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

Abstract

The cooperative O 2-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 O 2-affinities, respectively. Heterotropic allosteric effectors have been shown to interact not only with deoxy- but also oxy-Hbs causing significant reduction in their O 2-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 O 2-affinities ( K T = 0.0085 mmHg −1 and K R = 0.011 mmHg −1) and thus a very low cooperativity ( K R / K T = 1.3 and L 0 = 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 O 2-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 14 ppm, originated from inter- dimeric α 1β 2- (or α 2β 1-) hydrogen-bonds, and hyperfine-shifted (hfs) signals around 15–25 ppm, caused by high-spin heme-Fe(II)s. Upon addition of O 2, the hfs signals disappeared, reflecting that the heme-Fe(II)s are ligated with O 2, but the T-marker signals still remained, although slightly shifted and broadened, under the partial pressure of O 2 ( P O2 ) of 760 mmHg. 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 P O2 of 760 mmHg. 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 α 1G- and β 1G-helices, and the other weaker binding site causes the R → T transition without release of O 2. The fluctuation of the tertiary structure of Hb seems to be caused by both the structural perturbation of α 1β 1 (or α 2β 2) intra-dimeric interface, where the stronger L35-binding sites exist, and by the IHP-binding to the α 1α 2- (or β 1β 2-) cavity. The tertiary structural fluctuation induced by the allosteric effectors may contribute to the significant reduction of the O 2-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 O 2 -affinity state] and [ the oxy-state] = [ the R-quaternary structure] = [ the high O 2 -affinity state] and the O 2 -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.