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

Massimo ColettaDagger §, Mauro Angeletti, Paolo Ascenziparallel , Alberto Bertollini**, Stefano Della LongaDagger Dagger , Giampiero De Sanctis, Anna Maria Priori, Roberto SantucciDagger , and Gino Amiconi**

From the Dagger  Department of Experimental Medicine and Biochemical Sciences, University of Roma Tor Vergata, Via di Tor Vergata 135, I-00133 Roma, Italy, the  Department of Molecular, Cellular and Animal Biology, University of Camerino, Via F. Camerini 2, I-62032 Camerino (MC), Italy, the parallel  Department of Biology, III University of Roma, Viale G. Marconi 446, I-00146 Roma, Italy, the ** CNR Center for Molecular Biology and Department of Biochemical Sciences "Alessandro Rossi Fanelli," University of Roma "La Sapienza," Piazzale A. Moro 5, I-00185 Roma, Italy, and the Dagger Dagger  Department of Experimental Medicine, Biophysics Group, University of L'Aquila, Via Vetoio, I-67100 L'Aquila, Italy

    ABSTRACT
Top
Abstract
Introduction
References

The energetics of signal propagation between different functional domains (i.e. the binding sites for O2, inositol hexakisphospate (IHP), and bezafibrate (BZF)) of human HbA0 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 O2 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. pO2 = 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).

    INTRODUCTION
Top
Abstract
Introduction
References

Binding properties of gaseous ligands to human HbA01 are markedly influenced by the so-called third components (in addition to HbA0 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 HbA0 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 HbA0.

We have recently reported on the IHP binding properties to some heme ligated forms of ferrous HbA0 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 HbA0 (9), leading to the evidence that the inter-relationship between protons and IHP is regulated by the pKa shift of three classes of residues in HbA0, 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 O2-binding properties of HbA0, 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 of communication pathway(s) between different domains of HbA0, allowing the formulation of a series of linkage relationships among different heterotropic ligands and with respect to the ligand binding at the heme. Additional data have shown that the simultaneous presence of IHP and BZF brings about in human HbCO a dramatic shift of the allosteric equilibrium, such that even the fully liganded tetramer appears to be in a T-like conformation (13, 14); this phenomenon has also been observed on the carbonylated derivative of dromedary Hb (15).


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Fig. 1.   Structural formula of bezafibrate.

Therefore, starting from the detailed information on the thermodynamics of IHP binding to HbA0 (9), we have undertaken a quantitative study on the linkage between O2, IHP, and BZF. The investigation should give fairly detailed thermodynamic information on these inter-relationships for the first and the fourth O2 binding step, envisaging a somewhat different interaction mode between the two heterotropic ligands along the binding pathway. This is only the first step toward a deeper comprehension of the complex inter-relationships between O2, protons, IHP, and BZF, aiming at a better understanding of the intramolecular pathway(s) responsible for the energy transmission between different functional domains of a protein.

    MATERIALS AND METHODS

Human HbA0 has been prepared from freshly drawn blood according to the method reported by Williams and Tsay (16). Organic polyphosphates and other low-molecular mass contaminants have been removed, as reported for "stripping" of hemoglobin (17). Concentration of oxy-HbA0 solution was determined spectrophotometrically employing the extinction coefficient epsilon  = 13.8 mM-1 cm-1 (per heme) at 541 nm, assuming that the absorption spectrum of fully oxygenated HbA0 was the same in the absence and presence of allosteric effectors. The heme's iron oxidation state has been qualitatively checked on the basis of the absorbance ratio at 576/541 nm, taking 1.14 as a normal ratio value. In all experiments, the concentration of the ferric form has been monitored, as previously reported (18), and data displaying a percentage exceeding 1% have been discarded.

Spectral deconvolution of absorption spectra of HbO2 has been carried out with the software MATLAB (Mathworks, South Natick, MA) on a desktop computer. Spectral smoothing has been performed by using the singular value decomposition algorithm (19), and spectral deconvolution has been obtained starting from reference spectra (of oxy-, deoxy-, and ferric HbA0) by using the left division option, provided by MATLAB.

Circular dichroism spectra were obtained on a Jasco-500A spectropolarimeter equipped with a Jasco DP-500 processor. The molar ellipticity theta M is expressed as deg·cm2·dmol-1.

Resonance Raman spectra have been obtained at room temperature with CW excitation from an Ar+ laser (Coherent, Innova 90/5). The back-scattered light from a slowly rotating NMR tube was collected and focussed into a computer-controlled double monochromator (Jobin-Yvon HG2S/2000) equipped with a cooled photomultiplier (RCA C31034A) and photon counting electronics. The spectra were calibrated in frequency with indene and CCl4 as standards to an accuracy of ±1 cm-1 for the intense, isolated bands.

