COMMUNICATION
CO Ligation Intermediates and the Mechanism of Hemoglobin
Cooperativity*
Michele
Perrella
and
Enrico
Di Cera§¶
From the
Dipartimento di Scienze e Tecnologie
Biomediche, Universitá di Milano, 20090 Segrate, Italy and the
§ Department of Biochemistry and Molecular Biophysics,
Washington University School of Medicine,
St. Louis, Missouri, 63110
 |
ABSTRACT |
Direct experimental resolution of the ligation
intermediates for the reaction of human hemoglobin with CO reveals the
distribution of ligated states as a function of saturation. At low
saturation, binding of CO occurs with slightly higher affinity to the
chains, but pairwise interactions are more pronounced between the
chains. At high saturation, the two chains tend to behave
identically. The sequence of CO ligation reconstructed from the
distribution of intermediates shows that the overall increase in CO
affinity is 588-fold, but it is not distributed uniformly among the
ligation steps. The affinity increases 16.5-fold in the second ligation step, 4.6-fold in the third ligation step, and 7.7-fold in the fourth
ligation step. This pattern and the detailed distribution of ligated
states cannot be immediately reconciled with the predictions of either
the concerted allosteric model of Monod-Wyman-Changeux or the
sequential model of Koshland-Nemethy-Filmer and underscore a more
subtle mechanism for hemoglobin cooperativity.
 |
INTRODUCTION |
Hemoglobin has long served as a paradigm for cooperative proteins
(1, 2) and continues to provide new and important insights into how
allostery is exploited in processes of pathophysiological importance
(3). Two proposed models of hemoglobin cooperativity have made a
significant impact in our understanding of structure-function relations
in this and other proteins. The
MWC1 model (4) assumes that
hemoglobin exists in two quaternary structures, T and R, that differ in
their affinity for oxygen. The low affinity T state is predominant in
the absence of oxygen. Binding of oxygen progressively drives the
hemoglobin tetramer to the high affinity R state, thereby bringing out
the cooperative nature of the binding curve that is key to the function
in vivo. A key postulate of the MWC model is that site-site
interactions are not direct but are mediated indirectly through a
concerted allosteric transition from T to R involving the molecule as a whole. An alternative allosteric model, known as the KNF model (5),
assumes that each subunit is capable of tertiary conformational changes
upon oxygen binding. These changes are directly transmitted to neighbor
subunits through pairwise interactions and lead to sequential changes
in the affinity for oxygen. Cooperativity ensues as a result of direct communications.
There is much kinetic and structural evidence that the MWC model best
represents the behavior of hemoglobin under a variety of conditions
(6), but the validity of this model has been challenged (7, 8), and the
molecular code for hemoglobin cooperativity remains to be defined (9,
10). The highly cooperative nature of the oxygen binding isotherm
naturally suppresses the contribution of the singly, doubly, and triply
ligated species, making it very difficult to discriminate models of
cooperativity (11). It has long been recognized that the overall shape
of the oxygen binding curve is consistent with many models of
cooperativity (5) and does not provide conclusive discrimination. The
precise distribution of ligation intermediates that makes up the oxygen binding curve is more informative, but efforts to resolve these intermediates have been largely confined to model systems where hemoglobin or its ligands bear significant chemical perturbations (10).
Results from these model systems are not consistent with either the MWC
or the KNF model (8, 10), but their relevance to the properties of
native hemoglobin reacting with oxygen has been questioned (6, 9).
Resolution of the ligation intermediates of native hemoglobin reacting
with a gaseous ligand is therefore highly desirable. These measurements
are presented here for the first time and provide important new
information on the mechanism of hemoglobin cooperativity.
 |
EXPERIMENTAL PROCEDURES |
Equilibration of Hemoglobin with CO--
Weighted samples of
HbA0 (5 g/dl) in 0.1 M KCl, deoxygenated in a
tonometer using a stream of humidified nitrogen, were introduced into
Hamilton gas-tight syringes and mixed with weighted samples of the same
solutions exposed to CO. The syringes were kept for 20 h in a
closed cylinder filled with water containing dithionite and
thermostatted at 20 °C to let the hemoglobin solution equilibrate with CO. The CO saturation was calculated from the sample weights. The
methemoglobin content after equilibration was 1-2%. The pH of the
mixture was measured anaerobically after the attainment of equilibrium.
A constant value of 7.0 ± 0.05 was obtained by adjusting
the pH of the hemoglobin solution with 0.1 N KOH before equilibration. The PCO equilibrium value of the mixtures
was calculated from the CO binding isotherm obtained previously under
the same conditions of solvent, pH, protein concentration, and
temperature by equilibrating solutions of HbA0 with
CO/N2 mixtures of known composition (12).
Isolation of Intermediates--
Samples of hemoglobin solutions
(100 µl) equilibrated with CO were injected anaerobically into a
stirred cryosolvent (60% v/v ethylene glycol, 40% v/v 22 mM phosphate buffer, pH 7.5, at 20 °C) containing
ferricyanide and cooled at
30 °C. Ferricyanide oxidizes the
unliganded subunits of hemoglobin yielding a mixture of partially
oxidized species that can be separated by cryofocusing at
25 °C on
gel tubes. Only nine species are detected by this procedure because the
products of the oxidation of the two possible doubly ligated 
pairs (Fig. 1) have identical isoelectric points. Gels were then
removed from the glass tubes and sliced at the level of the colored
protein components, which were eluted and assayed. The procedures for
the separation and analysis of the isolated intermediates and all the
relevant controls are described in detail elsewhere (13). Resolution of
the intermediates
1CO
2
1CO
2
and
1CO
2
1
2CO
(Fig. 1) was obtained by noncryogenic focusing of the mixture of
oxidized species isolated as a single component by cryofocusing, as
described (14).
Theory and Data Analysis--
The hemoglobin subunits
and
assembled in the tetramer form two structurally nonequivalent

