Haemoglobin: Cooperativity

in ProteinLigand
Interactions
Chien Ho, Carnegie Mellon University, Pittsburgh, PA, USA
Jonathan A Lukin, Carnegie Mellon University, Pittsburgh, PA, USA
Haemoglobin is an essential component of the circulatory system of vertebrates. Its
chief physiological function is to transport oxygen from the lungs to the tissues.
Haemoglobinbinds oxygencooperatively, i.e. the affinity of the proteinfor the first oxygen
molecule is less than that for subsequent oxygen molecules. Human adult haemoglobin
was amongthe first proteins whose complete three-dimensional structure was determined
by X-ray crystallography.
Introduction
Haemoglobin is probably the most studied protein and has
served as a model or paradigm for the structurefunction
relationship in multimeric, allosteric proteins. The oxyge-
nation process of haemoglobin is cooperative, i.e. the
binding of the rst oxygen molecule enhances the binding
of the second, third, and fourth oxygen molecules. The
oxygen-binding properties of the haemoglobin molecule
are also modulated by interactions of individual amino
acid residues with solvent components, known as hetero-
tropic allosteric eectors. These eectors include hydrogen
ion, chloride, carbon dioxide, inorganic phosphate, and
organic polyanions such as 2,3-bisphosphoglycerate.
Concerted and sequential models have been proposed to
explain the mechanism of cooperative binding. The
detailed molecular mechanisms for the cooperative oxyge-
nation process of haemoglobin are now being revealed at
the atomic level as a result of mutagenesis of the
haemoglobin gene, combined with nuclear magnetic
resonance, kinetic, thermodynamic, and X-ray crystal-
lographic analyses.
Haemoglobin Structure
Haemoglobin (Hb) is a protein of molecular mass
~64 500 Da, consisting of four subunits: two identical a
chains of 141 amino acids each, and two identical b chains
of 146 aminoacids each. Eachpolypeptide subunit exhibits
a conformation similar to that of the related monomeric
protein myoglobin (Mb). Ab subunit of Hb includes eight
helical segments designated AH; an a subunit is very
similar, except that it lacks a D helix. The polypeptide
chain of each subunit forms a pocket that encloses and
binds a prosthetic haemgroup. Haemis an iron complex of
protoporphyrin IX whose iron atom in the ferrous state
can bind an oxygen molecule reversibly. In each subunit,
the haem is linked to the polypeptide by a covalent bond
betweenthe haemironatomandthe N
e2
of the imidazole of
a histidine residue (known as the proximal histidine (F8)).
The a
1
b
1
and a
2
b
2
dimers of Hb A are related by a twofold
axis of symmetry that runs down a water-lled cavity in the
centre of the protein (Figure 1a). Thus, the a
1
b
1
and a
2
b
2
subunit interfaces are identical, as are the a
1
b
2
and a
2
b
1
subunit interfaces. The arrangement of subunits (i.e. the
quaternary structure) of Hb depends on the ligation state
of the protein. The two classical quaternary structures are
the T (tense) form for the low-anity deoxy-Hb and the R
(relaxed) form for the high-anity oxy-Hb, rst reported
by Perutz (1970). Anewquaternary structure in the ligated
state, R2, was observedby Silva et al. (1992) andSmith and
Simmons (1994).
Haemoglobin is an essential component of the circula-
tory system of vertebrates. Its chief physiological function
is to transport oxygen fromthe lungs tothe tissues. Human
normal adult haemoglobin (Hb A) was among the rst
proteins whose complete three-dimensional structure was
determined by X-ray crystallography. This determination
of the structure of Hb to atomic resolution by Perutz and
others opened up the opportunity to correlate the structure
of a protein with its biological function. A major goal of
research is to understand how the four subunits of Hb
function at the molecular level to bind O
2
in the lungs and
release O
2
in the muscles. Hb is one of the most carefully
studied proteins to date, yet some aspects of its structure
function relationships remain controversial. We attempt
here to give a balanced view of the current state of
knowledge of the structurefunction relationship of this
physiologically vital protein.
Article Contents
Secondary article
. Introduction
. Haemoglobin Structure
. Cooperativity in O
2
Binding
. Mathematical Description of Cooperative Ligand
Binding
. Conformations and Conformational Changes
. Bohr Effect
. Concluding Remarks
. Acknowledgement
1 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
Figure 1 Illustrations of Hb A structure produced using the graphics programMOLMOL (Koradi et al., 1996). Coordinates of Hb A in the T (deoxy; Fermi
