Coupling of the Oxygen-linked Interaction Energy for Inositol
Hexakisphosphate and Bezafibrate Binding to Human HbA0*
Massimo
Coletta
§,
Mauro
Angeletti¶,
Paolo
Ascenzi
,
Alberto
Bertollini**,
Stefano
Della Longa
,
Giampiero
De
Sanctis¶,
Anna Maria
Priori¶,
Roberto
Santucci
, and
Gino
Amiconi**
From the
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
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

Department of Experimental Medicine,
Biophysics Group, University of L'Aquila, Via Vetoio,
I-67100 L'Aquila, Italy
 |
ABSTRACT |
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 |
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).
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
= 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
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
= 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,
|
(Eq. 1)
|
where
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,
|
(Eq. 2)
|
The relative percentage of five different populations have been
calculated according to the following relationships,
|
(Eq. 3)
|
where P = 1 +
1x +
2x2 +
3x3 +
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
i in Table
I).

View larger version (24K):
[in this window]
[in a new window]
|
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 = 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
 / logx, where x is the
O2 concentration at every step and 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 ( ) and 1 mM IHP and 10 mM BZF (+)) are reported as
ligand saturation degree versus log
pO2. The overall Adair constants values
employed for continuous curves in each panel are reported in Table I.
For further details, see text.
|
|
View this table:
[in this window]
[in a new window]
|
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 ( i =
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
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
16 kJ/mol
(1 joule = 4.184 cal).

View larger version (12K):
[in this window]
[in a new window]
|
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
( ), BZF alone (+), and BZF in the presence of 1 mM IHP
(×). [Hb] = 0.1 mM heme (after mixing). = 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 ( ),
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 ( ), 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
( ), 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
15-fold increase of BZF affinity constant when IHP is
already bound to its cleft in the
-dyad axis (thus resulting an
interaction energy
G
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
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)

View larger version (18K):
[in this window]
[in a new window]
|
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.
|
|
View this table:
[in this window]
[in a new window]
|
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
Trp
37(C3), located at the
1
2 contact surface and considered a
spectroscopic marker for the R
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.

View larger version (18K):
[in this window]
[in a new window]
|
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).