contacts and yield 10 possible ligation states (Fig.
1). These states define the
model-independent partition function for ligand binding to the four
hemes of hemoglobin as follows (11).
|
(Eq. 1)
|
where x is the ligand concentration or partial
pressure, K
and K
are the binding constants for the
and
subunits when all other
subunits are unligated, c
,
c
, c'
and
c
are the second order interaction constants between the four possible pairs of chains,
c

and c

are the third order interaction constants involving the two possible
triplets of chains and c


is the
fourth order interaction constants involving all chains. The
c values reflect the presence of positive (c > 1), negative (c < 1) or no (c = 1)
cooperativity. For example, a value of c
= 10 means that ligand binding to the second
chain is enhanced 10-fold when the first
chain is bound. The population of each intermediate is determined by the ratio of the term in the partition function related to the intermediate divided by
. These functions were used to analyze the distribution of CO ligation intermediates obtained experimentally.

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Fig. 1.
Manifold of distinct ligated states for the
interaction of hemoglobin with oxygen or CO (X).
The tetramer assembles from two identical  dimers
( 1 1 and
2 2). Symmetry of the
1 2 1 2
tetramer reduces the number of ligated states from 16 to 10. Each
intermediate is labeled according to the chain being ligated. The
complete site-specific description of the manifold requires nine
independent parameters that can be resolved from the distribution of
ligated states obtained experimentally as a function of
saturation.
|
|
The site-specific description of ligand binding to the four hemes of
hemoglobin is related to the classical Adair description (15) that
takes into account the five possible ligation states without
discriminating between hemoglobin chains. The partition function for
this description is
|
(Eq. 2)
|
where ki is the stepwise binding constant for
the ith ligation step. These constants can be written in
terms of the site-specific parameters as follows (11).
|
(Eq. 3)
|
|
(Eq. 4)
|
|
(Eq. 5)
|
|
(Eq. 6)
|
The distribution of CO-ligated intermediates was also analyzed
in terms of the MWC and KNF models (4, 5). The partition function of
the MWC model is as follows.
|
(Eq. 7)
|
where L is the allosteric constant reflecting the
population of the T state relative to the R state in the absence of
ligand, K
T and K
R
are the binding constants to the
chains in the T and R states, and
K
T and K
R are the
analogous binding constants for the
chains. The partition function
for the KNF model is as follows.
|
(Eq. 8)
|
where the K values are the binding constants of the
two chains and the c values are the pairwise interaction
constants between the possible pairs of chains. No distinction was
necessary to describe interactions involving the ligated 
pairs
(see "Results"). The two models have the same number of independent
parameters and could be compared directly.
 |
RESULTS AND DISCUSSION |
It follows from definition of the Adair constants in Equations
3-6 that different combinations of site-specific parameters reflecting the contribution of the ten ligated states of hemoglobin can translate in the same pattern of stepwise constants. This is because analysis of
the binding curve in terms of the Adair formalism contains four
independent parameters, as opposed to nine in the site-specific formalism (11). Therefore, the distribution of the 10 ligated intermediates in the native molecule reacting with a gaseous ligand provides a very accurate test of any proposed molecular code of hemoglobin cooperativity.
A high resolution cryogenic technique (13) has made it possible to
resolve the distribution of intermediates for the reaction of CO with
native human hemoglobin (Fig. 2). Because
CO shares with oxygen high binding affinity and cooperativity (11, 12), it represents an ideal ligand for dissecting hemoglobin cooperativity at the site-specific level, which cannot be achieved with oxygen in
view of its fast kinetics of dissociation (16). Analysis of the
distribution of CO-ligated intermediates as a function of saturation
reveals strong cooperativity already in the first two ligation steps
(Table I). Binding to the
chain is
slightly preferred in the singly ligated species, but such preference
disappears in the triply ligated species, indicating that
conformational changes taking place at high saturation tend to abolish
the differences between the chains. Once the first chain is ligated,
the second binding event takes place with significantly higher
affinity. Interactions are stronger between
chains and compensate
for the lower affinity of these chains compared with the
chains, thereby producing doubly ligated intermediates of comparable magnitude (Fig. 2). No significant difference is observed in the populations of
the mixed doubly ligated intermediates where the 
pair is ligated
within or across the dimer interface (c
= c'
). Interactions among the ligated chains
remain strong in the third and fourth ligation steps, as indicated by
the large values of the third and fourth order interaction
constants.