et al., 1984), R (oxy; Shaanan, 1983) and R2 (CO; Silva et al., 1992) conformations were obtained fromBrookhaven Protein Databank files 2HHB, 1HHO
and 1BBB, respectively. (a) Deoxy-Hb A, viewed along the twofold symmetry axis showing the water-filled central cavity. The haems are shown in a space-
filling representation, while the polypeptide chains are represented by ribbons colouredgreen, yellow, cyanandorange for a
1
, b
1
, a
2
, andb
2
, respectively.
(b) Backbonetrace of oxy-(R) anddeoxy-Hb(T), colouredredandblue, respectively. The a
1
b
1
dimers of the twoconformations, shownas thincoils towards
the rear of the figure, have beensuperimposedat the a
1
b
1
interface. Thicker, lighter-coloured coils towards the front of the figure represent the a
2
b
2
dimer
and illustrate the repositioning of this dimer upon quarternary structure rearrangement. (c) Individual a subunits of oxy- and deoxy-Hb with haems
superimposed. The haems are viewed edge-on, in a ball-and-stick representation. The protein chain is shown as a smooth coil passing through the
backbone Ca atoms, coloured red for oxy-Hb and blue for deoxy-Hb. Also shown are bonds of the proximal histidine and the distal ligand (O
2
). (d) Switch
regionof the a
1
b
2
interface of Hbinthe T, RandR2conformations, showninblue, redandgreen, respectively. Here, the backbone atoms of residues a
1
38
44 (shown in darker colours) have been superimposed, to emphasize the relative motion of b
2
His97.
Haemoglobin: Cooperativity in ProteinLigand Interactions
2 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
Cooperativity in O
2
Binding
Figure 2 shows plots of the oxygenation of Hb Aat dierent
values of pH in the absence and presence of 2,3-bispho-
sphoglycerate (2,3-BPG). The amount of oxygen bound to
Hb is determined by monitoring the optical spectrum and
comparing it to the characteristic spectra of the pure oxy-
and deoxy- forms of the protein. The free O
2
concentration
(i.e. the partial pressure of O
2
, pO
2
) of the Hb sample is
measured by an oxygen electrode. This measurement is
made automatically as the Hb is continuously oxygenated
(or deoxygenated), using a Hemox Analyzer (TCS Medical
Products, Huntington Valley, PA, USA). The sigmoidal
nature of the oxygen binding curve suggests that the
anity of the protein for the rst oxygen molecule is less
than that for subsequent oxygen molecules; that is, the
binding of O
2
toHbis cooperative. This is incontrast tothe
monomeric myoglobin, for which the O
2
binding curve is
hyperbolic. Thus, cooperativity resides in the oligomeric
nature of the Hb molecule. The oxygenation of Hb A can
be characterized by two parameters, namely the oxygen
anity at 50% saturation (i.e. p
50
in torr (mmHg)) and the
cooperativity for the oxygenation process as measured by
the Hill coecient (i.e. n
max
), obtained from the slope of a
plot of log (Y/(1 2Y) as a function of log pO
2
(inset in
Figure2). For HbAin0.1 mol L
21
phosphate at pH7.4 and
298C, p
50
is ~8 torr andn
max
is ~3.0. The fact that the Hill
coecient for oxygenation is less than 4 and greater than 1
suggests the existence of partially oxygenated species, i.e.
Hb molecules with one, two or three O
2
molecules bound.
The oxygenation of Hb A is regulated by intracellular
metabolites and ions, such as hydrogen ion, chloride,
carbon dioxide, phosphate, and 2,3-BPG. These serve as
heterotropic allosteric eectors, i.e. they aect the binding
of O
2
toHbwhile binding at other sites inthe molecule than
the O
2
binding site. These molecules do not aect the
binding of O
2
to Mb; the eect results from the oligomeric
structure of Hb. The functional consequences of these
eectors imply dierential interactions of each eector
with unligated and ligated forms of the Hb molecule.
Because they have a higher anity for deoxy-Hb A than
oxy-HbA, these eectors lower the oxygenanity of HbA
(Figure 2), and result in the release of O
2
in the tissues. 2,3-
BPG exerts the strongest eect among them. It binds to
bHis2, bLys82, and bHis143, located in the central cavity
of the Hb molecule, in the deoxy form. In oxy-Hb, the
central cavity is too narrow to accommodate the 2,3-BPG
molecule. Thus, the binding of 2,3-BPG to Hb A would
shift the equilibrium in favour of the deoxy (T) quaternary
structure, thus lowering the oxygen anity.
Extensive kinetic investigations on ligand binding to Hb
A have been carried out by Gibson, Olson, and others.
They provide valuable complementary information to
both thermodynamic and structural information. For a
reviewon kinetic studies of ligand binding toHb A, refer to
Mathews andOlson(1994) andthe references citedtherein.
1.0
0.8
0.6
0.4
0.2
0.0
S
a
t
u
r
a
t
i
o
n

