Coupling of the Oxygen-linked Interaction Energy for Inositol Hexakisphosphate and Bezafibrate Binding to Human HbA0

Abstract

The energetics of signal propagation between different functional domains (i.e. the binding sites for O 2 , inositol hexakisphospate (IHP), and bezafibrate (BZF)) of human HbA 0 was analyzed at different heme ligation states and through the use of a stable, partially heme ligated intermediate. Present data allow three main conclusions to be drawn, and namely: (i) IHP and BZF enhance each others binding as the oxygenation proceeds, the coupling free energy going from close to zero in the deoxy state to ؊3.4 kJ/mol in the oxygenated form; (ii) the simultaneous presence of IHP and BZF stabilizes the hemoglobin T quaternary structure at very low O 2 pressures, but as oxygenation proceeds it does not impair the transition toward the R structure, which indeed occurs also under these conditions; (iii) under room air pressure (i.e. pO 2 ‫؍‬ 150 torr), IHP and BZF together induce the formation of an asymmetric dioxygenated hemoglobin tetramer, whose features appear reminiscent of those suggested for transition state species (i.e. T-and R-like tertiary conformation(s) within a quaternary R-like structure).Binding properties of gaseous ligands to human HbA 0 1 are markedly influenced by the so-called third components (in addition to HbA 0 and heme's ligands), such as organic phosphates (e.g. 2,3-bisphosphoglycerate and inositol hexakisphosphate (IHP)), protons, and chloride ions (1-4), which interact with HbA 0 at sites topologically different (heterotropic sites) from the heme.These heterotropic functional effects have been previously investigated in thermodynamic (5, 6) and kinetic (7) terms, leading to the evidence of a network of interplays among different sites, such that linkage relationships can be put in evidence between different heterotropic ligands. However, a quantitative investigation of these effects has never been performed since the information is usually obtained through a differential influence of the non-heme binding properties for the deoxygenated and oxygenated derivatives of HbA 0 .We have recently reported on the IHP binding properties to some heme ligated forms of ferrous HbA 0 in the presence of 0.1 M chloride, showing the existence of two binding sites for IHP with a similar affinity independently of the heme ligand (8). In addition, a recent thermodynamic study has been carried out on the interaction of IHP with oxygenated HbA 0 (9), leading to the evidence that the inter-relationship between protons and IHP is regulated by the pK a shift of three classes of residues in HbA 0 , and it is characterized by a proton-linked balancing between the enthalpic and entropic contribution. Furthermore, in the last few years particular interest has been addressed toward the effect of bezafibrate (BZF) (see Fig. 1) on the O 2 -binding properties of HbA 0 , and mostly on the original observation that its effect is potentiated by the presence of organic phosphates (10, 11), indicating that the two heterotropic ligands bind to different sites (12). This observation is interesting, since it underlies the existence...

Crystal Structure of Horse Carbonmonoxyhemoglobin-Bezafibrate Complex at 1.55-Å Resolution

“…Our current functional data on the R-state-locked hemoglobin with and without BZF are consistent at least qualitatively with recent oxygen equilibrium studies of human hemoglobin (19,20) showing that the addition of BZF to stripped hemoglobin lowers the oxygen affinity of R-state hemoglobin by a factor of 8 -10 at pH 7.0.…”

“…Ala E6(␣57) and Leu E17(␣68), that are substituted by Gly and Asn, respectively. Furthermore, several lines of evidence indicate that BZF binds specifically to fully liganded R-state human hemoglobin in a functionally relevant way (17)(18)(19)(20). We do not yet fully understand the reason why the previous solution binding studies found only a nonspecific binding of BZF with human carbonmonoxyhemoglobin (11,12).…”

Global Allostery Model of Hemoglobin

“…Since the O 2 affinity is no longer directly related to the T/R quaternary states, there is no basis to assign any low affinity behaviors solely to the T state. Available kinetic data of low ligand affinity characteristics of ligated Hb in the presence of potent heterotropic effectors (30,31) indicates that kinetic techniques alone cannot distinguish low affinity behaviors of T-state Hb from those of R-state Hb. Coletta et al (31) integrated their kinetic, thermodynamic, and structural data and came to a conclusion that the observed low affinity kinetic behaviors of ligated (oxy and carbonmonoxy) Hb in the presence of BZF ϩ IHP at pH 7.0 are derived from the effectorinduced low affinity state of R (ligated) Hb.…”

“…Available kinetic data of low ligand affinity characteristics of ligated Hb in the presence of potent heterotropic effectors (30,31) indicates that kinetic techniques alone cannot distinguish low affinity behaviors of T-state Hb from those of R-state Hb. Coletta et al (31) integrated their kinetic, thermodynamic, and structural data and came to a conclusion that the observed low affinity kinetic behaviors of ligated (oxy and carbonmonoxy) Hb in the presence of BZF ϩ IHP at pH 7.0 are derived from the effectorinduced low affinity state of R (ligated) Hb. This is in full agreement with our present interpretation and is a direct contrast to the solely kinetics-based interpretation of Marden et al (30).…”

Conformational Dynamics Associated with Ligand Binding to Vertebrate Hexa-coordinate Hemoglobins

“…For example, the binding of BZF or IHP to CO-Hb complex decreases the CO association rate approximately four or eight times, respectively (Marden et al, 1990), and decreases the affinity of R state deoxy-Hb for oxygen . Coletta et al (1999a) have reported the α-and β-chain (Song et al, 2008).…”