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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

Hill’s plots of oxygen binding by Hb A, rHb(αH87G), rHb(βH92G) and sperm whale Mb.Hb A (-IHP) (blue closed triangle: ▲), Hb A (+IHP) (pink closed square: ■), rHb(αH87G) (black closed circle: ●), rHb(βH92G) (orange closed circle: ●) and sperm whale Mb (light green closed diamond: ◆). Y and pO2 are as in Fig 2. The symbols are the observed points. The hemoglobin concentration was 60 μM on a heme basis in 0.05 M bis-Tris buffer (pH 7.4) containing 0.1 M Cl-. In addition, rHb(αH87G) and rHb(βH92G) contained 5 mM imidazole and a metHb reducing system. The temperature was set at 25°C. IHP was added to a final concentration of 2 mM.
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pone.0135080.g004: Hill’s plots of oxygen binding by Hb A, rHb(αH87G), rHb(βH92G) and sperm whale Mb.Hb A (-IHP) (blue closed triangle: ▲), Hb A (+IHP) (pink closed square: ■), rHb(αH87G) (black closed circle: ●), rHb(βH92G) (orange closed circle: ●) and sperm whale Mb (light green closed diamond: ◆). Y and pO2 are as in Fig 2. The symbols are the observed points. The hemoglobin concentration was 60 μM on a heme basis in 0.05 M bis-Tris buffer (pH 7.4) containing 0.1 M Cl-. In addition, rHb(αH87G) and rHb(βH92G) contained 5 mM imidazole and a metHb reducing system. The temperature was set at 25°C. IHP was added to a final concentration of 2 mM.

Mentions: We expressed two mutant Hbs in E. coli and examined the effects of mutation on the O2 binding properties. As shown in Figs 2–4 and Table 1, both rHb(αH87G) and rHb(βH92G) exhibited O2 binding properties different from those of Hb A. Further, the O2 binding properties of these mutant Hbs were different from each other. We show UV-Vis absorption spectra of the mutant Hbs, rHb(αH87G) and rHb(βH92G), in S4 and S5 Figs, respectively.


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)

Hill’s plots of oxygen binding by Hb A, rHb(αH87G), rHb(βH92G) and sperm whale Mb.Hb A (-IHP) (blue closed triangle: ▲), Hb A (+IHP) (pink closed square: ■), rHb(αH87G) (black closed circle: ●), rHb(βH92G) (orange closed circle: ●) and sperm whale Mb (light green closed diamond: ◆). Y and pO2 are as in Fig 2. The symbols are the observed points. The hemoglobin concentration was 60 μM on a heme basis in 0.05 M bis-Tris buffer (pH 7.4) containing 0.1 M Cl-. In addition, rHb(αH87G) and rHb(βH92G) contained 5 mM imidazole and a metHb reducing system. The temperature was set at 25°C. IHP was added to a final concentration of 2 mM.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4526547&req=5

pone.0135080.g004: Hill’s plots of oxygen binding by Hb A, rHb(αH87G), rHb(βH92G) and sperm whale Mb.Hb A (-IHP) (blue closed triangle: ▲), Hb A (+IHP) (pink closed square: ■), rHb(αH87G) (black closed circle: ●), rHb(βH92G) (orange closed circle: ●) and sperm whale Mb (light green closed diamond: ◆). Y and pO2 are as in Fig 2. The symbols are the observed points. The hemoglobin concentration was 60 μM on a heme basis in 0.05 M bis-Tris buffer (pH 7.4) containing 0.1 M Cl-. In addition, rHb(αH87G) and rHb(βH92G) contained 5 mM imidazole and a metHb reducing system. The temperature was set at 25°C. IHP was added to a final concentration of 2 mM.
Mentions: We expressed two mutant Hbs in E. coli and examined the effects of mutation on the O2 binding properties. As shown in Figs 2–4 and Table 1, both rHb(αH87G) and rHb(βH92G) exhibited O2 binding properties different from those of Hb A. Further, the O2 binding properties of these mutant Hbs were different from each other. We show UV-Vis absorption spectra of the mutant Hbs, rHb(αH87G) and rHb(βH92G), in S4 and S5 Figs, respectively.

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