ALLOSTERY IN HEMOGLOBIN

BY DR S. M. BHATT (Asst. prof. University, Punjab India, drsmbhatt@gmail.com)

The term “allosteric” has been used to describe a ligand enzyme interaction, which results in a
measurable conformational change in proximal and distal regions of that protein. Cooperativity was
originally found by C. BOHR in hemoglobin, he observed the sigmoid binding curve of O2 to hemoglobin,
which he explained by saying that the binding of the first O2 molecule made it easier for the next O2 to
bind and hence could be called “cooperative.” THERE are two type of cooperativity positive and
negative.
This is a type of regulation mechanism through which eukaryotic cell can regulate the rate of
formation of product by regulating the binding of substrate. This is a life saving mechanism as in
hemoglobin in which availability of oxygen confers more binding of the oxygen. This is termed as
Positive cooperativity which confers the metabolic advantage of amplifying the sensitivity of a signal,
i.e. a small change in ligand L can have a far greater effect on the output response in a positively
cooperative system than in a Michaelis-Menten system. For example, a 3-fold increase in the ligand (L =
O2) concentration for hemoglobin changes the binding capacity 9-fold (from 10 to 90%), whereas in a
Michaelis-Menten system, it requires an 81-fold change in ligand concentration to go from 10% binding
capacity to 90% binding capacity. It can be understood since ligand concentration in michalis is S while in
case of cooperativity (S)N so if conc. Is 3 it will be (3)4 . Adair and Hills plot gives clearer picture of these
facts.
In general, cooperative interactions occur when ligands bind to proteins or protein complexes. If
the binding of ligands is regulated by different ligands at sites other than the heme, then we call this
allosteric interaction. Ligands function as effectors or modulators at different sites in the protein. If the
ligands are identical, this is known as homotropic effect, if they are different, it is known as heterotropic
effect. Molecular oxygen is a positive, homotropic effector for hemoglobin. The protein also exhibits
heterotropic effects which induce mostly negative cooperativity, i.e., they decrease the affinity of O2 to
hemoglobin. For example the generation of CO2 in the muscle and its conversion into bicarbonate
releases protons. These protons bind to Hb and reduce its affinity for O2 by stabilizing the T state. This
effect is known as Bohr Effect. In addition, CO2 binds reversibly to hemoglobin at its NH 2 terminal end to
form carbamates (R-NH-COO-). CO2 binds better to the N-terminal end of deoxyHb, and once bound,
stabilize the T state.
Oligomerization enables cooperativity between multiple binding sites, a property referred to as
allostery or allosteric regulation of enzymes. Protein complexes have defined quaternary structural
symmetries, if they are homo-oligomeric protein complexes where all subunits are identical. The
quaternary symmetry is pseudo-symmetries if the complex is a hetero-oligomer. Homo- and hetero-
oligomeric compositions allow for an additional regulatory hierarchy at the cellular, tissue, or organism
level, where different combinations of subunits of heteromeric complexes, due to cell specific gene
expression.
Oxygen binding to hemoglobin, where the first oxygen has four different sites that it can bind to.
This shows a relatively higher entropy compared with the binding the last oxygen will have, which has
only one site left that will bind. In going from an unbound to a bound state, the first oxygen must
overcome a larger entropy change versus the final binding oxygen. This entropy difference is the main
reason for the positive cooperativity in binding oxygen to hemoglobin.
 ADAIRS EQUATION

The four binding sites in hemoglobin show positive cooperative binding, meaning that after the
first binding site is occupied by an O2 molecule, the 3 remaining sites exhibit a largely increased affinity
for O2 molecules.

Fig.1 Positive cooperativity of binding of oxygen to hemoglobin

When O2 binds, it promotes the T R switch and it promotes more O2 binding. This happens best
at higher pO2, as occurs in the lungs When O2 is released, it promotes the R -------> T switch and it
promotes more O2 release. This happens best at lower pO2, as occurs in the peripheral tissues. It is
important, for example, that hemoglobin picks up the maximum amount of O2 in the lungs and unloads
the maximum amount of O2 to the tissues, a biological phenomenon for which positive cooperativity is
very important. In fact, humans with hemoglobin mutations that lack the cooperativity are very sick
people.

