrosylation of ferric human hemoglobin bound to human haptoglobin 1-1 and 2-2

  • Paolo Ascenzi
  • Giovanna De Simone
  • Fabio Polticelli
  • Magda Gioia
  • Massimo Coletta
Original Paper


Haptoglobin (Hp) sequesters hemoglobin (Hb) preventing the Hb-based damage occurring upon its physiological release into plasma. Here, reductive nitrosylation of ferric human hemoglobin [Hb(III)] bound to human haptoglobin (Hp) 1-1 and 2-2 [Hp1-1:Hb(III) and Hp2-2:Hb(III), respectively] has been investigated between pH 7.5 and 9.5, at T=20.0 °C. Over the whole pH range explored, only one process is detected reflecting NO binding to Hp1-1:Hb(III) and Hp2-2:Hb(III). Values of the pseudo-first-order rate constant for Hp1-1:Hb(III) and Hp2-2:Hb(III) nitrosylation (k) do not depend linearly on the ligand concentration but tend to level off. The conversion of Hp1-1:Hb(III)-NO to Hp1-1:Hb(II)-NO and of Hp2-2:Hb(III)-NO to Hp2-2:Hb(II)-NO is limited by the OH- and H2O-based catalysis. In fact, bimolecular NO binding to Hp1-1:Hb(III), Hp2-2:Hb(III), Hp1-1:Hb(II), and Hp2-2:Hb(II) proceeds very rapidly. The analysis of data allowed to determine the values of the dissociation equilibrium constant for Hp1-1:Hb(III) and Hp2-2:Hb(III) nitrosylation [K = (1.2 ± 0.1) × 10−4 M], which is pH-independent, and of the first-order rate constant for Hp1-1:Hb(III) and Hp2-2:Hb(III) conversion to Hp1-1:Hb(II)-NO and Hp2-2:Hb(II)-NO, respectively (k′). From the dependence of k′ on [OH], values of hOH– [(4.9 ± 0.6) × 103 M−1 s−1 and (6.79 ± 0.7) × 103 M−1 s−1, respectively] and of \( h_{{{\text{H}}_{ 2} {\text{O}}}} \) [(2.6 ± 0.3) × 10−3 s−1] were determined. Values of kinetic and thermodynamic parameters for Hp1-1:Hb(III) and Hp2-2:Hb(III) reductive nitrosylation match well with those of the Hb R-state, which is typical of the αβ dimers of Hb bound to Hp.


Ferric human hemoglobin Human haptoglobin 1-1 Human haptoglobin 2-2 Haptoglobin1-1:hemoglobin complex Haptoglobin2-2:hemoglobin complex Reductive nitrosylation Kinetics 



Complement control protein




Ferrous Hb


Ferric Hb


Nitrosylated Hb




Phenotype 1-1 of Hp


Phenotype 2-2 of Hp


Hp1-1:Hb complex


Hp1-1:Hb(II) complex


Hp1-1:Hb(III) complex


Hp1-1:Hb(II)-NO complex


Hp2-2:Hb complex


Hp2-2Hb(II) complex


Hp2-2:Hb(III) complex


Hp1-1:Hb(II)-NO complex




Ferric Mb



SP-like domain

Serine protease-like domain



The grant of Excellence Departments, MIUR (Legge 232/2016, Articolo 1, Comma 314-337), is gratefully acknowledged.


