Studies on nitrosyl hemes in Ni(II)–Fe(II) hybrid hemoglobins

Subunit heterogeneity within a particular subunit in hemoglobin A have been explored with electron paramagnetic resonance spectroscopy using the nitrosyl hemes in Ni–Fe hybrid Hb under various solution conditions. Our previous studies on the crystal structure of NiHb demonstrated the presence of subunit heterogeneity within α-subunit. To further cross check this hypothesis, we made a hybrid Hb in which either the α- or β-subunit contains iron, which alone can bind to NO. By this way dynamic exchange between penta- and hexa-coordinated forms within a subunit was confirmed. Upon the addition of inositol hexa phosphate (IHP) to these hybrids, R to T state transition is observed for [α2(Fe–NO)β2(Ni)] but such a direct transformation is less marked in [α2(Ni)β2(Fe–NO)]. Hence the bond between Nε and Fe is fundamental to the structure–function relation in Hb, as the motion of this nitrogen triggers the vast transformation, which occurs in the whole molecule on attachment of NO