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

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Abstract

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.

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Abbreviations

CCP:

Complement control protein

Hb:

Hemoglobin

Hb(II):

Ferrous Hb

Hb(III):

Ferric Hb

Hb(II)-NO:

Nitrosylated Hb

Hp:

Haptoglobin

Hp1-1:

Phenotype 1-1 of Hp

Hp2-2:

Phenotype 2-2 of Hp

Hp1-1:Hb:

Hp1-1:Hb complex

Hp1:Hb(II):

Hp1-1:Hb(II) complex

Hp1-1:Hb(III):

Hp1-1:Hb(III) complex

Hp1-1:Hb(II)-NO:

Hp1-1:Hb(II)-NO complex

Hp2-2:Hb:

Hp2-2:Hb complex

Hp2-2:Hb(II):

Hp2-2Hb(II) complex

Hp2-2:Hb(III):

Hp2-2:Hb(III) complex

Hp1-1:Hb(II)-NO:

Hp1-1:Hb(II)-NO complex

Mb:

Myoglobin

Mb(III):

Ferric Mb

Ngb:

Neuroglobin

SP-like domain:

Serine protease-like domain

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Acknowledgements

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

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Authors and Affiliations

  1. Interdepartmental Laboratory of Electron Microscopy, Roma Tre University, Via Della Vasca Navale 79, 00146, Rome, Italy

    Paolo Ascenzi

  2. Department of Sciences, Roma Tre University, Viale Guglielmo Marconi 446, 00146, Rome, Italy

    Giovanna De Simone & Fabio Polticelli

  3. Roma Tre Section, National Institute of Nuclear Physics, Via Della Vasca Navale 84, 00146, Rome, Italy

    Fabio Polticelli

  4. Department of Clinical Sciences and Translational Medicine, University of Roma “Tor Vergata”, Via Montpellier 1, 00133, Rome, Italy

    Magda Gioia & Massimo Coletta

  5. Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems, Via Celso Ulpiani 27, 70126, Bari, Italy

    Magda Gioia & Massimo Coletta

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  1. Paolo Ascenzi
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  2. Giovanna De Simone
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  3. Fabio Polticelli
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Correspondence to Paolo Ascenzi.

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Ascenzi, P., De Simone, G., Polticelli, F. et al. Reductive nitrosylation of ferric human hemoglobin bound to human haptoglobin 1-1 and 2-2. J Biol Inorg Chem 23, 437–445 (2018). https://doi.org/10.1007/s00775-018-1551-y

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  • DOI: https://doi.org/10.1007/s00775-018-1551-y

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