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

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, 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 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: αO₂^A (minor species) and αO ₂^B (major species); βO₂^A (major species) and βO₂^B (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.