(
Y
)
0 10 20 30 40 50 60 70 80
pO
2
(torr)
pO
2
(torr)
100
10
1
0.01
1 10 100
0.1
Y
/
(
1

Y
)
7.88
6.97
6.69
7.05
pH
0
0
0
5
2,3-BPG
(mmol L
1
)
3.8
14.5
17.2
20.9
p
50
(torr)
3.0
3.1
3.1
3.0
n
max
Figure 2 Measured oxygen-binding curves of Hb A in 0.1 mol L
21
phosphate at 298C at pH6.69, 6.97 and 7.88, and at pH7.05 in the presence of 2,3-
BPG. The inset shows Hill plots for the oxygenation data. We thank Ms Nancy T. Ho for providing the oxygen-binding data shown here.
Haemoglobin: Cooperativity in ProteinLigand Interactions
3 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
Mathematical Description of
Cooperative Ligand Binding
Hill equation
The cooperative binding of a ligand by an oligomeric
protein can be described by the Hill equation. Consider a
protein Pconsisting of n subunits, each of which can bind a
ligand L. If the ligand binds with innite cooperativity so
that either all or none of the binding sites are occupied, the
reaction is described by eqn [I].
P1nLPL
n
[I]
The dissociation constant for this reaction is given by eqn
[1],
K =
[P[[L[
n
[PL
n
[
[1[
while the fractional saturation (i.e. the fraction of binding
sites occupied by ligand) is given by eqn [2],
Y
L
=
n[PL
n
[
n [P[ [PL
n
[ ( )
[2[
Combining eqns [1] and [2] yields the Hill equation
Y
L
=
[L[
n
K [L[
n
[3[
In the case of Hb, the concentration of free ligand [L] is the
partial pressure of oxygen, pO
2
. Then K5(p
50
)
n
, where p
50
is the value of pO
2
when half of the O
2
-binding sites are
occupied. Making these substitutions and taking loga-
rithms, eqn [3] may be rearranged to yield
log
Y
O
2
1 Y
O
2
8
>
>
:
9
>
>
; = n log [ pO
2
[ n log p
50
[4[
If Hb bound four oxygen molecules in a single step, then a
plot of log [Y/(1 2Y)] vs log[pO
2
], called a Hill plot, would
be a straight line with a slope of n 54 and an intercept on
the log[pO
2
] axis of log p
50
. However, the Hill plot exhibits
a sigmoidal shape (Figure 2), indicating that binding occurs
in stages, such that the oxygen anity of Hb depends on
the number of subunits in the tetramer that are already
ligated. When the partial pressure of O
2
is very low, almost
all of the Hb molecules are unligated; on the other hand,
when pO
2
is very high, the vast majority of Hb tetramers
are fully ligated. Thus, the behaviour of the oxygenation
curve at opposite extremes of pO
2
indicates the binding
anity of the rst and fourth O
2
molecules. Extrapolating
the asymptotes of the Hill plot, we ndthat the fourthO
2
to
bind to Hb does so with 100-fold higher anity than the
rst. The Hill coecient n, taken as the maximum slope of
the Hill plot, indicates the degree of cooperativity of O
2
binding; n
max
~3.0 for normal human Hb.
Adair formulation
The derivation of the Hill equation presented above
assumes that the binding of all four oxygen molecules to
Hb takes place in a single step, i.e. that there are no
partially oxygenated molecules. This assumption must be
relaxed in the interpretation of the Hill coecient, n
max
,
and p
50
for Hb A, which vary along the Hill plot. A more
consistent and general model allows for sequential binding
of ligands with dierent dissociation constants. For a
protein with four ligand-binding sites (such as Hb), the
reaction sequence is as shown in eqn [II].
P1LPL
PL1LPL
2
[II]
PL
2
1LPL
3
PL
3
1LPL
4
The equilibrium of the ith ligand binding to the protein is
characterized by a macroscopic or apparent dissociation
constant (eqn [5]).
K
i
=
[PL
i1
[[L[
[PL
i
[
[5[
These constants are related to microscopic or intrinsic
dissociation constants k
i
by statistical factors that account
for the number of binding sites in the protein. For the
general equilibrium, PL
i 21
1LPL
i
, where 1 _i _4, the
concentration of free binding sites in PL
i 21
is
(4 2i 11)[PL
i 21
], while the concentration of bound
ligand on PL
i
is i[PL
i
]. Thus,
k
i
=
4 i 1
i
8
>
:
9
>
;
K
i
[6[
Solving eqns [II] sequentially for the concentration of each
ligated species, we obtain eqns [7].
[PL[ = [P[[L[=K
1
= 4[P[[L[=k
1
[PL
2
[ = [PL[[L[=K
2
=
3
2
[PL[[L[=k
2
= 6[P[[L[
2
=k
1
k
2
[PL
3
[ = [PL
2
[[L[=K
3
=
2
3
[PL
2
[[L[=k
3
= 4[P[[L[
3
=k
1
k
2
k
3
[PL
4
[ = [PL
3
[[L[=K
4
=
1
4
[PL
3
[[L[=k
4
= [P[[L[
4
=k
1
k
2
k
3
k
4
[7[
Now the fractional saturation of ligand binding is
Y
L
=
[PL[ 2[PL
2
[ 3[PL
3
[ 4[PL
4
[
4 [P[ [PL[ [PL
2
[ [PL
3
[ [PL
4
[ ( )
[8[
Haemoglobin: Cooperativity in ProteinLigand Interactions
4 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
Substituting eqns [7] into eqn [8] yields the Adair equation
[9].
Y
L
=
[L[
k
1

