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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

The Hill plots of oxygen binding by Hb A (▲) and rHb(αH92G) (●).Y and pO2 are as in Fig 2. The symbols are the observed points and lines were calculated from the best-fit values of the four stepwise Adair constants [3,7,62]. Adair constants, K1 and K4, used at fitting of rHb(βH92G) are 0.60 and 3.5 mmHg-1, those of Hb A are 0.014 and 8.0 mmHg-1, respectively. 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(βH92G) contained 5 mM imidazole and metHb reducing system. The temperature was 25°C.
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pone.0135080.g003: The Hill plots of oxygen binding by Hb A (▲) and rHb(αH92G) (●).Y and pO2 are as in Fig 2. The symbols are the observed points and lines were calculated from the best-fit values of the four stepwise Adair constants [3,7,62]. Adair constants, K1 and K4, used at fitting of rHb(βH92G) are 0.60 and 3.5 mmHg-1, those of Hb A are 0.014 and 8.0 mmHg-1, respectively. 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(βH92G) contained 5 mM imidazole and metHb reducing system. The temperature was 25°C.

Mentions: The Hill plot of rHb(βH92G) was compared with that of Hb A in Fig 3, where the symbols denote the observed points and the smooth curves were calculated from the best-fit values of the four stepwise Adair constants [3,7,62]. Recombinant Hb(βH92G) exhibited little cooperativity (Hill’s n = 1.2 − 1.3), in contrast with n = 3.0 for Hb A, and its Bohr effect was half of that of Hb A (Table 1). The Hill plot of rHb(βH92G) was further compared with those of rHb(αH87G), Mb, and Hb A in the presence and absence of IHP in Fig 4. The O2 equilibrium curve of rHb(βH92G) demonstrated that its O2 affinity was high (P50 = 1.9 mmHg), similar to that of Mb [64]. It was not influenced by allosteric effectors such as IHP, as seen in rHb(αH87G), shown in S7 Fig or S1 File. Thus, although both rHb(βH92G) and swMb show similar oxygen affinities, rHb(βH92G) has quaternary structure but swMb has a tertiary structure only. Comparison of oxygen affinity of rHb(βH92G) with that of swMb seems important for understanding the increase of oxygen affinity in rHb(βH92G) in spite of its T quaternary structure in deoxy form. The equilibrium constant associated with the fourth oxygenation step, K4, of rHb(βH92G) exhibited a value similar to that of Hb A, but the equilibrium constant for the first oxygenation step, K1, was 30 times larger than that of Hb A (Fig 3). If we accept the idea that Fe-Im heme has a higher affinity than that of Fe-His heme in consonance with the previous 1H NMR results on ligand binding to rHb(αH87G) [54], the observed K1 and K4 indicates the affinity of the β(Fe-Im) and α(Fe-His) subunits, respectively. This means that the detachment of the F-helix from heme in the β subunits by a disconnection of the Fe-His bond causes an increase of O2 affinity in the α subunits whose hemes are attached to the F-helix, to the same degree as that of Mb.


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)

The Hill plots of oxygen binding by Hb A (▲) and rHb(αH92G) (●).Y and pO2 are as in Fig 2. The symbols are the observed points and lines were calculated from the best-fit values of the four stepwise Adair constants [3,7,62]. Adair constants, K1 and K4, used at fitting of rHb(βH92G) are 0.60 and 3.5 mmHg-1, those of Hb A are 0.014 and 8.0 mmHg-1, respectively. 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(βH92G) contained 5 mM imidazole and metHb reducing system. The temperature was 25°C.
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Related In: Results  -  Collection

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

pone.0135080.g003: The Hill plots of oxygen binding by Hb A (▲) and rHb(αH92G) (●).Y and pO2 are as in Fig 2. The symbols are the observed points and lines were calculated from the best-fit values of the four stepwise Adair constants [3,7,62]. Adair constants, K1 and K4, used at fitting of rHb(βH92G) are 0.60 and 3.5 mmHg-1, those of Hb A are 0.014 and 8.0 mmHg-1, respectively. 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(βH92G) contained 5 mM imidazole and metHb reducing system. The temperature was 25°C.
Mentions: The Hill plot of rHb(βH92G) was compared with that of Hb A in Fig 3, where the symbols denote the observed points and the smooth curves were calculated from the best-fit values of the four stepwise Adair constants [3,7,62]. Recombinant Hb(βH92G) exhibited little cooperativity (Hill’s n = 1.2 − 1.3), in contrast with n = 3.0 for Hb A, and its Bohr effect was half of that of Hb A (Table 1). The Hill plot of rHb(βH92G) was further compared with those of rHb(αH87G), Mb, and Hb A in the presence and absence of IHP in Fig 4. The O2 equilibrium curve of rHb(βH92G) demonstrated that its O2 affinity was high (P50 = 1.9 mmHg), similar to that of Mb [64]. It was not influenced by allosteric effectors such as IHP, as seen in rHb(αH87G), shown in S7 Fig or S1 File. Thus, although both rHb(βH92G) and swMb show similar oxygen affinities, rHb(βH92G) has quaternary structure but swMb has a tertiary structure only. Comparison of oxygen affinity of rHb(βH92G) with that of swMb seems important for understanding the increase of oxygen affinity in rHb(βH92G) in spite of its T quaternary structure in deoxy form. The equilibrium constant associated with the fourth oxygenation step, K4, of rHb(βH92G) exhibited a value similar to that of Hb A, but the equilibrium constant for the first oxygenation step, K1, was 30 times larger than that of Hb A (Fig 3). If we accept the idea that Fe-Im heme has a higher affinity than that of Fe-His heme in consonance with the previous 1H NMR results on ligand binding to rHb(αH87G) [54], the observed K1 and K4 indicates the affinity of the β(Fe-Im) and α(Fe-His) subunits, respectively. This means that the detachment of the F-helix from heme in the β subunits by a disconnection of the Fe-His bond causes an increase of O2 affinity in the α subunits whose hemes are attached to the F-helix, to the same degree as that of Mb.

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