We found that recombinant human adult hemoglobin (rHb A) expressed in Escherichia coli showed heterogeneity of components with the intensity of a positive CD band at 260 nm and that it could be resolved into three components (SP-1, SP-2, and SP-3) by SP-Sepharose column chromatography. 1H NMR revealed that SP-1 is identical with native Hb A, while SP-2 and SP-3 largely contain the reversed heme isomer in both the alpha and beta subunits, with contents of approximately 50 and >80% in SP-2 and SP-3, respectively. Rotation of the heme 180 degrees about the 5,15-meso axis (reversed heme) causes an interexchange of the methyl groups at positions 2 and 7 with the vinyl groups at positions 8 and 3, respectively. To examine the effect of the modification of the heme-protein contact on the structure and function of Hb A, we compared the 1H NMR, CD, and oxygen binding properties of the three components with those of native Hb A. Native Hb A exhibits a distinct positive CD band in both the near-UV and Soret regions, but rHb A with reversed heme exhibits a very weak positive CD band at 260 nm and a prominent negative CD band in the Soret region. Cooperativity, as measured by Hill's n value, decreased from 3.18 (SP-1) to 2.94 (SP-2) to 2.63 (SP-3) with an increase in the reversed heme orientation. The effect of an allosteric effector, inositol hexaphosphate (IHP), on the oxygen binding properties was also reduced in rHb A with reversed heme. These results indicate that changes in the heme-globin contact exert a discernible influence on CD spectra and cooperative oxygen binding.

Human hemoglobin (Hb), which is an α2β2 tetramer and binds four O2 molecules, changes its O2-affinity from low to high as an increase of bound O2, that is characterized by ‘cooperativity’. This property is indispensable for its function of O2 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 O2-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 O2-binding with no cooperativity, indicating the coexistence of two independent hemes with different O2-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 O2-binding curve with no cooperativity. Resonance Raman, 1H NMR, and near-UV circular dichroism measurements revealed that the quaternary structure change did not occur upon O2-binding to rHb(αH87G), but it did partially occur with O2-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 O2-binding, but its absence in the β subunit simply enhances the O2-affinity of α subunit.