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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).Spectra are CO- and deoxyHb A (A, B), CO- and deoxy-rHb(αH87G) (C, D), and CO- and deoxy-rHb(βH92G) (E, F) between 10 and 16 ppm at pH 7.0 and 25°C. The hemoglobin concentrations of Hb A, rHb(αH87G) and 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.g005: 600 MHz 1H NMR spectra of Hb A, rHb(αH87G) and rHb(βH92G).Spectra are CO- and deoxyHb A (A, B), CO- and deoxy-rHb(αH87G) (C, D), and CO- and deoxy-rHb(βH92G) (E, F) between 10 and 16 ppm at pH 7.0 and 25°C. The hemoglobin concentrations of Hb A, rHb(αH87G) and 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: Fig 5 compares the 1H NMR spectra of the deoxy- and CO-forms of rHb(αH87G) and rHb(βH92G) with those of Hb A in the frequency region between 16 and 10 ppm. In deoxyHb A (Fig 5B), four proton signals were resolved between 16 and 10 ppm and assigned as indicated in the figures [65]. These four protons were ascribed to hydrogen bonded ones at the α1-β2 and α1-β1 subunit-interfaces. As the signal around 14.0 ppm derived from the hydrogen bond between Tyrα42 and Aspβ99 at the α1-β2 interface can be observed only in the deoxy-form, this signal has generally been used as a marker of the T quaternary structure. The signal observed around 11.0 ppm has also been observed only for the deoxy-form, and therefore, was thought to arise from the hydrogen bond between Aspα94 and Trpβ37 at the α1-β2 interface, although there is a report that does not regard the 11.0 ppm signal as a T-marker [54]. The fact that both the 14.0 and 11.0 ppm signals are observed for rHb(αH87G) (Fig 5D) and rHb(βH92G) (Fig 5F) in the deoxy-form, similar to deoxyHb A, suggests that both rHb(αH87G) and rHb(βH92G) take the T quaternary structure in the deoxy-form.


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).Spectra are CO- and deoxyHb A (A, B), CO- and deoxy-rHb(αH87G) (C, D), and CO- and deoxy-rHb(βH92G) (E, F) between 10 and 16 ppm at pH 7.0 and 25°C. The hemoglobin concentrations of Hb A, rHb(αH87G) and 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.g005: 600 MHz 1H NMR spectra of Hb A, rHb(αH87G) and rHb(βH92G).Spectra are CO- and deoxyHb A (A, B), CO- and deoxy-rHb(αH87G) (C, D), and CO- and deoxy-rHb(βH92G) (E, F) between 10 and 16 ppm at pH 7.0 and 25°C. The hemoglobin concentrations of Hb A, rHb(αH87G) and 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: Fig 5 compares the 1H NMR spectra of the deoxy- and CO-forms of rHb(αH87G) and rHb(βH92G) with those of Hb A in the frequency region between 16 and 10 ppm. In deoxyHb A (Fig 5B), four proton signals were resolved between 16 and 10 ppm and assigned as indicated in the figures [65]. These four protons were ascribed to hydrogen bonded ones at the α1-β2 and α1-β1 subunit-interfaces. As the signal around 14.0 ppm derived from the hydrogen bond between Tyrα42 and Aspβ99 at the α1-β2 interface can be observed only in the deoxy-form, this signal has generally been used as a marker of the T quaternary structure. The signal observed around 11.0 ppm has also been observed only for the deoxy-form, and therefore, was thought to arise from the hydrogen bond between Aspα94 and Trpβ37 at the α1-β2 interface, although there is a report that does not regard the 11.0 ppm signal as a T-marker [54]. The fact that both the 14.0 and 11.0 ppm signals are observed for rHb(αH87G) (Fig 5D) and rHb(βH92G) (Fig 5F) in the deoxy-form, similar to deoxyHb A, suggests that both rHb(αH87G) and rHb(βH92G) take the T quaternary structure in the deoxy-form.

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