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Fig. 2.
Distribution of intermediates for CO binding
to human hemoglobin under experimental conditions of 100 mM
KCl, pH 7.0 at 20 °C. a, unligated state
1 2 1 2 ( ),
and fully ligated state
1CO 2CO 1CO 2CO
( ). b, singly ligated states
1CO 2 1 2
( ) and
1 2 1CO 2
( ) and triply ligated states
1CO 2CO 1CO 2
( ) and
1CO 2 1CO 2CO
( ). c, doubly ligated states
1CO 2CO 1 2
( ),
1 2 1CO 2CO
( ), and
1CO 2 1CO 2 + 1CO 2 1 2CO
(+). The populations of
1CO 2 1CO 2
and
1CO 2 1 2CO
are comparable and are shown as a combined species. The continuous
curves were drawn according to the site-specific partition
function 1, with the best fit parameter values listed in Table I.
|
|
The distribution of ligated intermediates has peculiar features that
are difficult to reconcile with current models of hemoglobin cooperativity. The significant increase in affinity at the second step
of ligation populates the doubly ligated intermediates well beyond the
expected value (0.2-0.5%) in the absence of interactions. The MWC
model does not account for this important feature of CO ligation (Table
I). The best fit parameter values obtained from the analysis of the
distributions predict the T to R transition to take place upon binding
of the third CO molecule. This provides a satisfactory fit for all but
the doubly ligated intermediates, predicted to be only 1%, as opposed
to almost 5% found experimentally. The KNF model yields a better fit
but again fails to correctly reproduce the distribution of doubly
ligated intermediates and the high degree of cooperativity in the first
two ligation steps (Table I). Combinatorial switch mechanisms for
hemoglobin cooperativity, like the one implied by the "symmetry
rule," also fail to describe the distribution of doubly ligated
intermediates, because ligation of an 
pair is not dependent on
the location of the chains in the tetramer.
The discrepancies of the MWC and KNF models are also illustrated by the
cooperativity pattern embodied by the four stepwise Adair constants
(Table I), as shown in Fig. 3. Both
models significantly underestimate the affinity of the second ligation
step, but the MWC model also overestimates the affinity of the third
ligation step. However, the overall saturation curves predicted by
these models from the analysis of the distribution of intermediates are
practically indistinguishable from each other and from the curve
predicted by the model-independent analysis (Fig.
4). The excellent agreement of the models
with experimental data in Fig. 4 is deceiving, because the shape of the
binding isotherm is notoriously insensitive to the contribution of
poorly populated intermediates. The limitations of the MWC and KNF
models are observed only when the properties of these intermediates are
measured directly (Table I). Hence, overall binding isotherms cannot
and should not be used to assess the validity of mechanistic models or
to discriminate among them.