The iron K-edge x-ray spectra have been collected in the fluorescence mode at the LURE DCI synchrotron radiation facility (Orsay, France), operating at 1.8 GeV energy and a current of 270 mA. A Si(111) double crystal used as channel-cut single crystal was employed as a monochromator. The energy resolution at the iron K-edge is about 2 eV, and energy shifts of resolved absorption peaks of 0.5 eV can be detected. Harmonic contamination was rejected by using a total reflection mirror after the monochromator. Each spectrum represents a total signal averaging of 4 s/point, collected at room temperature by using a 7-element energy-resolving Ge detector from Canberra industries. The samples were mounted in a 1-mm thick Teflon cell with mylar windows, oriented with an angle of 45° with respect to the x-ray beam, with the detector positioned perpendicular to the beam direction. The specimen concentration was 8 mM heme, in the absence and presence of allosteric effectors, and no protein damage has been detected after x-ray exposure.

Kinetic experiments have been carried out on a Gibson-Durrum rapid-mixing stopped-flow apparatus interfaced with a fast data collection system (On Line Instrument Service, Jefferson, GA). Typical experiments have been undertaken mixing oxy-HbA0 (0.1 mM heme after mixing), in the absence and presence of varying amounts of allosteric effectors, with a solution of CO-saturated or degassed buffer containing the same amount of allosteric effectors and 20 mg/ml sodium dithionite. Such experiments, followed at lambda  = 543 nm, allowed the determination of the rate of the irreversible (because of the sodium dithionite) O2 replacement by CO. The observed rate did not depend either on the CO concentration (for [CO] = 50-500 µM) or on the sodium dithionite concentration (at least between 5 and 50 mg/ml), indicating that the observed process indeed refers to the O2 dissociation from the fully oxygenated HbA0.

O2 binding isotherms to HbA0, in the absence and presence of varying amounts of effectors, have been carried out employing a high-precision thin layer dilution technique (20). The experiments have been performed using [Hb] = 3 mM in heme at 293 K. Data have been analyzed according to the Adair formalism (21), employing the following equation,
Y=(&bgr;<SUB>1</SUB>x+2&bgr;<SUB>2</SUB>x<SUP>2</SUP>+3&bgr;<SUB>3</SUB>x<SUP>3</SUP>+4&bgr;<SUB>4</SUB>x<SUP>4</SUP>)/4(1+&bgr;<SUB>1</SUB>x+&bgr;<SUB>2</SUB>x<SUP>2</SUP>+&bgr;<SUB>3</SUB>x<SUP>3</SUP>+&bgr;<SUB>4</SUB>x<SUP>4</SUP>) (Eq. 1)
where beta i (i = 1, 2, 3, or 4) corresponds to the equilibrium association constant for the binding of i molecules of O2 to the HbA0 molecule, i.e. the reaction,
<UP>HbA</UP><SUB>0</SUB>+i<UP>O</UP><SUB>2</SUB> &cjs0813; <UP>HbA</UP><SUB>0</SUB>(O<SUB>2</SUB>)<SUB>i</SUB> (Eq. 2)

The relative percentage of five different populations have been calculated according to the following relationships,
   &agr;<SUB>0</SUB>=1/P, &agr;<SUB>1</SUB>=&bgr;<SUB>1</SUB>x/P; &agr;<SUB>2</SUB>=&bgr;<SUB>2</SUB>x<SUP>2</SUP>/P, &agr;<SUB>3</SUB>=&bgr;<SUB>3</SUB>x<SUP>3</SUP>/P, &agr;<SUB>4</SUB>=&bgr;<SUB>4</SUB>x<SUP>4</SUP>/P (Eq. 3)
where P = 1 + beta 1x + beta 2x2 + beta 3x3 + beta 4x4 is the O2 binding polynomial to HbA0 under different experimental conditions.

    RESULTS AND DISCUSSION

The present investigation has been focussed on the inter-relationships between O2 association and IHP and BZF binding to HbA0, trying to characterize (i) the spectroscopic properties of HbA0 in the presence of IHP or of BZF alone and when they are added together; (ii) the kinetic effect on the gaseous ligand dissociation exerted by IHP and BZF alone and when they are added together; and (iii) the individual and coupled effect of IHP and BZF on the thermodynamics of O2 binding.

