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

Roman Davydov, Viktoria Kofman, Judith M. Nocek, Robert W. Noble, Hilda Hui, Brian M. Hoffman*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

31 Scopus citations

Abstract

Exposure of frozen solutions of oxyhemoglobin to γ-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 βO2 and isolated αO2 and βO2 chains, as well as αO 2β(Zn), α(Zn)βO2, and αO 2β(Fe3+) 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 (g1 ≤ 2.25), denoted A and B. Averaged over many similar sites, the A-center has a rhombic EPR signal with a g-tensor, gA = [2.248(4), 2.146(1), 1.966(1)]; the B-center exhibits a less anisotropic EPR signal, gB = [2.216(3), 2.118(2), 1.966(1)]. Early measurement had suggested that, in the cryoreduced HbO2 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 αO2 and βO2 subunits: αO2A (minor species) and αO 2B (major species); βO2A (major species) and βO2B (minor species). Similar behavior is seen for MbO2 [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 αO2 and βO 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 α chains and that the glycol-induced changes in the substate populations of the R-state HbO2 tetramer are largely associated with the αO 2 subunit. 1H 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 αO2 and βO 2 subunits differ not only in their tertiary structures but in their affinities for O2.

Original languageEnglish (US)
Pages (from-to)6330-6338
Number of pages9
JournalBiochemistry
Volume43
Issue number20
DOIs
StatePublished - May 25 2004

ASJC Scopus subject areas

  • Biochemistry

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