Conformational substates of the oxyheme centers in alpha and beta subunits of hemoglobin as disclosed by EPR and ENDOR studies of cryoreduced protein

Biochemistry. 2004 May 25;43(20):6330-8. doi: 10.1021/bi036273z.


Exposure of frozen solutions of oxyhemoglobin to gamma-irradiation at 77 K yields EPR- and ENDOR-active, one-electron-reduced oxyheme centers which retain the conformation of the diamagnetic precursor. EPR spectra have been collected for the centers produced in human HbO(2) and isolated alphaO(2) and betaO(2) chains, as well as alphaO(2)beta(Zn), alpha(Zn)betaO(2), and alphaO(2)beta(Fe(3+)) hybrids, each in frozen buffer and in frozen glasses that form in the presence of glycols and sugars and also in the presence of IHP. These reveal two spectroscopically distinct classes of such ferriheme centers (g(1) <or= 2.25), denoted A and B. Averaged over many similar sites, the A-center has a rhombic EPR signal with a g-tensor, g(A) = [2.248(4), 2.146(1), 1.966(1)]; the B-center exhibits a less anisotropic EPR signal, g(B) = [2.216(3), 2.118(2), 1.966(1)]. Early measurement had suggested that, in the cryoreduced HbO(2) tetramer, the two centers corresponded to the two different chains [Symons, M. C. R., and Petersen, R. L. (1978) Proc. R. Soc. London, Ser. B 201, 285-300]. However, the present EPR and ENDOR results show that the two signals instead reflect the fact that the parent oxyhemes exist in two major conformational substates and that this is true for both alphaO(2) and betaO(2) subunits: alphaO(2)(A) (minor species) and alphaO(2)(B) (major species); betaO(2)(A)(major species) and betaO(2)(B) (minor species). Similar behavior is seen for MbO(2) [Kappl, R., Höhn-Berlage, M., Hüttermann, J., Bartlett, N., and Symons, M. C. R. (1985) Biochim. Biophys. Acta 827, 327-343]. The A/B g-tensors of alphaO(2) and betaO(2) chains vary little with the environment of the chains, while the relative populations of the substates depend greatly on glycols and IHP. These results suggest a quaternary influence on the oxyheme distal pocket of alpha chains and that the glycol-induced changes in the substate populations of the R-state HbO(2) tetramer are largely associated with the alphaO(2) subunit. (1)H ENDOR spectra from the distal histidine proton hydrogen-bonded to the peroxo ligand show very different isotropic coupling for the A- and B-centers. Analysis of the spectroscopic data suggests that the A- and B-centers represent different orientations of the oxyheme O(2) ligand relative to the distal histidine. It is likely that the A and B conformational substates in the alphaO(2) and betaO(2) subunits differ not only in their tertiary structures but in their affinities for O(2).

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Electron Spin Resonance Spectroscopy
  • Heme / chemistry*
  • Hemoglobins / chemistry*
  • Hemoglobins / metabolism
  • Humans
  • Myoglobin / chemistry
  • Oxidation-Reduction
  • Oxygen / chemistry*
  • Protein Conformation*
  • Protein Subunits / chemistry*
  • Protein Subunits / metabolism


  • Hemoglobins
  • Myoglobin
  • Protein Subunits
  • Heme
  • Oxygen