View larger version (27K):
[in this window]
[in a new window]
|
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, = 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
and
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 (
0), monoliganded
(
1), biliganded (
2), three-liganded
(
3), and fully liganded molecules (
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
1
1 and
1
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.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 7.
The relative percentage of populations of
unliganded ( 0), monoliganded
( 1), biliganded
( 2), three-liganded
( 3), and fully liganded molecules
( 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
4 as well. The knowledge of the intrinsic equilibrium
association constant for the fourth binding step (i.e.
K4 = 4·
4/
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
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
5.8 kJ/mol. Likewise,
the affinity of BZF alone for the three-ligated form (i.e.
0K3BZF) is somewhat larger (by
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
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
3.3 kJ/mol.
View this table:
[in this window]
[in a new window]
|
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,
|
(Eq. 4)
|
where the interaction energy is,
|
(Eq. 5)
|
(values of I are reported in Table
IV).
View this table:
[in this window]
[in a new window]
|
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
are lower than 1 (see Table IV), in the case of the first O2 binding step a value of
1 occurs for the
binding of BZF and/or IHP alone (and thus a negative value for
IIHP and IBZF), but a value of
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
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.
 |
REFERENCES |
-
Antonini, E.,
and Brunori, M.
(1971)
Hemoglobin and Myoglobin in Their Reaction with Ligands, Elsevier North-Holland Co., Amsterdam
-
Amiconi, G.,
Antonini, E.,
Brunori, M.,
Wyman, J.,
and Zolla, L.
(1981)
J. Mol. Biol.
152,
111-120[Medline]
[Order article via Infotrieve]
-
Antonini, E.,
Condo', S. G.,
Giardina, B.,
Ioppolo, C.,
and Bertollini, A.
(1982)
Eur. J. Biochem.
121,
325-328[Abstract]
-
Gill, S. J.,
Di Cera, E.,
Doyle, M. L.,
Bishop, G. A.,
and Robert, C. H.
(1987)
Biochemistry
26,
3995-4002[Medline]
[Order article via Infotrieve]
-
Imai, K.,
and Yonetani, T.
(1975)
J. Biol. Chem.
250,
2227-2231[Abstract]
-
Doyle, M. L.,
Di Cera, E.,
Robert, C. H.,
and Gill, S. J.
(1988)
J. Mol. Biol.
196,
927-934
-
Gray, R. D.
(1971)
J. Biol. Chem.
245,
2914-2921[Abstract/Free Full Text]
-
Coletta, M.,
Ascenzi, P.,
Bertollini, A.,
Santucci, R.,
and Amiconi, G.
(1993)
Biochim. Biophys. Acta
1162,
309-314[Medline]
[Order article via Infotrieve]
-
Messana, I.,
Angeletti, M.,
Castagnola, M.,
De Sanctis, G.,
Di Stasio, E.,
Giardina, B.,
Pucciarelli, S.,
and Coletta, M.
(1998)
J. Biol. Chem.
273,
15329-15334[Abstract/Free Full Text]
-
Perutz, M. F. & Poyart, C. (1983) Lancet 881-882
-
Marden, M. C.,
Bohn, B.,
Kister, J.,
and Poyart, C.
(1990)
Biophys. J.
57,
397-403[Abstract]
-
Perutz, M. F.,
Fermi, G.,
Abraham, D. J.,
Poyart, C.,
and Bursaux, E.
(1986)
J. Am. Chem. Soc.
108,
1064-1078
-
Marden, M. C.,
Kister, J.,
Bohn, B.,
and Poyart, C.
(1988)
Biochemistry
27,
1659-1664[Medline]
[Order article via Infotrieve]
-
Gill, S. J.,
Doyle, M. L.,
and Simmons, J. H.
(1989)
Biochem. Biophys. Res. Commun.
165,
226-233[Medline]
[Order article via Infotrieve]
-
Amiconi, G.,
Santucci, R.,
Coletta, M.,
Congiu Castellano, A.,
Giovannelli, A.,
Dell'Ariccia, M.,
Della Longa, S.,
Barteri, M.,
Burattini, E.,
and Bianconi, A.
(1989)
Biochemistry
28,
8547-8553[Medline]
[Order article via Infotrieve]
-
Williams, R. C.,
and Tsay, K.
(1973)
Anal. Biochem.
54,