3[L[
2
k
1
k
2

3[L[
3
k
1
k
2
k
3

[L[
4
k
1
k
2
k
3
k
4
1
4[L[
k
1

6[L[
2
k
1
k
2

4[L[
3
k
1
k
2
k
3

[L[
4
k
1
k
2
k
3
k
4
[9[
The dissociation constants k
i
can be determined by tting
eqn [9] to the oxygen-binding curve. For Hb,
k
1
4k
2
4k
3
4k
4
, indicating positive cooperativity of O
2
binding. The Adair equation is based on a phenomen-
ological treatment of binding that is independent of any
mechanistic model for the molecular basis of cooperativity.
Concerted and sequential models
The cooperativity of O
2
binding and the eects of
molecules such as H
1
ion and 2,3-BPG result from
interactions between sites that are distant on the Hb
molecule, i.e. the haems in the cooperative binding of O
2
(homotropic interactions) and the haems and other
binding sites in the eect of H
1
ion and 2,3-BPG on O
2
binding (heterotropic interactions). Such long-range inter-
actions, which must be mediated through the structure of
the Hb molecule, are termed allosteric interactions.
Monod et al. (1965) proposed a model for allosteric
interactions based on the conservation of symmetry in an
oligomeric protein at all stages of ligand binding. This
model, known as the MWC or concerted model, assumes
that the protein interconverts between two conformations:
a low-anity T form and a high-anity R form. All the
subunits of a particular molecule must have the tertiary
structure characteristic of one of these two forms; hybrids
are disallowed. Thus, the transition from T to R is
concerted in that all four subunits must change conforma-
tion in unison. The ligand-binding anity of a subunit
depends only on whether it is in the T or R state, and is
otherwise independent of whether neighbouring subunits
are ligated. With the notation T
i
and R
i
for the T-state and
R-state protein with i ligands bound, the equilibrium for
conformational conversion of ligand-free protein is
T
0
R
0
with the equilibrium constant L=[T
0
]/[R
0
]. Then,
from the ligand-binding equilibria, T
i
1LT
i 11
, eqns [7]
can be applied with k
1
5k
2
5k
3
5k
4
=k
T
to express each
concentration [T
i
] in terms of [T
0
], [L], and k
T
. Similarly,
[R
i
] can be written in terms of [R
0
], [L], and k
R
. Then, the
fractional saturation (eqn [10])
Y
L
=
P
4
i=1
i [R
i
[ [T
i
[ ( )
4
P
4
i=0
[R
i
[ [T
i
[ ( )
[10[
reduces to eqn [11],
Y
L
=
a(1 a)
3
Lca(1 ca)
3
(1 a)
4
L(1 ca)
4
[11[
with the denitions a =[L]/k
R
5pO
2
/k
R
in the case of O
2
binding, and c =k
R
/k
T
. Note that the MWC model for Hb
reduces the number of unknowns in the Adair equation by
one. In this model, the free energy of ligand binding
stabilizes the R state relative to the T state. Binding of
successive ligands increases the population of the high-
anity R-state molecules, giving rise to positive coopera-
tivity.
In contrast to the MWCmodel, the sequential (or KNF)
model of Koshlandet al. (1966) allows subunits of dierent
conformations to coexist within the same oligomeric
protein molecule. Each subunit is assumed to change its
conformation when it binds a ligand. This structural
change alters the anity of neighbouring subunits through
interactions at subunit interfaces. Thus, the deoxy to oxy
transition occurs sequentially through a sequence of
hybrids as induced by successive binding of ligand. The
algebraic form of the fractional saturation depends on the
specic model for interactions among subunits. Koshland
et al. (1966) found that the best t to observed binding data
for Hb resulted from a square geometry model that
assumes that stereochemical interaction occurs only
between unlike subunits (a and b).
The MWC and KNF models both provide an excellent
t to the measured O
2
binding curve of Hb; therefore, such
data alone cannot distinguish between the concerted and
sequential mechanisms for allostery.
Conformations and Conformational
Changes
The physiological function of a protein is determined by its
structure. Thus, in order to understand the molecular basis
for the cooperative oxygenation of Hb, we need to gain
detailed information regarding the nature of the structural
changes associated with the oxygenation process. Are the
structural changes during the oxygenation process con-
certed (Monod et al., 1965) or sequential (Koshland et al.,
1966) in nature? What roles do the partially oxygenated
species play in the cooperative oxygenation process? What
are the structures of partially oxygenated species? At-
tempts to answer these questions have resulted in extensive
research during the past ve decades, but we still do not
have precise answers. A substantial amount of early
published spectroscopic and crystallographic data has
been presented in support of a concerted two-state
description for the oxygenation of Hb. However, there
are also published data to suggest that the structural
changes during the oxygenation of Hb occur in a stepwise
Haemoglobin: Cooperativity in ProteinLigand Interactions
5 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
fashion. In this section, we summarize the current knowl-
edge regarding the role of partially oxygenated species, the
structures of these species, and the nature of the structural
changes (i.e. concerted or sequential) associated with the
cooperative oxygenation process.
A number of techniques, such as X-ray crystallography
and nuclear magnetic resonance (NMR) spectroscopy,
have been used to determine the conformation of the Hb
molecule and the changes in conformation that occur on
ligation. The a
1
b
1
interface is stabilized by a large number
of packing contacts, including hydrogen bonds, and is
almost identical in the oxy (R) and deoxy (T) conforma-
tions of Hb (Table 1). Thus, to a good approximation, the
a
1
b
1
and a
2
b
2
dimers move as rigid units during the deoxy-
to-oxy transition (Figure 1b). In contrast to the xed a
1
b
1
and a
2
b
2
interfaces, the a
1
b
2
and a
2
b
1
interfaces undergo
signicant changes upon ligation of Hb. During this
transition, the a
1
b
1
dimer rotates 158 with respect to the
a
2
b
2
dimer, about an axis passing through the a subunits.
The regions of the a
1
b
2
interface near this pivot point,
within the FGcorner of the a chain and the Chelix of the b
chain, remain in contact, and are termed the exible joint.
Farther from the pivot, the contact of the C helix of the a
chain (residues 3844) and the FG corner of the b chain
(residues 97100) is called the switch region. In the
transition from the T (deoxy) to the R (oxy) quaternary
structure, residue b
2
His97 is displaced by one turn of the C
helix of the a chain, from the groove between the side-
chains of a
1
Thr41 and a
1
Pro44 to between a
1
Thr38 and
a
1
Thr41 (Figure 1d). The T to R transition also disrupts
several hydrogenbonds andsalt bridges betweenthe a
1
and
b
2
subunits (Table 2).
The cooperativity of oxygen binding to Hb arises from
the eect of the ligation state of one haem on the ligand-
binding anity of another haem in the same Hb molecule.
However, the large distances (in excess of 25 A