Functional Behavior of HbA0 in the Absence and Presence of Allosteric Effectors-- In order to give a quantitative estimate of the various aspects of the energetic inter-relationships, high-precision O2 binding isotherms have been carried out on human HbA0 in the absence of allosteric effectors, as well as in the presence of IHP, BZF alone, and of both IHP and BZF (see Fig. 2). The analysis of these data has been carried out employing the phenomenological Adair equation (see Equation 1) (21). From the equilibrium parameters, reported in Table I, it comes out that IHP and BZF indeed seem to always alter the apparent association equilibrium constants for different O2 binding steps (see beta i in Table I).


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Fig. 2.   . O2 binding isotherms to human HbA0 at pH 7.0 (0.1 M HEPES) and 20 °C, in the absence of allosteric effectors (panel A), in the presence of 10 mM IHP alone (panel B), 35 mM BZF alone (panel C), and 1 mM IHP and 10 mM BZF together (panel D). Data are reported as optical density changes (DAi) at lambda  = 578 nm for stepwise variations in oxygen partial pressure for a thin layer (0.0025 cm) sample. Theoretical continuous curves correspond to the binding capacity partial <A><AC>Y</AC><AC>&cjs1171;</AC></A>/partial logx, where x is the O2 concentration at every step and <A><AC>Y</AC><AC>&cjs1171;</AC></A> is the ligand saturation degree (see Equation 1). Panel E, the same data reported in previous panels (i.e. in the absence of allosteric effectors (*), in the presence of 10 mM IHP alone (×), in the presence of 35 mM BZF (open circle ) and 1 mM IHP and 10 mM BZF (+)) are reported as ligand saturation degree <A><AC>Y</AC><AC>&cjs1171;</AC></A> versus log pO2. The overall Adair constants beta  values employed for continuous curves in each panel are reported in Table I. For further details, see text.

                              
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Table I
Thermodynamic parameters for O2 binding to human HbA0 (3 mM heme concentration) under different experimental conditions at pH 7.0 and 20 °C
We report the experimental binding constants obtained by data fitting according to Equation 1, and by parentheses the stepwise binding constants (expressed as M-1), as from the following relationship (beta i = <AR><R><C><IT>f</IT> = <IT>i</IT></C></R><R><C><B>&pgr;</B></C></R><R><C><IT>f</IT> = 1</C></R></AR>: Kf where  i  (= 1, 2, 3, 4) is the number of liganded hemes, n = 4 is the total number of binding sites of the macromolecule.

However, the possibility of describing quantitatively the energetics of the interaction between O2, IHP, and BZF binding is linked to the independent determination of allosteric effector(s) binding constants to either the fully deoxygenated and/or the fully oxygenated HbA0. However, in the case of deoxy-HbA0 we did not observe any spectroscopic variation (22), while an independent determination of BZF binding to deoxy-HbA0 has been undertaken by Perutz and co-workers (12) by anaerobic dialysis (23). On the other hand, this is possible for oxy-HbA0 by a kinetic approach, since the dependence of O2 dissociation rate constant from fully ligated HbA0 as a function of non-heme ligand concentration reflects the interaction of heterotropic ligand(s) with oxy-HbA0 (9). Fig. 3 displays the effect of IHP, BZF, and BZF added in the presence of saturating amounts of IHP (i.e. 1 mM IHP) on the O2 dissociation rate constant of fully oxygenated HbA0. It is worth outlining that under these conditions (i.e. no NaCl) no evidence of a second binding site for IHP is reported also in view of the much higher affinity for IHP in the absence of salt (8). In the presence of IHP the kinetic effect induced by BZF is exerted only on the slow phase (which amounts to approx 50% of the total ligand dissociation curve, see also Ref. 8), suggesting that under these conditions the interaction energy induces a structural transition that is transmitted from the binding pocket to only one type of subunit in the tetramer. Therefore, for the sake of clarity only the rate constant calculated from the slow phase is reported in Fig. 3 also for the interaction of BZF and IHP alone. Unfortunately, BZF cannot be used at concentrations exceeding 50 mM, since, aside from its relatively low solubility, beyond this value it begins to induce a very slow but detectable irreversible denaturation of HbA0. Therefore, even though we are aware that under these conditions HbO2 is not fully saturated with BZF alone, we never used concentrations above 40 mM, a value at which the functional behavior of HbA0 is stable for at least 1 day. From the experiments reported in Fig. 3 we can derive the binding constants to oxy-HbA0 of BZF, IHP, and BZF in the presence of saturating amounts of IHP (see Table III). The enhancement of the BZF binding constant when IHP is already present appears evident (see Fig. 3 and Table III), envisaging a relevant free energy increase of approx 16 kJ/mol (1 joule = 4.184 cal).