137-145[Medline]
[Order article via Infotrieve]
-
Riggs, A.
(1981)
Methods Enzymol.
76,
5-29[Medline]
[Order article via Infotrieve]
-
Giardina, B.,
and Amiconi, G.
(1981)
Methods Enzymol.
76,
417-427[Medline]
[Order article via Infotrieve]
-
Henry, E. R.,
and Hofrichter, J. H.
(1992)
Methods Enzymol.
210,
129-192
-
Dolman, D.,
and Gill, S. J.
(1978)
Anal. Biochem.
87,
127-134[Medline]
[Order article via Infotrieve]
-
Adair, G. S.
(1925)
J. Biol. Chem.
63,
529-545[Free Full Text]
-
Coletta, M., Angeletti, M., Ascone, I., Boumis, G., Congiu Castellano,
A., Dell'Ariccia, M., Della Longa, S., De Sanctis, G., Priori, A. M., Santucci, R., Feis, A. & Amiconi, G. (1998) Biophys. J.,
in press
-
Benesch, R. E.,
Benesch, R.,
Kwong, S.,
and Baugh, C. M.
(1983)
Proc. Natl. Acad. Sci. U. S. A.
80,
6202-6205[Abstract]
-
Ascenzi, P.,
Bertollini, A.,
Coletta, M.,
Desideri, A.,
Giardina, B.,
Polizio, F.,
Santucci, R.,
Scatena, R.,
and Amiconi, G.
(1993)
J. Inorg. Biochem.
50,
263-272[CrossRef][Medline]
[Order article via Infotrieve]
-
De Sanctis, G.,
Priori, A. M.,
Polizio, F.,
Ascenzi, P.,
and Coletta, M.
(1998)
J. Biol. Inorg. Chem.
3,
135-139[CrossRef]
-
Perutz, M. F.,
Fersht, M. R.,
Simon, F. R.,
and Roberts, G. C. K.
(1974)
Biochemistry
13,
2174-2186[Medline]
[Order article via Infotrieve]
-
Ackers, G. K.,
Doyle, M. L.,
Myers, D.,
and Daugherty, M. A.
(1992)
Science
255,
54-63[Medline]
[Order article via Infotrieve]
-
Gelin, R. G.,
Lee, A. W.-M.,
and Karplus, M.
(1983)
J. Mol. Biol.
171,
489-559[Medline]
[Order article via Infotrieve]
-
Wyman, J.
(1984)
Quart. Rev. Biophys.
17,
453-488[Medline]
[Order article via Infotrieve]
-
Pin, S.,
Alpert, B.,
Congiu Castellano, A.,
Della Longa, S.,
and Bianconi, A.
(1994)
Methods Enzymol.
232,
266-292[Medline]
[Order article via Infotrieve]
-
Abraham, D. J.,
Peascoe, R. A.,
Randad, R. S.,
and Panikker, J.
(1992)
J. Mol. Biol.
227,
480-492[Medline]
[Order article via Infotrieve]
-
Benesch, R. E.,
Benesch, R.,
and Kwong, S.
(1986)
J. Mol. Biol.
190,
481-485[CrossRef][Medline]
[Order article via Infotrieve]
-
Schõnert, H.,
and Stoll, B.
(1988)
Eur. J. Biochem.
176,
319-325[Abstract]
-
Monod, J.,
Wyman, J.,
and Changeux, J.-P.
(1965)
J. Mol. Biol.
12,
88-118[Medline]
[Order article via Infotrieve]
-
Borgstahl, G. F. O.,
Rogers, P. H.,
and Arnone, A.
(1994)
J. Mol. Biol.
236,
831-843[CrossRef][Medline]
[Order article via Infotrieve]
-
Sawicki, C. A.,
and Gibson, Q. H.
(1977)
J. Biol. Chem.
252,
7538-7547[Medline]
[Order article via Infotrieve]
-
Wilson, J.,
Phillips, K.,
and Luisi, B.
(1996)
J. Mol. Biol.
264,
743-756[CrossRef][Medline]
[Order article via Infotrieve]
-
Hopfield, J. J.
(1973)
J. Mol. Biol.
77,
207-222[Medline]
[Order article via Infotrieve]
-
Mouawad, L.,
and Perahia, D.
(1996)
J. Mol. Biol.
258,
393-410[CrossRef][Medline]
[Order article via Infotrieve]
-
Liddington, R.,
Derewenda, Z.,
Dodson, E.,
Hubbard, R.,
and Dobson, G.
(1992)
J. Mol. Biol.
228,
551-579[Medline]
[Order article via Infotrieve]
-
Holt, J. M.,
and Ackers, G. K.
(1995)
FASEB J.
9,
210-218[Abstract/Free Full Text]
-
Eaton, W. A.,
Henry, E. R.,
and Hofrichter, J.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
4472-4475[Abstract]
-
Hofrichter, J.,
Henry, E. R.,
Szabo, A.,
Murray, L. P.,
Ansari, A.,
Jones, C. M.,
Coletta, M.,
Falcioni, G.,
Brunori, M.,
and Eaton, W. A.
(1991)
Biochemistry
30,
6583-6598[Medline]
[Order article via Infotrieve]
-
Jayaraman, V.,
Rodgers, K. R.,
Mukerji, I.,
and Spiro, T. G.
(1995)
Science
269,
1843-1848[Medline]
[Order article via Infotrieve]
-
Lumry, R.,
and Eyring, H.
(1954)
J. Phys. Chem.
58,
110-120
-
Sharma, V. S.,
Schmidt, M. R.,
and Ranney, H. M.
(1976)
J. Biol. Chem.
251,
4267-4272[Abstract]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.