) separating
the haems preclude any direct interaction between them.
Thus, any haemhaem interactions must be mechanically
transmitted by the protein molecule. By comparing the
crystal structures of Hb in the deoxy (T) and ligated (R)
states, Perutz (1970) proposed a stereochemical mechan-
ism for the cooperative oxygenation of Hb. In deoxy-Hb,
the haem porphyrins are domed, so that each iron atom is
displaced ~0.6 A

out of the haem plane, towards the

proximal His (F8). Upon oxygenation, the bonds between
the iron and porphyrin nitrogens contract, so that the iron
moves into the haem plane. To avoid bumping into the
haem, the proximal histidine (F8) must tilt slightly, so that
the F helix moves laterally ~1 A

across the haem

(Figure1c). According toPerutzs model, changes intertiary
structure propagate to the C helix and the FG segment,
which are involved in the a
1
b
2
interface. Because of steric
constraints, the subunits of Hb cannot adopt the tertiary
structure of oxy-Hb while they remain in the quaternary
arrangement of the T conformation. As successive oxygen
molecules bind to T-state Hb, strain accumulates in the
ligated subunits until the molecule snaps into the R
conformation, undergoing a quaternary rearrangement
at the switch region as described above. It has been
proposed that this quaternary shift must occur simulta-
neously at the a
1
b
2
and a
2
b
1
interfaces, because of the
rigidity of the a
1
b
1
and a
2
b
2
interfaces. A continuous
transition from the T to the R quaternary structure is
hindered by the stereochemistry of the switch region
(Figure 1d). The existence of two discrete, stable quaternary
structures of Hb, as proposed by Perutz, is consistent with
the concerted (MWC) model of cooperativity.
Ackers and co-workers (1992) have reasoned that
cooperativity in ligand binding in Hb A is mediated
throughinteractions at the intersubunit contacts withinthe
tetrameric Hb molecule. They have developed an experi-
mental approach to determine the free energies of subunit
assembly (dimers to tetramers) and cooperativity for each
of the 10 tetrameric species. They have observed three
distinct levels of free energy of dimertetramer assembly
for intermediate-state species, namely, 258.5 kJ for the
fully unligated species; 246.8 kJ for the singly ligated
species and the species with one ab dimer doubly ligated
and the other unligated; and 235.1 kJ for the fully ligated
species as well as the other three doubly ligated and two
triply ligatedspecies. Their data suggest that there is a third
allosteric structure during the cooperative ligation
process and that the T-to-R quaternary transition occurs
whenever the haem-site binding creates a tetramer with at
least one ligated subunit on each dimeric half-molecule
(a
1
b
1
and a
2
b
2
). Based on a systematic investigation, they
have proposed a molecular code for cooperativity in Hb.
Their model suggests that cooperativity arises from both
concerted quaternary switching and sequential modula-
tion of binding within each quaternary state, T and R
(Ackers et al., 1992).
Eaton, Mozzarelli, and co-workers have carried out
detailed studies on the oxygenation properties of crystal-
line Hb A in poly(ethylene glycol), i.e. where the TR
transition cannot take place, using a microspectrophot-
ometer (Rivetti et al., 1993; Bettati et al., 1996; Mozzarelli
et al., 1997). The conclusions from these studies are as
follows: (i) oxygen binding to the crystal of Hb A is
noncooperative; (ii) the achainhas ~5-foldhigher oxygen
anity than the b-chain; (iii) allosteric eectors, such as
chloride and inositol hexaphosphate (IHP), do not aect
the oxygen anity of Hb A crystals; and (iv) there is no
observable Bohr eect of crystals of Hb A. Similar
conclusions have also been reached from oxygen-binding
studies of the T-state of Hb A encapsulated in silica gels
(Bettati andMozzarelli, 1997). Thus, there are no allosteric
interactions without the TR transition. These research-
ers have concluded that their results are consistent with the
MWCmodel and Perutzs mechanism, but are inconsistent
with the KNF model and the molecular code proposed by
Ackers and co-workers.
Haemoglobin: Cooperativity in ProteinLigand Interactions
6 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
Table 1 The
1

1
interface contacts in T (Fermi et al., 1984), R (Shaanan, 1983) and R2 (Silva et al., 1992) conformations
The position of each amino acid residue in the helix or nonhelical segment to which it belongs is listed in brackets. The letters T, R or R2 printed
in a particular row and column of a table indicate a contact between the corresponding amino acid residues of the designated conformation: con-
tacts between (a) the B or C helix of
1
and the GH segment or H helix of
1
; (b) the GH segment or H helix of
1
and the B helix of
1
; (c) the
H helix of
1
and the C helix of
1
; and (d) the G-GH-H region of
1
and the corresponding region of
1
.
(a)
1