HILLS PLOT

To understand cooperativity of O2 binding we have to define the fractional saturation, YO2, of globin with
O2 molecules. The fractional saturation can experimentally be determined by measuring the partial
oxygen pressure, pO2, in solution. The fractional saturation and the partial oxygen pressure are related
as shown in this equation:
Y(O2) =
p(50) is defined as partial pressure of O2 to upon 50% occupation of all heme binding sites by ligands.
The cooperativity factor is indicated by the factor n; for hemoglobin n = 3; for myoglobin n = 1.
 MECHANISM OF COOPERATIVITY OF O2 BINDING

Hemoglobin is a tetrameric protein complex composed of two alpha and two beta subunits with
an overall subunit stoichiometry of α2β2. The hemoglobin tetramer is a spheroidal protein complex with
the dimension 6.4 x 5.5 x 5.0 nm. The tetramer has a pseudo-D2 symmetry and the association between
subunits is stabilized by hydrophobic interactions as well as hydrogen bonds. The overall sequences and
structures of the hemoglobin subunits are homologous to myoglobin. Their arrangement into a
tetramer, built from the association of two αβ dimers has important consequences with respect to
oxygen binding affinity and regulation. Hemoglobin contains 4 heme groups, all of which are bound to
each individual subunits.
The structural changes in Hb
subunits can be analyzed and a kinetic
model for those structural changes has
been described. In this model, the
quaternary structure of hemoglobin with
no molecular oxygen bound, deoxy Hb, is
designated as T-state and the fully
oxygenated form, oxy Hb, as R-state.
Similarly the subunit forms are
designated t and r states.
It is important to understand that
the occupancy state of one heme cannot
be sensed by another heme by direct
electronic interaction. They are too far
away (>2.5nm). The interaction is a
mechanical one, transmitted through the
conformational change induced by the
binding of one O2 to a heme and the
consequent displacement of the F-Helix.
This helix displacement brings
upon a change in the quaternary
structure such that the dimer (a1b1)
rotates 15° relative to the dimer. The
dimers α1-β1 and α2-β2 relative subunit
orientation is unaffected. If two or three
sites are vacant, the cumulative force may
be sufficient to allow the switch back to the T-state. The binding of the first O2 to any of the hemes
induces a shift in the corresponding F-helix. This structural change within a subunit is sensed by the
neighboring subunits in a way that the binding of O2 to their hemes is favored as compared to the initial
conformational situation. Thus, the binding of a first O2 induces a decrease in the binding energy for
subsequent O2 binding in the other three subunits. This mechanism is called cooperative effect.
It is the movement of the Fe(II) towards the heme plane that triggers the T to R conformational
change. The relative displacement between subunits has only two stable conformations, i.e., the T and
the R state. Intermediate positions are energetically unfavorable, therefore the change in quaternary
structure provides two conformational endstates, one with a low O2 affinity, the other with an increased
affinity to the heme groups. These two states are stabilized by two energetically equivalent sets of
hydrogen bonds at the subunit interfaces. After the binding of the first oxygen, all subunits are
converted from the t to the r state whether or not they bind a ligand.
Holt et al. 1992 using spin labels and NMR have shown clear examples of a stepwise change in
the case of hemoglobin structure that is in agreement with the KNF model but also shows a switch
between two quaternary structures, which is in agreement with the MWC model. Perutz 1998, an initial
advocate of the MWC model for Hb, has recently said the changes induced by oxygen indicated that
both the MWC model and the KNF model are partly right, and Holt and Ackers 1995 have proposed a
sequential model with a switch in quaternary structure at the 2O2 bound state. In the case of aspartyl
transcarbamoylase, Stevens and Lipscomb 1992 showed a combination of sequential intrasubunit
conformational changes on a broad background of a quaternary change.