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Fig. 3.   Dependence of the O2 dissociation rate constant by CO displacement in the presence of sodium dithionite from fully liganded human HbA0 at pH 7.0 (0.1 M HEPES) and 20 °C as a function of the concentration of IHP alone (open circle ), BZF alone (+), and BZF in the presence of 1 mM IHP (×). [Hb] = 0.1 mM heme (after mixing). lambda  = 563 nm. Continuous lines have been obtained by least-squares fitting of data according to the kobs = k0/(1 + K[AE]) + kb·K[AE]/(1 + K[AE]), where kobs is the experimentally observed rate constant, k0 and kb are the rate constants in the absence and presence of saturating amounts of the allosteric effector, respectively, [AE] is the concentration of the allosteric effector, and K is the equilibrium binding constant of the allosteric effector to fully ligated HbA0 (i.e. 0K4IHP for IHP alone (open circle ), 0K4BZF for BZF alone (+), IHPK4BZF for BZF in the presence of 1 mM IHP (×)). The parameters employed for obtaining the continuous lines are: k0 = 4.4 ± 0.2 s-1 for IHP alone (open circle ), 4.4 ± 0.2 s-1 for BZF alone (+), 13.9 ± 0.7 s-1 for BZF in the presence of 1 mM IHP (×), kb = 13.9 ± 0.7 s-1 for IHP alone (open circle ), 13.9 ± 0.7 s-1 for BZF alone (+), 27.0 ± 1.3 s-1 for BZF in the presence of 1 mM IHP (×). Values of effector binding equilibrium constants are K = 5.9(±1.2) × 104 M-1 for IHP alone (×), 2.5(±0.3) × 101 M-1 for BZF alone (+), 1.8(±0.3) × 104 M-1 for BZF in the presence of 1 mM IHP (×). For further details, see text.

In the case of the ferrous nitrosylated form of HbA0 (24), we detected a approx 15-fold increase of BZF affinity constant when IHP is already bound to its cleft in the beta -dyad axis (thus resulting an interaction energy Delta G congruent  7.5 kJ/mol). It definitely indicates that, unlike IHP binding to ligated HbA0 (which is scarcely affected by the type of heme's ligand, see Ref. 8), the intramolecular interaction energy between the two heterotropic binding sites is markedly dependent on the heme's ligand (i.e. O2 versus NO), underlying a preferential ligand-linked communication pathway between the different portions of the macromolecule. This occurrence has been observed also in the case of IHP and BZF on ferrous nitrosylated iron-cobalt hybrids of HbA0. Thus, in this case the two allosteric effectors have been shown to affect in a drastically different fashion the intersubunit communication pathway, envisaging a different mode for the synergistic effect in the NO-bound Fe-Co hybrids (25).

Spectroscopic Studies-- An additional effect concerns the absorption spectroscopic behavior of oxy-HbA0 upon addition of IHP and/or BZF. Thus, while the individual addition of one of the two effectors either does not bring about any meaningful change in the absorption spectra of oxy-HbA0 (e.g. for IHP) or it shows a very limited influence (e.g. for BZF), the simultaneous presence of IHP and BZF leads to a variation of the absorption spectra (Fig. 4), which can be accounted for by a progressive partial deoxygenation of HbA0 molecule even at atmospheric oxygen pressure (11). Table II reports the calculated O2 saturation degrees at several concentrations of IHP and BZF, showing that at 20 °C and pO2 congruent  150 torr a progressive deoxygenation of oxy-HbA0 takes place when BZF is added in the presence of IHP (e.g. 0.5 mM), tending to level off at about 40% of deoxygenation (see Table II). Such behavior is in keeping with the previous observation that IHP and BZF together act synergistically, bringing about a tertiary and/or a quaternary shift toward a low-affinity conformation (13, 14, 24), provided that the pH is held constant. This behavior is shared by all vertebrate hemoglobins in the heme-ligated state (e.g. dromedary Hb, see Ref. 15)


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Fig. 4.   Spectra of oxy-HbA0 at atmospheric oxygen pressure obtained by absorption spectroscopy in the absence of allosteric effectors (spectrum a) and in the presence of 1 mM IHP and 10 mM BZF (spectrum b). The spectrum of deoxy-HbA0 (spectrum c) is also shown. Spectra have been obtained at pH 7.0 and 20 °C. For further details, see text.

                              
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Table II
Allosteric effector-induced oxygen release at equilibrium from oxy-HbA0 (59 µM heme) at atmospheric pressure at pH 7.0 and 20 °C
The fraction of oxidized HbA0 was always << 0.01.