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Fig. 3.
Stepwise Adair constants for CO binding to
native human hemoglobin. Shown are the values derived from the
site-specific parameters using Equations 3-6 in the text (black
bars) compared with the predictions of the MWC (white
bars) and KNF (hatched bars) models.
|
|

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Fig. 4.
CO binding isotherm obtained from the
distribution of ligated intermediates shown in Fig. 2. Also shown
are the binding curves obtained from the Adair constants
derived from analysis of these intermediates using the MWC
(continuous line) and KNF (dashed line) models.
The two models give an excellent fit of the data, although they are
inconsistent with the distribution of doubly ligated intermediates in
Fig. 2 and predict Adair constants significantly different from those
derived from the model-independent analysis (Table I and Fig. 3).
|
|
The distribution of CO ligation intermediates obtained experimentally
and the pattern of stepwise constants derived from them reveal a more
subtle mechanism for hemoglobin cooperativity than those predicted by
the MWC and KNF models. The KNF model fits the data significantly
better than the MWC model, but it does not capture the large
enhancement in binding affinity in the first two ligation steps. An
unambiguous interpretation of the pattern in Fig. 3 is not possible,
because of the limitations intrinsic to the Adair description. It
should also be pointed out that the pattern in Fig. 3 applies
specifically to the experimental conditions in the present study and
may be modified by allosteric effectors and physical variables. For
these reasons, we limit ourselves to emphasize the phenomenological
significance of the results. A modified MWC model with direct pairwise
interactions within the T state would be consistent with the
experimental data, but so would a modified KNF model with interactions
higher than second order. More information must be gathered on the
ligation intermediates of native hemoglobin reacting with a ligand that
closely mimics oxygen, before the code of hemoglobin cooperativity can
be formulated conclusively. The results reported here represent an
important first step in this direction.
 |
FOOTNOTES |
*
This work was supported by a grant from MURST (to M. P.) and National Institutes of Health Grants HL49413 and HL58141 (to E. D. C.).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 correspondence should be addressed. Tel.:
314-362-4185; Fax: 314-362-7183; E-mail: enrico{at}caesar.wustl.edu.
The abbreviations used are:
MWC, Monod-Wyman-Changeux; KNF, Koshland-Nemethy-Filmer.
 |
REFERENCES |
-
Perutz, M. F.
(1970)
Nature
228,
726-739[Medline]
[Order article via Infotrieve]
-
Perutz, M. F.
(1989)
Q. Rev. Biophys.
22,
139-236[Medline]
[Order article via Infotrieve]
-
Gow, A. J.,
and Stamler, J. S.
(1998)
Nature
391,
169-173[CrossRef][Medline]
[Order article via Infotrieve]
-
Monod, J.,
Wyman, J.,
and Changeux, J. P.
(1965)
J. Mol. Biol.
12,
88-118[Medline]
[Order article via Infotrieve]
-
Koshland, D. E.,
Némethy, G.,
and Filmer, D.
(1966)
Biochemistry
5,
365-385[Medline]
[Order article via Infotrieve]
-
Henry, E. R.,
Jones, C. M.,
Hofrichter, J.,
and Eaton, W. A.
(1997)
Biochemistry
36,
6511-6528[CrossRef][Medline]
[Order article via Infotrieve]
-
Ackers, G. K.,
Doyle, M. L.,
Myers, D.,
and Dougherty, M. A.
(1992)
Science
255,
54-63[Medline]
[Order article via Infotrieve]
-
Ho, C.
(1992)
Adv. Protein Chem.
43,
153-312[Medline]
[Order article via Infotrieve]
-
Perutz, M. F.,
Wilkinson, A. J.,
Paoli, M.,
and Dodson, G. G.
(1998)
Annu. Rev. Biophys. Biomol. Struct.
27,
1-34[CrossRef][Medline]
[Order article via Infotrieve]
-
Ackers, G. K.
(1998)
Adv. Protein Chem.
51,
185-253[Medline]
[Order article via Infotrieve]
-
Di Cera, E.
(1995)
Thermodynamic Theory of Site-specific Binding Processes in Biological Macromolecules, Cambridge University Press, Cambridge, UK
-
Perrella, M.,
Colosimo, A.,
Benazzi, L.,
Ripamonti, M.,
and Rossi-Bernardi, L.
(1990)
Biophys. Chem.
37,
211-223[CrossRef][Medline]
[Order article via Infotrieve]
-
Perrella, M.,
and Rossi-Bernardi, L.
(1994)
Methods Enzymol.
232,
445-460[Medline]
[Order article via Infotrieve]
-
Perrella, M.,
Ripamonti, M.,
and Caccia, S.
(1998)
Biochemistry
37,
2017-2028[CrossRef][Medline]
[Order article via Infotrieve]
-
Adair, G. S.
(1925)
J. Biol. Chem.
63,
529-545[Free Full Text]
-
Olson, J. S.,
Anderson, M. E.,
and Gibson, Q. H.
(1971)
J. Biol. Chem.
246,
5919-5923[Abstract/Free Full Text]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.