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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 229-nm excited UVRR spectra of Hb A, rHb(αH87G) and rHb(βH92G).Spectra are deoxyHb A (A), deoxy rHb(αH87G) (B) and deoxy rHb(βH92G) (C), and the difference between Hb A (deoxy–CO) (D), rHb(αH87G) (deoxy–CO) (E) and rHb(βH92G) (deoxy–CO) (F). The hemoglobin concentration was 200 μM (in heme) in a 0.05 M phosphate buffer (pH 7.0) containing 0.2 M SO42- as the internal intensity standard. In addition, rHb(αH87G) and rHb(βH92G) contained 10 mM imidazole. The difference spectra were obtained so that the Raman band of SO42- (980 cm-1) could be abolished. The spectra shown are an average of 13 scans.
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pone.0135080.g007: The 229-nm excited UVRR spectra of Hb A, rHb(αH87G) and rHb(βH92G).Spectra are deoxyHb A (A), deoxy rHb(αH87G) (B) and deoxy rHb(βH92G) (C), and the difference between Hb A (deoxy–CO) (D), rHb(αH87G) (deoxy–CO) (E) and rHb(βH92G) (deoxy–CO) (F). The hemoglobin concentration was 200 μM (in heme) in a 0.05 M phosphate buffer (pH 7.0) containing 0.2 M SO42- as the internal intensity standard. In addition, rHb(αH87G) and rHb(βH92G) contained 10 mM imidazole. The difference spectra were obtained so that the Raman band of SO42- (980 cm-1) could be abolished. The spectra shown are an average of 13 scans.

Mentions: Fig 7 shows the 229-nm excited UVRR spectra of the deoxy-form and the deoxy-minus-CO difference spectra for Hb A (A, D), rHb(αH87G) (B, E) and rHb(βH92G) (C, F) in the frequency region from 1700 to 650 cm-1. The Raman bands of Tyr and Trp are marked by Y and W, respectively, followed by their mode number [68–71]. The band at 980 cm-1 arises from the SO42- ions added as an internal intensity standard. In the raw spectra, the intensities of the W3, W16, W17, and W18 bands of Trp are weaker in COHb A than in deoxyHb A, while the peak positions remain unaltered. Accordingly, positive peaks appear in the deoxy-minus-CO difference spectra. For the Tyr bands, however, the frequencies of the Y8a and Y9a bands of deoxyHb A are shifted lower in COHb A, and therefore, differential patterns appeared in the deoxy-minus-CO difference spectra (Fig 7D). These spectral differences have been ascribed to certain alterations in the hydrogen bonding and the surrounding hydrophobicity of the Trp and Tyr residues upon ligand binding [68–72]. Therefore, the deoxy-minus-CO difference spectrum of Hb A (Fig 7D) will serve hereafter as the standard for the T–R difference spectrum.


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 229-nm excited UVRR spectra of Hb A, rHb(αH87G) and rHb(βH92G).Spectra are deoxyHb A (A), deoxy rHb(αH87G) (B) and deoxy rHb(βH92G) (C), and the difference between Hb A (deoxy–CO) (D), rHb(αH87G) (deoxy–CO) (E) and rHb(βH92G) (deoxy–CO) (F). The hemoglobin concentration was 200 μM (in heme) in a 0.05 M phosphate buffer (pH 7.0) containing 0.2 M SO42- as the internal intensity standard. In addition, rHb(αH87G) and rHb(βH92G) contained 10 mM imidazole. The difference spectra were obtained so that the Raman band of SO42- (980 cm-1) could be abolished. The spectra shown are an average of 13 scans.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0135080.g007: The 229-nm excited UVRR spectra of Hb A, rHb(αH87G) and rHb(βH92G).Spectra are deoxyHb A (A), deoxy rHb(αH87G) (B) and deoxy rHb(βH92G) (C), and the difference between Hb A (deoxy–CO) (D), rHb(αH87G) (deoxy–CO) (E) and rHb(βH92G) (deoxy–CO) (F). The hemoglobin concentration was 200 μM (in heme) in a 0.05 M phosphate buffer (pH 7.0) containing 0.2 M SO42- as the internal intensity standard. In addition, rHb(αH87G) and rHb(βH92G) contained 10 mM imidazole. The difference spectra were obtained so that the Raman band of SO42- (980 cm-1) could be abolished. The spectra shown are an average of 13 scans.
Mentions: Fig 7 shows the 229-nm excited UVRR spectra of the deoxy-form and the deoxy-minus-CO difference spectra for Hb A (A, D), rHb(αH87G) (B, E) and rHb(βH92G) (C, F) in the frequency region from 1700 to 650 cm-1. The Raman bands of Tyr and Trp are marked by Y and W, respectively, followed by their mode number [68–71]. The band at 980 cm-1 arises from the SO42- ions added as an internal intensity standard. In the raw spectra, the intensities of the W3, W16, W17, and W18 bands of Trp are weaker in COHb A than in deoxyHb A, while the peak positions remain unaltered. Accordingly, positive peaks appear in the deoxy-minus-CO difference spectra. For the Tyr bands, however, the frequencies of the Y8a and Y9a bands of deoxyHb A are shifted lower in COHb A, and therefore, differential patterns appeared in the deoxy-minus-CO difference spectra (Fig 7D). These spectral differences have been ascribed to certain alterations in the hydrogen bonding and the surrounding hydrophobicity of the Trp and Tyr residues upon ligand binding [68–72]. Therefore, the deoxy-minus-CO difference spectrum of Hb A (Fig 7D) will serve hereafter as the standard for the T–R difference spectrum.

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