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Hemoglobin 1
General Properties of Complex
Binding
Function - Structure
Relationships
Review of last lecture - myoglobin structure
We noted that the oxygen binding required an unusual symbiosis of an inorganic ion (Fe 2+)
and a non-proteinaceous hydrophobic group (protoporphyrin IX) and the protein chain.
Oxygen binding by Fe2+ is not very strong and even if it were strong would be of not much use
as an oxygen carrier since it would diffuse rapidly to regions where it was not needed or be cleared
by the kidney.
So the Fe2+ ion associates to the protoporphyrin IX - this ultimately becomes the prosthetic group
of the protein.
Question 1: Why would a Fe2+ ion bind to a heme group?
Answer: The Fe2+ has the ability to produce a d2sp3 octahedral hybrid for electron pairs from donors
to fill. The heme group can provide 4 electron pairs in this way, leaving two orbitals empty
and ready for other donors.
However, in aqueous solution the free heme group has very low affinity for oxygen.
Question 2: If there are two orbitals left in the hybrid to be filled why does not oxygen bind to these
two sites?
Answer:
It is likely that the oxygen of the water molecule can effectively compete with oxygen
to donate electron pairs to the empty Fe2+ orbitals because the molar concentration of water
is enormous.
We noted in our last lecture that despite differences in the primary structure, myoglobin and the two
hemoglobin chains folded in the same fashion as eight helical segments A - H
Therefore, a hydrophobic cavity is present and heme being largely hydrophobic will have
a tendency to bind into the pocket.
But many proteins have hydrophobic cavities so what is so particular about this one that
causes heme to bind with high affinity?
Role of the conserved histidines:
The cavities to which heme binds in myoglobin and hemoglobin have two conserved histidines
one in the E-helical segment (distal histidine) and one in the F-helix (proximal histidine).
Proximal Histidine. One of the N atom of the imidazole ring of the proximal histidine donates a lone pair
to the heme anchoring it in the cavity and leaving one remaining empty orbital to be filled.
The net effect of the binding into the hydrophobic cavity is the heme is now shielded from
the aqueous medium by the protein and oxygen can diffuse and reach it to donate its electron pair.
Distal Histidine. This histidine does not form any coordination bond with the heme but it is close
enough to prevent the linear oxygen molecule to attach perpendicularly to the plane of the heme.
In this way, the oxygen binding is weakened slightly to allow for its displacement when the free
oxygen concentration falls to a critical value.
This effect on the oxygen binding by the distal his is not so apparent
in the stick and ball model representation shown above.
distal his
It is more obvious in this space filling representation.
Hemoglobin. Tetrameric protein made of four subunits (chains) a2b2 made up of two
ab dimers. The quarternary structure shows that these subunits are arranged
tetrahedrally. The molecule is essentially spheroidal (64 x 55 x 50 Å)
The binding sites are the four hemes associated with each subunit
From the oxygen binding studies we know that the binding behavior is complex.
That is the direct plot of oxygen binding by hemoglobin is sigmoidal and not
hyperbolic.
This signifies that the sites while identical are not independent, meaning that the
binding of the first oxygen facilitates the binding of the next oxygen to the molecule.
Normally, if there is going to be dependence between the sites this usually signifies
a direct effect implying that the sites are close enough to affect one another.
We can examine the structure and measure the distances between the Fe atoms of the
hemes by using the DTMM modeler (make sure the auto valency in the toggle menu
(style) is unchecked) and then deleting the protein chains leaving the hemes and some
water molecules . Then by using the calculate menu we can get the distances.
If the hemes are too far apart to influence the binding of one another,
then how does this dependency occur?
oxy-hemoglobin
deoxy-hemoglobin
Binding of oxygen produces a change in the position of the F-helix in the subunit and this
propogates and this possibly alters the interactions between the other subunits thereby
altering their conformations slightly. In some fashion this makes the remaining hemes
easier to bind too.
If the hemes are too far apart to influence the binding of one another, then how does
this dependency occur?
We have earlier examined how we can analyze the binding data using the Hill analysis,
but as we saw then, the Hill mechanism is not a valid molecular model. Why not?
The mechanism for binding is that c oxygen molecules all bind at the same time or dissociate
at the same time:
Hb (O2)c = Hb
+ c O2
Since c does not have to be integral, this is not a valid molecular mechanism but it does
provide a way to analyze cooperative binding.
The Hill equation and the graphical representation allows for the determination of c,
the Hill coefficient, from the slope and the dissociation constant, Kd, for the above
mechanism from the y axis intercept.
log Y = - c log [B50] + c log [B]
1-Y
There are two molecular models that can explain the cooperative binding effect:
1. The sequential model
2. The concerted model
The sequential model for the binding suggests a conformational change in the subunit containing the
heme binding the oxygen and as a consequence, this change is propogated to the other subunits which
have no oxygen bound to them but these changes increase the affinity for subsequent binding of oxygen
to the other sites.
Hb + O2
= Hb O2
Hb O2 + O2 = Hb (O2)2
Hb(O2)2 + O2 = Hb (O2)3
Hb(O2)3 + O2 = Hb(O2)4
k1
k2
k3
k4
In this model, the intrinsic binding constants, k’s (while identical initially) change as oxygen loads
one at the time to the hemoglobin.
Recall, that we have to represent these as dissociation reactions and, therefore, the equilibrium
reactions should be shown as the reverse of those given above.
The concerted model is very different in that it can explain the sigmoidal direct curve by
assuming that the sites are identical and independent.
This seems to be defying what we know about the anticipated behavior for multiple identical
and independent sites, since we know that this type of binding gives hyperbolic and not
sigmoidal direct plots. If the intrinsic equilibrium dissociation constants are the same and do
not change with binding, then how can one get a sigmoidal direct plot?
The reason we can get sigmoidal binding behavior is that we make the assumption that
the protein exists in two different conformations in equilibrium with one another.
Furthermore, we also assume that the ligand (oxygen) binds preferentially only to one form.
Ro = To
Therefore,
L =
[To]
[Ro]
where the subscript o indicates the equilibrium when there is no ligand (in this case oxygen)
present. L is called the allosteric constant.
We assume that binding can only takes place to the R form and that the binding to the sites
in R occurs as independent and identical.
Let the intrinsic dissociation constant for the binding of ligand to any of these sites be
kR
With this information you must be able to derive the binding function relating Y to the
concentration of free ligand [B], the intrinsic dissociation constant, kR, and the allosteric
constant L.
The function is:
Y = a(1 + a) / {(1 + a)2 + L}
where a = [B] / kR
Some features of oxygen binding by hemoglobin:
1. Only the ferro-form binds oxygen - the ferri-form sometimes referred to as
methemoglobin or metmyoglobin does not bind oxygen.
2. Carbon dioxide does not bind to the heme group - most of it is in the form of
dissolved CO2 ( and H2CO3) or in a modified form involved in the carbamylation
of the a-NH2 of the chains.
3. Carbon monoxide can bind to ferro-hemoglobin - the binding is strong but because
of the distal histidine the CO must bind at an angle and this is thought to lower its
affinity somewhat.
4. Oxygen-binding causes a red shift in the spectrum of the bound heme. This spectral
change in the visible range can be used to measure the amount of oxygen bound.
5. The binding of oxygen is affected by effectors such as H+ (pH) and this is called
the Bohr effect.
6. The binding is also affected by certain small organo phosphate molecules such
as 2,3-bisphosphoryl glycerate (BPG)
Rotation of the molecule reveals a large cavity