Heme-bound Tyrosine Vibrations in Hemoglobin M: Resonance Raman, Crystallography and DFT Calculation.

Hemoglobins M (Hbs M) are human hemoglobin variants in which either α or β subunit contains a ferric heme in the α2 β2 tetramer. Though the ferric subunit cannot bind O2 , it regulates O2 affinity of its counterpart ferrous subunit. We have investigated resonance Raman spectra of two Hbs M Iwate (α87His → Tyr) and M Boston (α58His → Tyr), having tyrosine as a heme axial ligand at proximal and distal positions, respectively, that exhibit unassigned resonance Raman bands arising from ferric (not ferrous) hemes at 899 and 876 cm-1 . Our quantum chemical calculations using density functional theory (DFT) on Fe-porphyrin models with p-cresol and/or 4-methylimidazole showed that the unassigned bands correspond to the breathing-like modes of Fe3+ -bound tyrosine (Tyr) and are sensitive to the Fe-O-C(Tyr) angle. Based on the frequencies of the Raman bands, the Fe-O-C(Tyr) angles of Hbs M Iwate and M Boston were predicted to be 153.5o and 129.2o , respectively. Consistent with this prediction, X-ray crystallographic analysis showed that the Fe-O-C(Tyr) angles of Hbs M Iwate and M Boston in the T quaternary structure were 153.6o and 134.6o , respectively. It also showed a similar Fe-O bond length (1.96 and 1.97 Å) and different tilting angles.