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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

CD spectra of Hb A, rHb(αH87G) and rHb(βH92G).Spectra are Hb A (A), rHb(αH87G) (B) and rHb(βH92G) (C) in the deoxy (blue spectra) and oxy forms (red spectra) in the wavelength region between 275 nm and 320 nm. Hemoglobin concentration, 45 μM (in heme) in a 0.05 M phosphate buffer (pH 7) containing 5 mM imidazole and a metHb reducing system. The broken black lines in (B) and (C) indicate the curve of deoxy Hb A.
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pone.0135080.g009: CD spectra of Hb A, rHb(αH87G) and rHb(βH92G).Spectra are Hb A (A), rHb(αH87G) (B) and rHb(βH92G) (C) in the deoxy (blue spectra) and oxy forms (red spectra) in the wavelength region between 275 nm and 320 nm. Hemoglobin concentration, 45 μM (in heme) in a 0.05 M phosphate buffer (pH 7) containing 5 mM imidazole and a metHb reducing system. The broken black lines in (B) and (C) indicate the curve of deoxy Hb A.

Mentions: Fig 9 shows the near-UV CD spectra of the deoxy- and oxy-forms of Hb A (A), rHb(αH87G) (B) and rHb(βH92G) (C) in the wavelength region from 275 to 310 nm. As shown in Fig 9A, oxyHb A yields small positive CD bands around 287 and 300 nm, while deoxyHb A gives a distinct negative CD band at 287 nm. Both rHb(αH87G) (Fig 9B) and rHb(βH92G) (Fig 9C) yielded a negative CD band in both the oxy- and deoxy-forms, although the negative band in the deoxy form was smaller than that of Hb A, as shown by broken black line. Oxy-rHb(βH92G) exhibits two troughs at 285 and 292 nm, and since the latter is ascribed to the tertiary ‘t’ structure as explained later, it is likely that the quaternary and tertiary structures of oxy-rHb(βH92G) are in part T and t, 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)

CD spectra of Hb A, rHb(αH87G) and rHb(βH92G).Spectra are Hb A (A), rHb(αH87G) (B) and rHb(βH92G) (C) in the deoxy (blue spectra) and oxy forms (red spectra) in the wavelength region between 275 nm and 320 nm. Hemoglobin concentration, 45 μM (in heme) in a 0.05 M phosphate buffer (pH 7) containing 5 mM imidazole and a metHb reducing system. The broken black lines in (B) and (C) indicate the curve of deoxy Hb A.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0135080.g009: CD spectra of Hb A, rHb(αH87G) and rHb(βH92G).Spectra are Hb A (A), rHb(αH87G) (B) and rHb(βH92G) (C) in the deoxy (blue spectra) and oxy forms (red spectra) in the wavelength region between 275 nm and 320 nm. Hemoglobin concentration, 45 μM (in heme) in a 0.05 M phosphate buffer (pH 7) containing 5 mM imidazole and a metHb reducing system. The broken black lines in (B) and (C) indicate the curve of deoxy Hb A.
Mentions: Fig 9 shows the near-UV CD spectra of the deoxy- and oxy-forms of Hb A (A), rHb(αH87G) (B) and rHb(βH92G) (C) in the wavelength region from 275 to 310 nm. As shown in Fig 9A, oxyHb A yields small positive CD bands around 287 and 300 nm, while deoxyHb A gives a distinct negative CD band at 287 nm. Both rHb(αH87G) (Fig 9B) and rHb(βH92G) (Fig 9C) yielded a negative CD band in both the oxy- and deoxy-forms, although the negative band in the deoxy form was smaller than that of Hb A, as shown by broken black line. Oxy-rHb(βH92G) exhibits two troughs at 285 and 292 nm, and since the latter is ascribed to the tertiary ‘t’ structure as explained later, it is likely that the quaternary and tertiary structures of oxy-rHb(βH92G) are in part T and t, 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