The electronic absorption spectroscopic feature is mirrored by circular dichroism spectroscopic changes both in the near ultraviolet range and in the Soret region. The very small spectral variations observed in the 280-300-nm region (Fig. 5A) indicate that at atmospheric pressure the environment of Trpbeta 37(C3), located at the alpha 1beta 2 contact surface and considered a spectroscopic marker for the R iff  T quaternary transition (26), is very slightly affected (if any) by the simultaneous addition of IHP and BZF, despite the presumed interaction of this residue with BZF in the deoxygenated form (12) (but not necessarily in the oxygenated species), suggesting therefore that the quaternary R structure of the tetramer is mostly maintained. On the other hand, the ellipticity measured in the Soret region (Fig. 5B) in the presence of both heterotropic ligands corresponds mainly to that of the deoxygenated form of HbA0, and, accordingly, the heme conformation should conform to that typical of a low-affinity structure. As a whole, these results indicate that IHP and BZF induce only a tertiary conformational change, suggesting the (at least partial) stabilization of a tertiary T-like conformation within a quaternary R-like intermediate structure (hereafter referred as rRt state, according to the standard terminology (27)). Therefore, these data seem to support the idea that, in the presence of IHP and BZF, oxy-HbA0 is shifted toward a structure different from that observed for the carbonylated HbA0 and dromedary Hb (13-15). Such behavior is well in keeping with the concept of allosteric core (28), as well as allosteric nesting (29), suggesting that IHP and BZF do not alter to a significant extent the intersubunit contacts, at least in the oxygenated form, but they only exert their effect on the tertiary conformations of subunits, inducing a shift toward a low-affinity form of the R quaternary state.


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Fig. 5.   Spectra of oxy-HbA0 at atmospheric oxygen pressure obtained by circular dichroism spectroscopy in near ultraviolet region (panel A) and in the Soret region (panel B), in the absence of allosteric effectors (spectrum a) and in the presence of 1 mM IHP and 10 mM BZF together (spectrum b). Spectrum of deoxy-HbA0 (spectrum c) is also shown. Panel C, XANES spectra of oxy-HbA0 at atmospheric oxygen pressure obtained in the absence of allosteric effectors (spectrum a), and in the presence of 1 mM IHP and 10 mM BZF together (spectrum b). Spectrum of deoxy-HbA0 (spectrum c) is also shown. Spectra have been obtained at pH 7.0 and 20 °C. For further details, see text.

This effect is also documented by XANES spectroscopy observations, a technique uniquely suited (30) for probing structural modifications around the iron atom (Fig. 5C). In the presence of 1 mM IHP and 10 mM BZF XANES spectra of oxy-HbA0 under atmospheric air pressure (i.e. pO2 = 150 torr) display a fraction of unliganded hemes, which is fully consistent with observations by electronic absorption spectroscopy (see Fig. 4), and they can be accounted for simply by a linear combination of oxygenated and deoxygenated hemes, without implying any distortion of the local geometry.

Likewise, resonance Raman spectra in the high frequency region of oxy-HbA0 in the presence of 1 mM IHP and 10 mM BZF display a combination of frequencies belonging to either oxy- or deoxy-HbA0 without any variation for the respective frequencies (data not shown), clearly indicating that no relevant changes are involving the stereochemistry of the porphyrin core.

As a whole, reported data allow two main considerations to be drawn on the aggregation state of human HbO2 when IHP and BZF are present together, namely (i) the dichroic features at 287 nm strongly indicate that only O2-bound R-like tetramers (or dimers) are present, and this despite previous observations that allosteric effectors with molecular structure reminiscent of that of BZF stabilize a tetraoxygenated T-state in crystals (31); and (ii) deoxy-type stereochemical changes of the heme (as from XANES and resonance Raman observations) allow to rule out a significant contribution from dimers under our experimental conditions, even though it is known that polyanions may stabilize oxygenated dimers (32, 33).

The general outcome of this spectroscopic investigation may be summarized by stating that (i) the quaternary structure is not the unique dominant factor determining the tertiary environment around the heme, and (ii) the transition from a tense to a relaxed conformation (34) is in line with a multistep model in which a quaternary transition may be associated to several tertiary relaxations (35).

Kinetic Properties of Partially Oxygenated HbA0-- The kinetic behavior of the stabilized intermediate species (i.e. HbA0 in the presence of both IHP and BZF) has been investigated, and Fig. 6 reports the O2 dissociation kinetics by sodium dithionite, in the absence and presence of 1 mM IHP and 10 mM BZF. In the presence of both effectors the autocatalytic kinetic behavior is abolished, somewhat reducing also the amplitude of the total process (as expected in view of the partial deoxygenation of the protein, see above). Furthermore, the deoxygenation process becomes much faster and biphasic (see Fig. 6). Such behavior indicates that kinetic properties of the hybrid rRt state, which has been shown to be favored by the interaction of IHP and BZF with oxy-HbA0 (see Fig. 5), are intermediate between those of the R quaternary conformation, observed in the absence of heterotropic ligands, and of the T quaternary state, as observed at very low oxygen saturation degrees (36). The biphasicity of the deoxygenation kinetics suggests that (i) in the rRt form the kinetic heterogeneity between the two subunits is more marked than in the R-state (36), but, more importantly, that (ii) the equilibrium affinity for oxygen of the two chains in the rRt form should not be very diverse, since they look to have a similar ligand occupancy probability, as expected from the amplitude of the two phases (see Fig. 6).