  1. 1.
    Bunn HF, Forget BG (1986) Hemoglobin: molecular, genetic and clinical aspects. WB Saunders Company, PhiladelphiaGoogle Scholar
  2. 2.
    Perutz MF (1990) Annu Rev Physiol 52:1–25CrossRefPubMedGoogle Scholar
  3. 3.
    Gow AJ, Luchsinger BP, Pawloski JR, Singel DJ, Stamler JS (1999) Proc Natl Acad Sci USA 96:9027–9032CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Imai K (1999) Nature 401:437–439CrossRefPubMedGoogle Scholar
  5. 5.
    McMahon TJ, Moon RE, Luschinger BP, Carraway MS, Stone AE, Stolp BW, Gow AJ, Pawloski JR, Watke P, Singel DJ, Piantadosi CA, Stamler JS (2002) Nat Med 8:711–717CrossRefPubMedGoogle Scholar
  6. 6.
    Ascenzi P, Brunori M (2016) J Porphyrins Phthalocyanines 20:134–149CrossRefGoogle Scholar
  7. 7.
    Ascenzi P, Bocedi A, Visca P, Altruda F, Tolosano E, Beringhelli T, Fasano M (2005) IUBMB Life 57:749–759CrossRefPubMedGoogle Scholar
  8. 8.
    Smith A, McCulloh RJ (2015) Front Physiol 6:187CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Andersen CBF, Stødkilde K, Sæderup KL, Kuhlee A, Raunser S, Graversen JH, Moestrup SK (2017) Antioxid Redox Signal 26:814–831CrossRefPubMedGoogle Scholar
  10. 10.
    Wang Y, Kinzie E, Berger FG, Lim SK, Baumann H (2001) Redox Rep 6:379–385CrossRefPubMedGoogle Scholar
  11. 11.
    Polticelli F, Bocedi A, Minervini G, Ascenzi P (2008) FEBS J 275:5648–5656CrossRefPubMedGoogle Scholar
  12. 12.
    Andersen CB, Torvund-Jensen M, Nielsen MJ, de Oliveira CL, Hersleth HP, Andersen NH, Pedersen JS, Andersen GR, Moestrup SK (2012) Nature 489:456–459CrossRefPubMedGoogle Scholar
  13. 13.
    Stødkilde K, Torvund-Jensen M, Moestrup SK, Andersen CB (2014) Nat Commun 5:5487CrossRefPubMedGoogle Scholar
  14. 14.
    Kristiansen M, Graversen JH, Jacobsen C, Sonne O, Hoffman HJ, Law SK, Moestrup SK (2001) Nature 409:198–201CrossRefPubMedGoogle Scholar
  15. 15.
    Nagel RL, Gibson QH (1966) J Mol Biol 22:249–255CrossRefPubMedGoogle Scholar
  16. 16.
    Chiancone E, Antonini E, Brunori M, Alfsen A, Lavialle F (1973) Biochem J 133:205–207CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Azarov I, He X, Jeffers A, Basu S, Ucer B, Hantgan RR, Levy A, Kim-Shapiro DB (2008) Nitric Oxide 18:296–302CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Antonini E, Brunori M (1971) Hemoglobin and myoglobin in their reactions with ligands. North-Holland Publishing Co, AmsterdamGoogle Scholar
  19. 19.
    Brunori M, Alfsen A, Saggese U, Antonini E, Wyman J (1968) J Biol Chem 243:2950–2954PubMedGoogle Scholar
  20. 20.
    Nagel RL, Whittenberg JB, Ranney HM (1965) Biochim Biophys Acta 100:286–289CrossRefPubMedGoogle Scholar
  21. 21.
    Chiancone E, Wittenberg JB, Wittenberg BA, Antonini E, Wyman J (1966) Biochim Biophys Acta 117:379–386CrossRefPubMedGoogle Scholar
  22. 22.
    Ascenzi P, Brunori M, Pennesi G, Ercolani C, Monacelli F (1987) J Chem Soc Dalton Trans 369–371Google Scholar
  23. 23.
    Hoshino M, Ozawa K, Seki H, Ford PC (1993) J Am Chem Soc 115:9568–9575CrossRefGoogle Scholar
  24. 24.
    Hoshino M, Maeda M, Konishi R, Seki H, Ford PC (1996) J Am Chem Soc 118:5702–5707CrossRefGoogle Scholar
  25. 25.
    Boffi A, Sarti P, Amiconi G, Chiancone E (2002) Biophys Chem 98:209–216CrossRefPubMedGoogle Scholar
  26. 26.
    Herold S, Fago A, Weber RE, Dewilde S, Moens L (2004) J Biol Chem 279:22841–22847CrossRefPubMedGoogle Scholar
  27. 27.
    Herold S, Puppo A (2005) J Biol Inorg Chem 10:946–957CrossRefPubMedGoogle Scholar
  28. 28.
    Ascenzi P, Bocedi A, Antonini G, Bolognesi M, Fasano M (2007) FEBS J 274:551–562CrossRefPubMedGoogle Scholar
