UV Resonance Raman Studies of α-Nitrosyl Hemoglobin Derivatives:  Relation between the α1−β2 Subunit Interface Interactions and the Fe−Histidine Bonding of α Heme

Human α-nitrosyl β-deoxy hemoglobin A, αNOβdeoxy, is considered to have a T (tense) structure with the low O2 affinity extreme and the Fe−histidine (His87) (Fe−His) bond of α heme cleaved. The Fe−His bonding of α heme and the intersubunit interactions at the α1−β2 contact of αNO-Hbs have been examined under various conditions with EPR and UV resonance Raman (UVRR) spectra excited at 235 nm, respectively. NOHb at pH 6.7 gave the UVRR spectrum of the R structure, but in the presence of inositol-hexakis-phosphate (IHP) for which the Fe−His bond of the α heme is broken, UVRR bands of Trp residues behaved half-T-like while Tyr bands remained R-like. The half-ligated nitrosylHb, αNOβdeoxy, in the presence of IHP at pH 5.6, gave T-like UVRR spectra for both Tyr and Trp, but binding of CO to its β heme (αNOβCO) changed the UVRR spectrum to half-T-like. Binding of NO to its β heme (NOHb) changed the UVRR spectrum to 70% T-type for Trp but almost R-type for Tyr. When the pH was raised to 8.2 in the presence of IHP, the UVRR spectrum of NOHb was the same as that of COHb. EPR spectra of these Hbs indicated that the Fe−His bond of αNO heme is partially cleaved. On the other hand, the UVRR spectra of αNOβdeoxy in the absence of IHP at pH 8.8 showed the T-like UVRR spectrum, but the EPR spectrum indicated that 40−50% of the Fe−His bond of α hemes was intact. Therefore, it became evident that there is a qualitative correlation between the cleavage of the Fe−His bond of α heme and T-like contact of Trp-β37. We note that the behaviors of Tyr and Trp residues at the α1−β2 interface are not synchronous. It is likely that the behaviors of Tyr residues are controlled by the ligation of β heme through His-β92(F8)→Val-β98(FG5)→Asp-β99(G1)→Tyr-α42(C7) or Tyr-β145(HC2).