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Fig. 6.   Kinetic progress curves of O2 dissociation from human HbA0 at pH 7.0 (0.1 M HEPES) and 20 °C in the absence of allosteric effectors (curve a), and in the presence of 1 mM IHP and 10 mM BZF together (curve b). [HbA0] = 10 µM heme after mixing, lambda  = 430 nm. For further details, see text.

Therefore, altogether these data seem to indicate that (i) the effect of individual heterotropic ligands, such as IHP and BZF, is to mainly perturb the tertiary liganded conformation; (ii) in the presence of both allosteric effectors biliganded species appear to be stabilized within a quaternary structure in which interdimer rotation has already occurred; (iii) the intermediate species is a hybrid structure not included in the classical two-state model (34); (iv) the globin moiety places little mechanical tension on the deoxygenated hemes of both chains in the R-state, since alpha  and beta  subunits show similar functional properties, a view in line with both a flexible structure of the R quaternary form (being able to accommodate equally well ligated and unligated hemes (37)) and a strain energy distributed throughout the tetramer in small deformations (38).

Energetics of Interactions between Allosteric Effectors and HbA0 at Different Heme Ligation States-- A quantitative description of the inter-relationship between O2 binding and the tertiary conformational changes related to IHP and BZF association requires a quantitative analysis of the oxygen binding isotherms under saturating concentrations of IHP and/or BZF. Thermodynamic parameters which describe the data in Fig. 2 are reported in Table I, and in Fig. 7 we report the relative percentage of unliganded (alpha 0), monoliganded (alpha 1), biliganded (alpha 2), three-liganded (alpha 3), and fully liganded molecules (alpha 4) as a function of log pO2. It appears immediately obvious that in the absence of effectors the monoliganded, biliganded, and three-liganded species are significantly populated (see Fig. 7, panel A), while the addition to either IHP and/or BZF alone greatly reduces the percentage of intermediate populations but in a different fashion. Thus, in the case of IHP alone the relative amount of biliganded species is enhanced and that of three-liganded one is very much depressed (see Fig. 7, panel B), whereas in the presence of BZF alone the relative amount of both biliganded and three-liganded molecules is dramatically reduced (see Fig. 7, panel C). Therefore, the separate effects of IHP and BZF are markedly different on the intermediate populations, but their simultaneous presence brings about a stabilization of both biliganded and three-liganded tetramers (see Fig. 7, panel D), probably reflecting thermodynamic properties of the rRt tertiary conformation. The existence of at least two sets of structures along the transition pathway from T to R quaternary form(s), as revealed by normal mode analysis and energy minimization (39), can help in interpreting these observations on the fraction of intermediate species in the presence of different allosteric effectors (see Fig. 7). Thus, in some structures of one set the contacts at the alpha 1beta 1 and alpha 1beta 2 interfaces are R-like, with the heme environment being in a T-like arrangement, while in another set the intersubunit contacts are different from both T and R structures (39). Therefore, on the basis of these considerations, IHP and BZF appear to affect the fraction of partially oxygenated species by simply stabilizing different native conformational substates, which are scarcely populated at room temperature.


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Fig. 7.   The relative percentage of populations of unliganded (alpha 0), monoliganded (alpha 1), biliganded (alpha 2), three-liganded (alpha 3), and fully liganded molecules (alpha 4) are plotted as a function of log pO2 for HbA0 in the absence of allosteric effectors (panel A), in the presence of 10 mM IHP (panel B), 35 mM BZF (panel C), and 1 mM IHP and 10 mM BZF together (panel D). For further details, see text.

