T-quaternary structure of oxy human adult hemoglobin in the presence of two allosteric effectors, L35 and IHP

T-quaternary structure of oxy human adult hemoglobin in the presence of two allosteric effectors, L35 and IHP,10.1016/j.bbabio.2011.06.004,Biochimica

T-quaternary structure of oxy human adult hemoglobin in the presence of two allosteric effectors, L35 and IHP  
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The cooperative O2-binding of hemoglobin (Hb) have been assumed to correlate to change in the quaternary structures of Hb: T(deoxy)- and R(oxy)-quaternary structures, having low and high O2-affinities, respectively. Heterotropic allosteric effectors have been shown to interact not only with deoxy- but also oxy-Hbs causing significant reduction in their O2-affinities and the modulation of cooperativity. In the presence of two potent effectors, L35 and inositol hexaphosphate (IHP) at pH 6.6, Hb exhibits extremely low O2-affinities (KT=0.0085mmHg−1 and KR=0.011mmHg−1) and thus a very low cooperativity (KR/KT=1.3 and L0=2.4). 1H-NMR spectra of human adult Hb with these two effectors were examined in order to determine the quaternary state of Hb in solution and to clarify the correlation between the O2-affinities and the structural change of Hb caused by the heterotropic effectors. At pH 6.9, 1H-NMR spectrum of deoxy-Hb in the presence of L35 and IHP showed a marker of the T-quaternary structure (the T-marker) at 14ppm, originated from inter- dimeric α1β2- (or α2β1-) hydrogen-bonds, and hyperfine-shifted (hfs) signals around 15–25ppm, caused by high-spin heme-Fe(II)s. Upon addition of O2, the hfs signals disappeared, reflecting that the heme-Fe(II)s are ligated with O2, but the T-marker signals still remained, although slightly shifted and broadened, under the partial pressure of O2 (PO2) of 760mmHg. These NMR results accompanying with visible absorption spectroscopy and visible resonance Raman spectroscopy reveal that oxy-Hb in the presence of L35 and IHP below pH 7 takes the ligated T-quaternary structure under the PO2 of 760mmHg. The L35-concentration dependence of the T-marker in the presence of IHP indicates that there are more than one kind of L35-binding sites in the ligated T-quaternary structure. The stronger binding sites are probably intra-dimeric binding sites between α1G- and β1G-helices, and the other weaker binding site causes the R→T transition without release of O2. The fluctuation of the tertiary structure of Hb seems to be caused by both the structural perturbation of α1β1 (or α2β2) intra-dimeric interface, where the stronger L35-binding sites exist, and by the IHP-binding to the α1α2- (or β1β2-) cavity. The tertiary structural fluctuation induced by the allosteric effectors may contribute to the significant reduction of the O2-affinity of oxy-Hb, which little depends on the quaternary structures. Therefore, the widely held assumptions of the structure-function correlation of Hb — [the deoxy-state]=[the T-quaternary structure]=[the low O2-affinity state] and [the oxy-state]=[the R-quaternary structure]=[the high O2-affinity state] and the O2-affiny of Hb being regulated by the T/R-quaternary structural transition — are no longer sustainable. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.
Journal: Biochimica Et Biophysica Acta-bioenergetics - BBA-BIOENERGETICS , vol. 1807, no. 10, pp. 1253-1261, 2011
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