1
Phe122
[GH5]
Thr123
[H1]
Pro124
[H2]
Pro125
[H3]
Gln127
[H5]
Ala128
[H6]
Gln131
[H9]
Glu27[B8] T R R2
Glu30[B11] T R R2
Arg31[B12] T R R2 T R R2 T R R2 T R R2
Leu34[B15] T R R2 T R R2 T R R2
Ser35[B16] R T R R2 T R R2 T R R2
Phe36[C1] R2 T R R2
(b)

1
Glu26
[B8]
Arg30
[B12]
Val33
[B15]
Val34
[B16]
Tyr35
[C1]
Phe117[GH5] T R R2
Thr118[H1] T R R2
Pro119[H2] R R2 T R R2 T R R2 T R R2
Ala120[H3] T R R2
His122[H5] T R R2 T R R2
Ala123[H6] T R R2 T R R2
Asp126[H9] T R R2 T R R2
(c)

1
Pro51
[D2]
Met55
[D6]
Pro119[H2] T R R2 T R R2
Ala120[H3] T R R2
(d)

1
Glu101
[G3]
Asn108
[G10]
Val109
[G11]
Val111
[G13]
Cys112
[G14]
Ala115
[G17]
His116
[G18]
Gly119
[GH2]
Lys120
[GH3]
Phe122
[GH5]
Gln127
[H5]
Gln131
[H9]
Lys99[G6] R R2
His103[G10] T R R2 R T R R2 T R R2 T R R2 T R R2
Cys104[G11] T R R2
Leu106[G13] T R R2
Val107[G14] T R R2 T R R2 T R R2 T R R2 T R R2
Ala110[G17] T R R2 T R R2 T R R2
Ala111[G18] T R R2 T R R2 T R R2 T R R2 T R R2
His112[G19] R R2
Leu113[GH1] T
Pro114[GH2] T R R2
Ala115[GH3] T R R2
Phe117[GH5] T R R2 T R R2
His122[H5] T R R2 T R R2
Haemoglobin: Cooperativity in ProteinLigand Interactions
7 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
Table 2 The
1

2
interface contacts in T (Fermi et al., 1984), R (Shaanan, 1983) and R2 (Silva et al., 1992) conformations
The position of each amino acid residue in the helix or nonhelical segment to which it belongs is listed in brackets. The letters T, R or R2 printed
in a separate row and column of a table indicate a contact between the corresponding amino acid residues of the designated conformation: (a)
contacts between the C helix and CD segment of
1
and the corresponding region of
2
; (b) contacts between the H helices of
1
and
2
; (c) the
switch region; (d) the flexible joint region; (e) contacts between the C helix of
1
and the carboxy-terminus of
2
; and (f) contacts between the
carboxy-terminus of
1
and the C helix of
2
.
(a)

1
Arg40 [C6]
Thr41[C6] R R2
Tyr42[C7] T R R2
Pro44[CD2] R
(b)

1
Asp99[G1] Glu101[G3] Asn102[G4] Leu105[G7]
Asp94[G1] T R R2 T T R R2 T
Val96[G3] T R R2 T
Asn94[G4] T R
Leu100[G7] R
(c)

1
Cys93[F9] His97[FG4] Val98[FG5] Asp99[G1] Pro100[G2]
Pro37[C2] T R
Thr38[C3] R R2 R T R T
Thr41[C6] T R R2 T T T
Tyr42[C7] T
Pro44[CD2] T
(d)

1
Pro36[C2] Trp37[C3] Gln39[C5] Arg40[C6] Glu43[CD2]
Leu91[FG3] T R R2
Arg92[FG4] T R R2 T R R2 T R R2 T R R2 T R2
Val93[FG5] T R R2 T
Asp94[G1] T R R2 T
Pro95[G2] T R R2
(e)

