An Origin of Cooperative Oxygen Binding of Human Adult Hemoglobin: Different Roles of the α and β Subunits in the α(2)β(2) Tetramer

Human hemoglobin (Hb), which is an α(2)β(2) tetramer and binds four O(2) molecules, changes its O(2)-affinity from low to high as an increase of bound O(2), that is characterized by ‘cooperativity’. This property is indispensable for its function of O(2) transfer from a lung to tissues and is accounted for in terms of T/R quaternary structure change, assuming the presence of a strain on the Fe-histidine (His) bond in the T state caused by the formation of hydrogen bonds at the subunit interfaces. However, the difference between the α and β subunits has been neglected. To investigate the different roles of the Fe-His(F8) bonds in the α and β subunits, we investigated cavity mutant Hbs in which the Fe-His(F8) in either α or β subunits was replaced by Fe-imidazole and F8-glycine. Thus, in cavity mutant Hbs, the movement of Fe upon O(2)-binding is detached from the movement of the F-helix, which is supposed to play a role of communication. Recombinant Hb (rHb)(αH87G), in which only the Fe-His in the α subunits is replaced by Fe-imidazole, showed a biphasic O(2)-binding with no cooperativity, indicating the coexistence of two independent hemes with different O(2)-affinities. In contrast, rHb(βH92G), in which only the Fe-His in the β subunits is replaced by Fe-imidazole, gave a simple high-affinity O(2)-binding curve with no cooperativity. Resonance Raman, (1)H NMR, and near-UV circular dichroism measurements revealed that the quaternary structure change did not occur upon O(2)-binding to rHb(αH87G), but it did partially occur with O(2)-binding to rHb(βH92G). The quaternary structure of rHb(αH87G) appears to be frozen in T while its tertiary structure is changeable. Thus, the absence of the Fe-His bond in the α subunit inhibits the T to R quaternary structure change upon O(2)-binding, but its absence in the β subunit simply enhances the O(2)-affinity of α subunit.