Elsevier

Nitric Oxide

Volume 13, Issue 4, December 2005, Pages 226-231
Nitric Oxide

Studies on nitrosyl hemes in Ni(II)–Fe(II) hybrid hemoglobins

https://doi.org/10.1016/j.niox.2005.07.001Get rights and content

Abstract

Subunit heterogeneity within a particular subunit in hemoglobin A have been explored with electron paramagnetic resonance spectroscopy using the nitrosyl hemes in Ni–Fe hybrid Hb under various solution conditions. Our previous studies on the crystal structure of NiHb demonstrated the presence of subunit heterogeneity within α-subunit. To further cross check this hypothesis, we made a hybrid Hb in which either the α- or β-subunit contains iron, which alone can bind to NO. By this way dynamic exchange between penta- and hexa-coordinated forms within a subunit was confirmed. Upon the addition of inositol hexa phosphate (IHP) to these hybrids, R to T state transition is observed for [α2(Fe–NO)β2(Ni)] but such a direct transformation is less marked in [α2(Ni)β2(Fe–NO)]. Hence the bond between Nε and Fe is fundamental to the structure–function relation in Hb, as the motion of this nitrogen triggers the vast transformation, which occurs in the whole molecule on attachment of NO.

Section snippets

Experimental procedures

Human blood was processed and purified by well-established methods to obtain electrophoretically pure Hb. Hybrids namely [α2(Fe)β2(Ni)] and [α2(Ni)β2(Fe)] were made according to previously reported methods [6]. The nitrosyl derivatives of the hybrids were prepared according to earlier workers [11]. X-band EPR measurements were carried out on a Varian E-112 EPR spectrometer operating with 100 kHz field modulations and phase sensitive detection to obtain the first derivative signal. All

Types of EPR signal detected for hybrid Hb’s

The X-band EPR spectrum of [α2(Ni)β2(Fe–NO)] at 77 K exhibits a roughly rhombic line shape with g-values approximately 2.080, 2.045, and 1.988 (Fig. 1D). Upon addition of an allosteric effector inositol hexa phosphate (IHP) a poorly resolved three-line hyperfine splitting is observed as a result of breaking of the Fe–N (imidazole) of the proximal histidine (F8) bond (Fig. 1H) or at least lengthening of the proximal histidine–iron (Nε–Fe) bond (vide infra for details). Unlike in the case of [α2

Discussion

Nitric oxide has been widely used as a spectroscopic probe of Hb structure linked to its binding properties [8], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20]. The EPR work of on model compounds [12] (Figs. 1A and E) and on peroxidases [21] allowed the complete assignment of the 77 K EPR spectra of nitrosyl heme-proteins. It has been shown by earlier workers that the EPR spectrum of isolated α(Fe–NO) exhibits a rhombically distorted line shape unlike β(Fe–NO). It is not quite

Conclusions

The main conclusion emerging so far from these studies are that there is no indication of any T-state in [α2(Fe–NO)β2(Ni)] hybrid which easily gets driven to the T-state during the addition of the allosteric regulator, IHP, possibly indicating the loosening of the Fe–Nε bond while such a drive towards the T-state is less so pronounced in [α2(Ni)β2(Fe–NO)]. In a sense this is similar to the observation of four- and five-coordination in the T-structured and only the five-coordination in the

Acknowledgments

P.T.M. and B.V. thanks the DST, Government of India for a research scheme (SP/S1/F-18/2000) and S.V. thanks the CSIR for a fellowship. P.T.M. also thanks the JNCASR, Banglore and the INSA, New Delhi.

References (31)

  • J.A. Mc cleverty

    Chemistry of nitric oxide relevant to biology

    Chem. Rev.

    (2004)
  • M.F. Perutz

    Stereochemistry of cooperative effects in hemoglobin: heme–heme interaction and problem of allostery

    Nature

    (1970)
  • M.F. Perutz

    Structure and mechanism of haemoglobin

    Brit. Med. Bull.

    (1976)
  • W.E. Blumberg

    Methods Enzymol.

    (1981)
  • M.F. Perutz

    Mechanism of cooperativity and allosteric regulation in proteins

    Q. Rev. Bioph.

    (1989)
  • N. Shibayama et al.

    Proton nuclear magnetic resonance and spectrophotometric studies of nickel(II)–iron(II) hybrid hemoglobins

    Biochemistry

    (1987)
  • B. Venkatesh et al.
  • Y. Henry et al.

    Electron paramagnetic studies of nitric oxide hemoglobin derivatives: isolated subunits and nitric oxide hybrids

    J. Mol. Biol.

    (1973)
  • K. Nagai et al.

    The influence of quaternary structure on the EPR spectra of ferric haemoglobin

    FEBS Lett.

    (1978)
  • K. Nagai et al.

    The effect of quaternary structure on the state of the α and β subunits within nitrosyl hemoglobin

    Biochim. Biophys. Acta

    (1978)
  • T. Yonetani et al.

    Electron paramagnetic resonance and oxygen binding studies of α-nitrosyl hemoglobin: a novel oxygen carrier having no-assisted allosteric functions

    J. Biol. Chem.

    (1998)
  • H. Kon

    Paramagnetic resonance study of nitric oxide hemoglobin

    J. Biol. Chem.

    (1968)
  • H. Kon et al.

    Electron paramagnetic resonance of nitric oxide-protoheme complexes with some nitrogenous base. Model systems of nitric oxide hemoproteins

    Biochemistry

    (1969)
  • J.C.W. Chein

    Electron paramagnetic resonance study of the stereochemistry of nitrosyl hemoglobin

    J. Chem. Phys.

    (1969)
  • T. Shiga et al.

    Electron paramagnetic resonance studies of nitric oxide hemoglobin derivatives. I. Human hemoglobin subunits

    Biochemistry

    (1969)
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