  29. 29.
    Ascenzi P, di Masi A, Gullotta F, Mattu M, Ciaccio C, Coletta M (2010) Biochem Biophys Res Commun 393:196–200CrossRefPubMedGoogle Scholar
  30. 30.
    Ascenzi P, Yu C, di Masi A, Gullotta F, De Sanctis G, Fanali G, Fasano M, Coletta M (2010) FEBS J 277:2474–2485CrossRefPubMedGoogle Scholar
  31. 31.
    Ascenzi P, Pesce A, Nardini M, Bolognesi M, Ciaccio C, Coletta M, Dewilde S (2013) Biochem Biophys Res Commun 430:1301–1305CrossRefPubMedGoogle Scholar
  32. 32.
    Ascenzi P, Marino M, Ciaccio C, Santucci R, Coletta M (2014) IUBMB Life 66:438–447CrossRefPubMedGoogle Scholar
  33. 33.
    Ascenzi P, di Masi A, Tundo GR, Pesce A, Visca P, Coletta M (2014) PLoS One 9:e102811CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Ascenzi P, Bocedi A, Gioia M, Fanali G, Fasano M, Coletta M (2017) J Inorg Biochem 177:63–75CrossRefPubMedGoogle Scholar
  35. 35.
    Ascenzi P, Ciaccio C, De Simone G, Santucci R, Coletta M (2017) J Porphyrins Phthalocyanines 21:1–9CrossRefGoogle Scholar
  36. 36.
    Antonini E, Ascenzi P, Menegatti E, Guarneri M (1983) Biopolymers 22:363–375CrossRefPubMedGoogle Scholar
  37. 37.
    Bolli A, Ciaccio C, Coletta M, Nardini M, Bolognesi M, Pesce A, Guertin M, Visca P, Ascenzi P (2008) FEBS J 275:633–645CrossRefPubMedGoogle Scholar
  38. 38.
    Moore EG, Gibson QH (1976) J Biol Chem 251:2788–2794PubMedGoogle Scholar
  39. 39.
    Perutz MF (1979) Annu Rev Biochem 48:327–386CrossRefPubMedGoogle Scholar
  40. 40.
    Perutz MF (1989) Trends Biochem Sci 14:42–44CrossRefPubMedGoogle Scholar
  41. 41.
    Bolognesi M, Bordo D, Rizzi M, Tarricone C, Ascenzi P (1997) Prog Biophys Mol Biol 68:29–68CrossRefPubMedGoogle Scholar
  42. 42.
    Pesce A, Dewilde S, Nardini M, Moens L, Ascenzi P, Hankeln T, Burmester T, Bolognesi M (2003) Structure 11:1087–1095CrossRefPubMedGoogle Scholar
  43. 43.
    Hamdane D, Kiger L, Dewilde S, Green BN, Pesce A, Uzan J, Burmester T, Hankeln T, Bolognesi M, Moens L, Marden MC (2003) J Biol Chem 278:51713–51721CrossRefPubMedGoogle Scholar
  44. 44.
    Brucker EA, Olson JS, Ikeda-Saito M, Phillips GN Jr (1998) Proteins 30:352–356CrossRefPubMedGoogle Scholar
  45. 45.
    Beetlestone JG, Adeosun OS, Goddard JE, Kushimo JB, Ogunlesi MM, Ogunmola GB, Okonjo KO, Seamonds B (1976) J Chem Soc Dalton Trans 1251–1278Google Scholar
  46. 46.
    Miele AE, Santanché S, Travaglini-Allocatelli C, Vallone B, Brunori M, Bellelli A (1999) J Mol Biol 290:515–524CrossRefPubMedGoogle Scholar
  47. 47.
    Giacometti GM, Ascenzi P, Bolognesi M, Brunori M (1981) J Mol Biol 146:363–374CrossRefPubMedGoogle Scholar
  48. 48.
    Antonini E, Ascenzi P, Bolognesi M, Menegatti E, Guarneri M (1983) J Biol Chem 258:4676–4678PubMedGoogle Scholar
  49. 49.
    Ascenzi P, Gianni S (2013) IUBMB Life 65:836–844CrossRefPubMedGoogle Scholar

Copyright information

© SBIC 2018

Authors and Affiliations

  • Paolo Ascenzi
    • 1
  • Giovanna De Simone
    • 2
  • Fabio Polticelli
    • 2
    • 3
  • Magda Gioia
    • 4
    • 5
  • Massimo Coletta
    • 4
    • 5
  1. 1.Interdepartmental Laboratory of Electron MicroscopyRoma Tre UniversityRomeItaly
  2. 2.Department of SciencesRoma Tre UniversityRomeItaly
  3. 3.Roma Tre SectionNational Institute of Nuclear PhysicsRomeItaly
  4. 4.Department of Clinical Sciences and Translational MedicineUniversity of Roma “Tor Vergata”RomeItaly
  5. 5.Interuniversity Consortium for the Research on the Chemistry of Metals in Biological SystemsBariItaly

Personalised recommendations