A comparison study of the Mössbauer spectra of deoxy Hb-A (low oxygen affinity) and its isolated α and β subunits (high oxygen affinity) was carried out over the temperature range 77–200°K. Within experimental error, no difference was detected between these three proteins, either in the isomeric shifts or in the quadrupole splittings. These results show that the characteristically different oxygen affinity of deoxy Hb-A and its isolated subunits is not a consequence of different electronic states for the ferrous ions in Hb-A and its isolated subunits. The electronic structure of the ferrous ion in hemoglobin was determined using a crystal field approximation. The adjustable parameters in the crystal field model were systematically searched for an electronic level configuration that would give good agreement with the experimental data of the temperature-dependent quadrupole splitting and magnetic susceptibility of deoxy Hb-A. The resulting low lying energy levels in order of increasing energy were 5B2, 1A1, 5E, and 3E. The spin and orbital degeneracy of these levels were removed by the spin-orbit interaction and the rhombic perturbation of the crystalline field. The electronic ground state obtained produces an electric field gradient at the iron nucleus with a principal component of 0.11 e<r−3> parallel to the heme plane and an asymmetry parameter η = 0.51.
The Mössbauer spectra of dehydrated αSH and βSH deoxyhemoglobin subunits were obtained over a temperature range from 77 to 200°K and compared with anhydrous hemoglobin spectra. The Mössbauer data for both subunits show a superposition of a ferrous high spin (S = 2) and a ferrous low spin (S = 0) quadrupole doublet of approximately equal intensity. No differences either in the isomer shifts or the quadrupole splittings were detected between the three hemoproteins. Consequently, the model for AHb–A with the α and β chains in different spin states proposed by previous investigators is probably incorrect. The observed quadrupole splittings, ΔEQ(T), are analyzed in a ligand field approximation, which gives the electronic structure of the low lying eigenstates of the ferrous ion at the two spin sites. The principal components of the EFG for the low and high spin iron sites calculated from the ligand field model are normal to the heme plane and have the values at T = 80°K of −0.09e 〈r−3〉 and 0.21e 〈r−3〉, respectively. At T = 80°K the asymmetry parameter, η, is zero for the low spin iron site and 0.39 for the high spin site. A possible interpretation of the origin of the spin state mixture based on the disruption of the hydrophobic interaction upon dehydration is discussed.
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