An Origin of Cooperative Oxygen Binding of Human Adult Hemoglobin: Different Roles of the α and β Subunits in the α2β2 Tetramer

Abstract

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.

Heme Orientation of Cavity Mutant Hemoglobins (His F8 → Gly) in Either α or β Subunits: Circular Dichroism, 1H NMR, and Resonance Raman Studies

“…As shown in the previous article, rHb(αH87G) displayed a biphasic O 2 binding curve consisting of a high O 2 affinity component (mutated α subunits) and a low O 2 affinity one (native β subunits). No cooperativity, no Bohr effect, and no apparent effect by an allosteric effector (inositol hexaphosphate, IHP) were observed.…”

“…The plasmids for rHb(αH87G) and rHb(βH92G) were produced using an amplification procedure for closed circular DNA in vitro and transformed into E. coli JM109. Culture of cells and expression of rHb were the same as reported previously . Recombinant Hbs were purified according to the methods described before .…”

“…Culture of cells and expression of rHb were the same as reported previously . Recombinant Hbs were purified according to the methods described before . We separated the rHb A with reversed heme (in both the α and β subunits with contents of >80%) from that with normal heme through SP‐Sepharose column chromatography .…”

“…The CD measurements were carried out with a Jasco (Tokyo, Japan) J‐820 spectropolarimeter with the same conditions as reported previously . Absorption spectra were measured with a double‐beam spectrophotometer (Hitachi, model U‐3010).…”

“…Thus, His F8 might be involved in determination of the heme orientation in the pocket. In order to examine the effect of substitution of glycine (Gly) for His F8 on the CD spectrum, we prepared two mutant Hbs, rHb(αH87G) and rHb(βH92G), in the presence of imidazole and their heme orientations were investigated by CD, 1 H NMR, and resonance Raman (RR) spectroscopies.…”