1 Tyr145[HC2] His146[HC3]
Pro37[C2] T T
Thr38[C3] T R
Lys40[C5] T
Thr41[C6] T
(f)

1
Val34[B16] Tyr35[C1] Pro36[C2] Trp37[C3]
Lys139[HC1] R2
Tyr140[HC2] T R T R
Arg141[HC3] T T T T
Haemoglobin: Cooperativity in ProteinLigand Interactions
8 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
The Perutz mechanism implicitly assumes that the
structures of oxy-Hbanddeoxy-Hbpresent inerythrocytes
under physiological conditions are the same as the Rand T
structures determined by X-ray crystallography. However,
the observationof a thirdquaternary structure of Hb(Silva
et al., 1992) has cast doubt on the two-structure concerted
model for the oxygenation of Hb A. This new conforma-
tion, designated R2, has been seen not only in carbonmo-
noxy-Hb A in low salt conditions at pH 5.8 (Silva et al.,
1992), but also in cyanomet-Hb A crystallized under
physiological salt andpHconditions (Smith andSimmons,
1994). Thus, the R2 structure may be more relevant to Hb
function than is the R structure, which was crystallized at
high salt concentration (Shaanan, 1983). A detailed
geometric analysis of the three conformations suggests
that the R structure is an intermediate form, which lies on
the path between T and R2 (Srinivasan and Rose, 1994).
Figure1dillustrates the switch region of the a
1
b
2
interface in
the T, R and R2 conformations. Note the displacement of
b
2
His97 across one turn of the a
1
C helix in the T-to-R
transition, and its continuing motion across and away
from the helix in the R to R2 transition. Further studies,
using techniques suchas NMRspectroscopy, are neededto
determine whether the Ror R2 structure predominates for
oxy-Hb in solution, as well as the nature of the T structure
under physiological conditions.
A major feature of Perutzs model is the coupling
between the rearrangements of the haem and of the globin
structure, mediated by the proximal histidine (F8). It is the
movement of the proximal histidyl residue (whichbinds the
haem iron atom) to the centre of the porphyrin that pulls
the F helix away from its T-structure position, thus
initiating the tertiary and quaternary structural changes
of the Hb molecule. This model has been tested by deleting
the bonds between the proximal histidyl imidazole side-
chain and the polypeptide. This was accomplished by
expressing three proximal histidyl-detached recombinant
Hbs, rHb(aH87G), rHb(bH92G), and rHb(aH87G/
bH92G), using an Escherichia coli expression system(Shen
et al., 1993; Barrick et al., 1997). The ligand binding data
for these three rHbs show that the proximal histidyl
coupling mechanism contributes approximately two-
thirds to the total interaction energy between haems; there
must be alternative coupling pathways for the remaining
third (Barrick et al., 1997).
Other experimental evidence for multiple, conforma-
tionally distinct intermediates comes from
1
H NMR
spectroscopy. This technique can shed light on the
behaviour of Hb in solution under varied conditions by
following the response of assigned
1
Hresonance lines; here
their chemical shifts are reported relative to the methyl
proton resonance of the sodium salt of 2,2-dimethyl-2-
silapentane-5-sulfonate (DSS). Useful NMR lines include
the two hyperne-shifted proton resonances at ~23 ppm
(assigned to the b-haem) and at ~17 ppm (assigned to the
a-haem) as well as the exchangeable proton resonance at
~14 ppm (assigned to the intersubunit H-bond between
aTyr42 and bAsp99, a characteristic feature of the deoxy
(T)-quaternary structure of Hb A). From monitoring of
the intensity of these resonances, the following conclusions
can be made: (i) in the absence of organic phosphate, there
is no preferential O
2
binding to the a or b chains of Hb A;
(ii) in the presence of organic phosphates, such as 2,3-BPG
andIHP, achains have a higher anity for O
2
comparedto
b chains of Hb A; (iii) the ligand-induced structural
changes in the Hb molecule are not concerted; and (iv)
some cooperativity in the oxygenation process must be
present within the deoxy (or T) quaternary state during the
oxygenation process (Viggiano and Ho, 1979).
Another experimental approach using NMR spectro-
scopy involves the use of crosslinked mixed-valence
hybrids, in which tetramers with one or two cyanomet
chains serve as models for singly and doubly ligated species
during the cooperative oxygenation of Hb (Miura and Ho,
1982). From monitoring of the exchangeable proton
resonance at ~14 ppm and the hyperne-shifted proton
resonances of the cyanomet chain of these hybrid Hbs in
the deoxy and carbonmonoxy forms, the following
conclusions have been reached: (i) the T-quaternary
structure of the two singly ligated species in the deoxy
formis altered as manifested by a reduction of the intensity
of the T-structural marker at ~14 ppm, compared to that
of deoxy-Hb A; (ii) hybrid Hbs with two cyanomet chains
per Hb tetramer have no observable exchangeable proton
resonance at ~14 ppm, suggesting that they are not in the
T-quaternary state; and (iii) the structural changes of these
crosslinked mixed-valence hybrid Hbs as a function of
ligation are not concerted and cannot be explained simply
by two-structure concerted models (Miura and Ho, 1982).
Bohr Effect
The oxygen anity of Hb A depends on pH; that is, H
1
ions are heterotropic allosteric eectors in the oxygenation
of Hb (Figure 2). The higher anity of deoxy-Hb Afor H
1
ions as pHincreases above 6.5 leads to the thermodynamic
consequence of increasing oxygen anity with increasing
pH, with oxygenation of Hb resulting in a release of H
1
ions by the Hb molecule, known as the alkaline Bohr eect.
This physiologically relevant eect is fundamentally
important in the ability of the Hb molecule to deliver O
2
to the tissues, particularly to working muscles, where lactic
acid is produced. Below pH 6.5, ligated Hb has a higher
anity for H
1
ions, O
2
anity increases with decreasing
pH, and the Hb molecule absorbs H
1
ions upon
oxygenation, known as the acid Bohr eect. The Bohr
eect can be measured by two independent methods. First,
it can be determined from the dierence between the H
1
ion binding curves of HbO
2
(or HbCO) and deoxy-Hb.
Second, it canbe measuredfromthe change inO
2
anity as