Furthermore, since concentrations of IHP used in these experiments are saturating both for deoxy- and oxy-HbA0, as indicated by the kinetic observations of their effect on the oxygenated derivative (see Fig. 3), and the same is almost true for BZF (see above), we can look somewhat more in depth at the energetic interactions between these allosteric effectors and O2 binding to HbA0, building a thermodynamic cube for the synergistic interaction of IHP and BZF at pH 7 in the three-liganded and fully liganded HbA0 (see Scheme I). Even though the simultaneous presence of IHP and BZF brings about a partial deoxygenation of HbA0 (see Fig. 4), this is possible because there is a low but finite amount of tetraligated species, and the fitting of data in Fig. 2D is able to define beta 4 as well. The knowledge of the intrinsic equilibrium association constant for the fourth binding step (i.e. K4 = 4·beta 4/beta 3), in the absence and presence of saturating amounts of allosteric effectors (see Table I), as well as the information on the equilibrium association constant for both IHP and BZF (see above and Fig. 3), allows extraction of complete information on the thermodynamic square for the synergistic phenomenon in the three-ligated species. The outcome of this approach is reported in Table III, in which the values of different parameters appearing in Scheme I are reported. The affinity of IHP for the three-ligated form (i.e. 0K3IHP) is only slightly larger (by approx 2.4 kJ/mol of free energy of interaction) than in the fully ligated HbA0 (i.e. 0K4IHP, see Table III). On the other hand, in the presence of BZF, IHP affinity for the three-ligated HbA0 (i.e. BZFK3IHP) is much larger than for the fully oxygenated molecule (i.e. BZFK4IHP, see Table III) with an enhancement of the negativity for the binding free energy by approx 5.8 kJ/mol. Likewise, the affinity of BZF alone for the three-ligated form (i.e. 0K3BZF) is somewhat larger (by approx 5.7 kJ/mol of interaction free energy) than in the fully oxygenated HbA0 (i.e. 0K4BZF, see Table II and Table III), while a much larger O2-linked effect is observed in the presence of IHP (by approx 9.0 kJ/mol of coupling free energy). Therefore, although the binding affinity of IHP and BZF is different, the O2-linked effect on their affinity when they are both present is closely similar, amounting to approx 3.3 kJ/mol.


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Scheme I.  

                              
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Table III
Effector binding parameters in 0.1 M HEPES pH 7.0 and 20 °C for the three- and four-ligated HbA0 (see Scheme 1 and Table I)

Therefore, it appears evident that the fourth O2 binding step brings about a conformational change which only moderately affects the individual binding sites for IHP and BZF, altering instead to a large extent the mode of energy transmission between the two binding pockets. Furthermore, the close similarity for the affinity enhancement of the IHP binding in the presence of BZF (i.e. BZFK4IHP/BZFK3IHP, see Scheme I and Table III) and of BZF binding in the presence of IHP (i.e. IHPK4BZF/IHPK3BZF, see Scheme I and Table III) indeed suggests that the BZF- and IHP-linked conformational change has essentially the same features and the energetic contribution is similar for the two heterotropic ligands for the fourth O2 binding step.

Similar information can be obtained for the first binding step of oxygenation, even though the lack of an accurate knowledge of the IHP and BZF affinity constant to deoxy-HbA0 impairs the calculation of the binding constant also to the monoligated species (see Scheme II). Nonetheless, the variation of the IHP and BZF equilibrium association constant upon binding the first O2 molecule can be calculated according to the following relationships,
&dgr;<SUB><UP>IHP</UP></SUB>=<SUP><UP>IHP</UP></SUP>&bgr;<SUB>1</SUB>/<SUP>0</SUP>&bgr;<SUB>1</SUB>=<SUP><UP>0</UP></SUP><UP>K<SUB>1IHP</SUB>/<SUP>0</SUP>K<SUB>0IHP</SUB>; &dgr;<SUB>BZF</SUB></UP>=<SUP><UP>BZF</UP></SUP><UP>&bgr;<SUB>1</SUB>/<SUP>0</SUP>&bgr;<SUB>1</SUB></UP>=<SUP><UP>0</UP></SUP><UP>K<SUB>1BZF</SUB>/<SUP>0</SUP>K<SUB>0BZF</SUB>; &dgr;<SUB>IHPBZF</SUB></UP> (Eq. 4)
=<SUP><UP>IHPBZF</UP></SUP><UP>&bgr;<SUB>1</SUB>/<SUP>IHP</SUP>&bgr;<SUB>1</SUB></UP>=<SUP><UP>IHP</UP></SUP><UP>K<SUB>1BZF</SUB>/<SUP>IHP</SUP>K<SUB>0BZF</SUB>; &dgr;<SUB>BZFIHP</SUB></UP>=<SUP><UP>IHPBZF</UP></SUP><UP>&bgr;<SUB>1</SUB>/<SUP>BZF</SUP>&bgr;<SUB>1</SUB></UP>
=<SUP><UP>BZF</UP></SUP><UP>K<SUB>1IHP</SUB>/<SUP>BZF</SUP>K<SUB>0IHP</SUB></UP>
where the interaction energy is,
<UP>I<SUB>IHP</SUB></UP>=<UP>RTln&dgr;<SUB>IHP</SUB>; I<SUB>BZF</SUB></UP>=<UP>RTln&dgr;<SUB>BZF</SUB>; I<SUB>IHPBZF</SUB></UP>=<UP>RTln&dgr;<SUB>IHPBZF</SUB>; I<SUB>BZFIHP</SUB></UP>=<UP>RTln&dgr;<SUB>BZFIHP</SUB></UP> (Eq. 5)
(values of I are reported in Table IV).


