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Home / Equine Research Bank / Biochem J / The pH dependence of naturally occurring low-spin forms of m...
The Biochemical journal2000; 351 Pt 3(Pt 3); 595-605;

The pH dependence of naturally occurring low-spin forms of methaemoglobin and metmyoglobin: an EPR study.

Abstract: The paramagnetic species in human metHb and horse metmyoglobin (metMb) have been studied at low temperature using EPR spectroscopy. The high-spin (HS) haem signal in aquometMb has a greater rhombic distortion than the HS metHb signal. Nevertheless, the individual line width (g=6) is smaller in metMb than in metHb, consistent with non-identical signals from the alpha and beta Hb subunits. Three low-spin (LS) haem forms are present in metHb, while metMb has only two. The major LS form in both proteins is the alkaline species (with OH(-) at the sixth co-ordination position). The minor LS forms are assigned to different histidine hemichromes in equilibrium with the normal HS species at low temperature. LS forms disappear when the haem is bound by a ligand, such as fluoride, which ensures 100% occupancy of the HS state both at room temperature and at 25 K. The small differences in effective g-factors of the histidine hemichromes are interpreted in terms of different distances between the distal histidine and haem iron. The pH dependence of the inter-conversion of the different paramagnetic species is consistent with a model whereby protonation of a residue with a pK of 5.69 (metHb) or 6.12 (metMb), affects ligand binding and transformation from the HS to the LS form. Chemical and spectroscopic considerations suggest that the residue is unlikely to be the proximal or distal histidine. We therefore propose a model where protonation of this distant amino acid causes a conformational change at the iron site. Identical effects are seen in frozen human blood, suggesting that this effect may have physiological significance.
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Publication Date: 2000-10-24 PubMed ID: 11042113PubMed Central: PMC1221398
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  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The study uses Electron Paramagnetic Resonance (EPR) spectroscopy to examine the paramagnetic species in human metHb and horse metmyoglobin (metMb) at low temperatures, comparing the distinct differences and behaviors of high-spin (HS) and low-spin (LS) forms of haem and their human and equine counterparts. The research provides both chemical and spectroscopic evidence suggesting that the transformation from the HS to the LS form is influenced by the protonation of a particular residue, theorizing this change could be important for human physiology.

EPR Spectroscopy and Paramagnetic Species

  • The research makes use of EPR spectroscopy, a method used to study materials with unpaired electrons, like paramagnetic substances.
  • Paramagnetic species examined were those found in human methaemoglobin (metHb) and horse metmyoglobin (metMb), molecules relevant to oxygen transport in the blood.
  • These species were studied in a low-temperature environment, as cold temperatures can provide more accurate results by slowing down molecular motion.

Specifications of High-Spin and Low-Spin Haem Forms

  • The high-spin (HS) haem signal in aquometMb had greater distortion than the HS metHb signal, with the individual line width smaller in metMb.
  • In metHb, three low-spin (LS) haem forms are present, while metMb has only two. The major LS form in both proteins is the alkaline species, which has OH(-) at the sixth coordination position.
  • The minor LS forms have been associated with different histidine hemichromes, which are in equilibrium with the normal HS species at low temperate. LS forms disappear when they are bound by a ligand, such as fluoride, ensuring 100% occupancy of the HS state at both room temperature and 25 K.

Differences in Effective g-factors and pH Dependence

  • The small differences in effective g-factors in the histidine hemichromes suggest different distances between the distal histidine and haem iron.
  • The conversion between the different paramagnetic species is influenced by the pH, consistent with a model whereby protonation of a residue with a pK of 5.69 effects ligand binding and transformation in metHb, while a pK of 6.12 effects this in metMb.

Implications and Physiological Significance

  • The study suggests that the residue is unlikely to be the proximal or distal histidine due to chemical and spectroscopic considerations. The authors proposed a model where protonation of a distant amino acid causes a conformational change at the iron site.
  • Identical effects appear in frozen human blood, suggesting that these findings could be of physiological significance regarding how the human body internally processes and circulates oxygen.

Cite This Article

APA
Svistunenko DA, Sharpe MA, Nicholls P, Blenkinsop C, Davies NA, Dunne J, Wilson MT, Cooper CE. (2000). The pH dependence of naturally occurring low-spin forms of methaemoglobin and metmyoglobin: an EPR study. Biochem J, 351 Pt 3(Pt 3), 595-605.

Publication

The Biochemical journal
ISSN: 0264-6021
NlmUniqueID: 2984726R
Country: England
Language: English
Volume: 351 Pt 3
Issue: Pt 3
Pages: 595-605

Researcher Affiliations

Svistunenko, D A
  • Department of Biological Sciences, Central Campus, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, UK.
Sharpe, M A
    Nicholls, P
      Blenkinsop, C
        Davies, N A
          Dunne, J
            Wilson, M T
              Cooper, C E

                MeSH Terms

                • Animals
                • Electron Spin Resonance Spectroscopy
                • Horses
                • Humans
                • Hydrogen-Ion Concentration
                • Methemoglobin / chemistry
                • Metmyoglobin / chemistry

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