Haemoglobin: Cooperativity in ProteinLigand Interactions
9 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
a function of pH. According to Wymans linkage relation-
ship, there is anexact relationshipbetweenthe change inO
2
anity and the number of H
1
ions released as a function
of pH (Wyman, 1964). The maximum number of H
1
ions
released per O
2
bound for Hb Ais ~0.5 at pH ~ 7.4 under
physiological conditions.
There are two schools of thought regarding the
molecular basis of the Bohr eect. One believes that there
are only a limited number of amino acid residues (such as
three to four per ab dimer) in the Hb molecule responsible
for the alkaline Bohr eect. The other believes that a large
number of amino acid residues in Hb can change their pK
values in going from the deoxy to the oxy form, and thus
are potential Bohr groups. In addition, any amino acid
residues that have a dierential anity for anions between
deoxy- and oxy-Hb could also contribute to the Bohr
eect, the so-called anion Bohr eect. In fact, anions, such
as chloride, phosphate, and 2,3-BPG, exert a profound
inuence on the Bohr eect. The implication of the second
model is that dierent sets of amino acid residues are
involved in the Bohr eect under dierent experimental
conditions and that electrostatic interactions in the Hb
molecule play an important role in the Bohr eect. There
has been considerable debate about these two alternative
models for the Bohr eect since the 1980s.
Alkaline Bohr groups can be classied into three
categories: (i) the imidazoles of surface histidyl residues;
(ii) the a-amino groups from the amino termini; and (iii)
other proton-binding sites whose pK values are signi-
cantly shifted into the physiological pH range owing to
unique structural and conformational arrangements in the
Hb molecule. There are 38 histidyl residues in Hb A, of
which about 26 are located on the surface of the molecule
and are likely to be involved in the Bohr eect.
1
H NMR
spectroscopy is ideally suitedtoinvestigating the molecular
basis of the Bohr eect because the C2 proton resonances
of the histidyl residues are usually separated from other
proton resonances, and are sensitive to the protonation
state of the histidyl residues. Using mutant Hbs (especially
recombinant mutant Hbs in which a specic histidyl
residue is replaced by a glutamyl residue) and chemically
modied Hbs, a large number of the C2 proton resonances
of surface histidyl residues have been assigned to specic
residues. From monitoring of the
1
H chemical shifts of
these C2 protons in the deoxy and CO states as a function
of pHinthe absence andpresence of various anions, the pK
values of these histidyl residues can be determined. The
recent experimental results can be summarized as follows
(Sun et al., 1997): (i) a large number of histidyl residues
contribute to the Bohr eect and some oppose the net Bohr
eect; and (ii) in some histidyl groups, the addition of
anions can diminish or reverse the contributions of specic
histidyl residues to the overall Bohr eect. The Bohr eect,
a heterotropic eect, depends on the intricate arrangement
and interactions of all hydrogen and anion binding sites in
the Hb molecule. It is an excellent example in which the
global network of electrostatic interactions, rather than a
fewspecic aminoacid residues in the Hb molecule, plays a
dominant role in an important physiological function.
Concluding Remarks
Haemoglobin is a truly remarkable molecule whose
oligomeric structure is designed in such a way that it can
facilitate the loading and unloading of appropriate
amounts of oxygen molecules that are needed for the
physiological functions of vertebrates. Accumulated ex-
perimental results indicate that perhaps no single, simple
model can account for all aspects of the structurefunction
relationship in Hb. Recent experimental results strongly
suggest that allosteric proteins, such as Hb, require
multiple pathways for signal communication. This point
is illustrated in the cooperative oxygenation of Hb and in
the Bohr eect. In spite of the extensive research eorts
devoted to the Hb molecule, the detailed molecular basis
for the cooperative oxygenation process remains contro-
versial (Ho, 1992; Rivetti et al., 1993; Srinivasan and Rose,
1994; Bettati et al., 1996; Edelstein, 1996; Barrick et al.,
1997; Bettati and Mozzarelli, 1997; Mozzarelli et al., 1997;
Sun et al., 1997; Ackers, 1998; Perutz et al., 1998).
Additional research is needed to understand the struc-
turefunction relationship of this interesting molecule. In
particular, if a detailed structure and structural changes
associatedwiththe oxygenationof HbAcanbe obtainedin
solution under physiological conditions, these data can be
directly compared with those obtained by single-crystal X-
ray diraction. The recent development of high-eld
multinuclear, multidimensional NMR techniques oers a
realistic possibility to determine the complete three-
dimensional structure of proteins of the size of Hb A and
their dynamic properties in solution (Gardner and Kay,
1998; Wu thrich, 1998). This opens up the possibility not
only of making a direct comparison between the crystal
and solution structures of Hb A but also of correlating the
information derived from kinetic, thermodynamic and
spectroscopic studies with that obtained from structural
investigations. Many of the controversial issues discussed
in this reviewmay then be resolved. Hence, haemoglobin is
an excellent example to illustrate how far we can correlate
the function of a protein molecule with its detailed atomic
structure in both solution and crystalline states.
Acknowledgement
The haemoglobin research in our laboratory is supported
by research grants from the National Institutes of Health
(HL-24525 and HL-58249).
Haemoglobin: Cooperativity in ProteinLigand Interactions
10 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
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Wyman J and Gill SJ (1990) Binding and Linkage: Functional Chemistry
of Biological Macromolecules. Mill Valley, CA: University Science
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Haemoglobin: Cooperativity in ProteinLigand Interactions
11 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net