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Scheme II.  

                              
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Table IV
Interaction energy for the O2-linked binding of allosteric effectors at the first ligation step, at pH 7.0 and 20 °C, calculated according to the following relationship (joule = 4.184 cal)

Interestingly, while for the fourth O2 binding step all values of delta  are lower than 1 (see Table IV), in the case of the first O2 binding step a value of delta  << 1 occurs for the binding of BZF and/or IHP alone (and thus a negative value for IIHP and IBZF), but a value of delta  approx  1 results for the binding of either effector in the presence of the other one (see IIHPBZF and IBZFIHP in Table IV). This behavior suggests that the O2-linked effect for the first binding step is significantly different according to whether only one type of effector is bound and/or both heterotropic ligands are present.

In particular, a closer look at Schemes I and II with the help of data reported in Tables III and IV allows identifying more quantitatively this different behavior. Thus, the individual binding sites for IHP and BZF indeed are strongly affected by the first O2 binding step (with a decrease of affinity corresponding to 9.6 kJ/mol for IHP and to 10.4 kJ/mol for BZF), while the effect is much less pronounced for the last oxygen binding step (with a decrease of affinity corresponding to 2.4 kJ/mol for IHP and to 5.7 kJ/mol for BZF). On the other hand, when either IHP or BZF are already present in their binding pocket the effect of the first O2 binding step on the allosteric effector binding affinity is much less pronounced (with a decrease of affinity corresponding to 0.3 kJ/mol for IHP binding in the presence of BZF and to 1.1 kJ/mol for BZF binding in the presence of IHP). The reverse is true for the last binding step. Thus, in the presence of IHP or BZF, the addition of the fourth oxygen molecule brings about a more marked affinity decrease for the other allosteric effector binding affinity (corresponding to 5.7 kJ/mol for IHP binding in the presence of BZF and to 9.1 kJ/mol for BZF binding in the presence of IHP). It is worth outlining that the oxygen-linked effect both for the first and fourth binding step is less marked for IHP than for BZF (see Table III and Table IV), suggesting that O2 binding always brings about a larger perturbation of the BZF-binding site.

Concluding Remarks-- The most significant conclusions to be drawn from the reported results may be summarized as follows.

The coupling free energy between IHP and BZF is always negative and increases from a value around zero in the deoxygenated form to a maximum in the tetraoxygenated HbA0. This means that IHP and BZF promote each others binding as a function of heme ligation and their simultaneous presence favors the stabilization of the same fully oxygenated conformation.

At room air pressure (i.e. pO2 congruent  150 torr) and in the presence of both allosteric effectors, in the average half of HbA0 chains in the tetramer are oxygenated in a R-like quaternary structure with both T and R tertiary conformations. In such a hybrid molecule O2 is not distributed preferentially on one type of subunit, as observed in the T-state (40), but the structure is more reminiscent of one of the possible diligated intermediates within a scheme which does not obey the symmetry principle of the classical two-state model (41). Such an intermediate shows the structural characteristics of the species possibly involved in the transition path (35, 42-44), corresponding to a tetramer in a quaternary R-like conformation associated to a tertiary T-like structure (at least for some subunit).

There is a good correlation between the allosteric effector-induced structural change(s) in HbA0 and the O2 dissociation kinetic process (see Fig. 6). This observation is in line with the idea that the protein conformation is not only an active element in the ligand binding mechanism, but it also provides most of the machinery by interconverting conformational free energy into electronic potential energy (45). Thus, an important role has to be played by electronic factors in determining the rate-limiting step of the heme ligand(s) dissociation process (46).

    ACKNOWLEDGEMENTS

We thank Prof. M. Brunori, Prof. B. Giardina, and Prof. G. Smulevich for several stimulating discussions.

    FOOTNOTES

* This work was supported in part by funds from the Ministero dell'Universita' e della Ricerca Scientifica e Tecnologica (MURST) and the Consiglio Nazionale delle Ricerche (CNR) of Italy.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom all correspondence should be addressed. Tel.: 39-6-72596365; Fax: 39-6-72596353; E-mail: coletta{at}seneca.uniroma2.it.

    ABBREVIATIONS

The abbreviations used are: HbA0, human adult hemoglobin; IHP, inositol hexakisphosphate; BZF, bezafibrate.

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