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

Nagatomo S, Nagai Y, Aki Y, Sakurai H, Imai K, Mizusawa N, Ogura T, Kitagawa T, Nagai M - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Tsukuba, Tsukuba, Ibaraki, Japan.

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.

No MeSH data available.


Related in: MedlinePlus

600 MHz 1H NMR spectra of Hb A, rHb(αH87G) and rHb(βH92G) in the deoxy-form.Spectra are between 10 and 30 ppm at pH 7.0 and 25°C. The hemoglobin concentrations of Hb A, rHb(αH87G) an rHb(βH92G) were 1 mM, 800 and 500 μM, respectively, on a heme basis in 0.05 M phosphate buffer (pH 7.0). In addition, rHb(αH87G) and rHb(βH92G) contained 10 mM imidazole.
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pone.0135080.g006: 600 MHz 1H NMR spectra of Hb A, rHb(αH87G) and rHb(βH92G) in the deoxy-form.Spectra are between 10 and 30 ppm at pH 7.0 and 25°C. The hemoglobin concentrations of Hb A, rHb(αH87G) an rHb(βH92G) were 1 mM, 800 and 500 μM, respectively, on a heme basis in 0.05 M phosphate buffer (pH 7.0). In addition, rHb(αH87G) and rHb(βH92G) contained 10 mM imidazole.

Mentions: The 1H NMR spectra in the heme methyl region (between 30 and 10 ppm) of Hb A, rHb(αH87G) and rHb(βH92G) in the deoxy-form are displayed in Fig 6, where the paramagnetically shifted heme methyl proton signals are observed at 23.2 and 19.4 ppm for the β heme and at 20.4 and 17.3 ppm for the α heme, respectively [26,27,65]. In the spectra of rHb(αH87G) only the methyl signals of heme derived from the normal β heme were observed at the same position as those of Hb A, and similarly in rHb(βH92G), only the methyl signals of heme from the normal α heme were observed. The fact that the chemical shifts of these heme methyl proton signals observed for rHb(αH87G) and rHb(βH92G) are very similar to those of deoxyHb A strongly suggests that the heme structures of the normal α subunits in rHb(βH92G) and normal β subunits in rHb(αH87G) have the Fe-His bond similar to native deoxyHb A. These paramagnetically shifted heme methyl proton signals disappeared upon CO binding to heme due to diamagnetism shown in S1 Fig or S1 File.


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

Nagatomo S, Nagai Y, Aki Y, Sakurai H, Imai K, Mizusawa N, Ogura T, Kitagawa T, Nagai M - PLoS ONE (2015)

600 MHz 1H NMR spectra of Hb A, rHb(αH87G) and rHb(βH92G) in the deoxy-form.Spectra are between 10 and 30 ppm at pH 7.0 and 25°C. The hemoglobin concentrations of Hb A, rHb(αH87G) an rHb(βH92G) were 1 mM, 800 and 500 μM, respectively, on a heme basis in 0.05 M phosphate buffer (pH 7.0). In addition, rHb(αH87G) and rHb(βH92G) contained 10 mM imidazole.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526547&req=5

pone.0135080.g006: 600 MHz 1H NMR spectra of Hb A, rHb(αH87G) and rHb(βH92G) in the deoxy-form.Spectra are between 10 and 30 ppm at pH 7.0 and 25°C. The hemoglobin concentrations of Hb A, rHb(αH87G) an rHb(βH92G) were 1 mM, 800 and 500 μM, respectively, on a heme basis in 0.05 M phosphate buffer (pH 7.0). In addition, rHb(αH87G) and rHb(βH92G) contained 10 mM imidazole.
Mentions: The 1H NMR spectra in the heme methyl region (between 30 and 10 ppm) of Hb A, rHb(αH87G) and rHb(βH92G) in the deoxy-form are displayed in Fig 6, where the paramagnetically shifted heme methyl proton signals are observed at 23.2 and 19.4 ppm for the β heme and at 20.4 and 17.3 ppm for the α heme, respectively [26,27,65]. In the spectra of rHb(αH87G) only the methyl signals of heme derived from the normal β heme were observed at the same position as those of Hb A, and similarly in rHb(βH92G), only the methyl signals of heme from the normal α heme were observed. The fact that the chemical shifts of these heme methyl proton signals observed for rHb(αH87G) and rHb(βH92G) are very similar to those of deoxyHb A strongly suggests that the heme structures of the normal α subunits in rHb(βH92G) and normal β subunits in rHb(αH87G) have the Fe-His bond similar to native deoxyHb A. These paramagnetically shifted heme methyl proton signals disappeared upon CO binding to heme due to diamagnetism shown in S1 Fig or S1 File.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Tsukuba, Tsukuba, Ibaraki, Japan.

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.

No MeSH data available.


Related in: MedlinePlus