PBIO-534 CELL & MOLECULAR PHYSIOLOGY

Structure/Function Relationships of Protein: Myoglobin & Hemoglobin

Myoglobin
Heme group/iron center: Iron in its Fe2+, ferrous, form giving it 6 positions for chelating, one of
which is available for binding oxygen. If the heme is doing the work, why do we need the protein?
Looking at the figure below, we see that there is a proximal histidine on the left that keeps the position
on the right available for Oxygen binding. Secondly, the abundance of alpha helix creates a hydrophobic
center. If you take heme and put it in water, it switches from its ferrous form and turns into its ferric
form, making it no longer capable binding oxygen. Therefore, creating this hydrophobic center keeps
water away from the heme group and maintains its oxygen binding ability. Lastly, the distal histidine
(the one shown above the heme ring in the figure on the right) also helps to stabilize oxygen in the ring
via H-bonding.

Equilibrium, Association, Dissociation constants and θ

These constants are derived from the equation

Mb + O2 ↔ MbO2

The equilibrium constant, Keq and the association constant, Ka, are equal to the ratio of formed
myoglobin bound to oxygen, over the concentration of the unbound oxygen and Myoglobin. The
Dissociation constant, Kd is the inverse of Ka which gives us a Molar value. This ration describes
the extent to which something wants to dissociate. A smaller number represents a strong desire to
dissociate.

There is also another way to describe the reaction of oxygen binding to myoglobin and that is
using θ/Y. This seems to be more intuitive.

[MbO2]
! = Y = [Mb] + [MbO
2]
PBIO-534 Myoglobin & Hemoglobin CELL & MOLECULAR PHYSIOLOGY

Theta/Y represents a numerical fraction of the concentration of Myoglobin bound to oxygen and
gives a simple hyperbolic curve. This fraction will range anywhere between 0-1 with one
meaning that myoglobin is completely saturated with oxygen and 0 means that no oxygen is
bound. At 50% bound, this is where Kd is and can also be called the P50 value. Myoglobin
would look something like this curve.

Now, let’s look at the way oxygen binds the heme group schematically. In the first figure we see
that Oxygen actually binds at an angle whereas CO, carbon monoxide, binds the heme group at
an affinity 25,000X higher than oxygen and does not bind at an angle.

One of the things that prevent CO from binding is something that

was mentioned earlier. The distal histidine group that helps stabilize
oxygen also prevents CO from binding at 90 degrees and instead forces it to bind at an angle.
This lowers CO’s affinity to bind heme to only 200X that of oxygen.

Hemoglobin
Hemoglobin is a tetramer made up of 4 subunits: 2 alpha subunits and 2 beta subunits.
Each subunit has the ability to bind oxygen and instead of seeing a simple hyperbolic curve, we
see a sigmoidal curve. This sigmoidal curve represents cooperativity and implies a multimer.
Also, proteins that are cooperative also tend to have lower affinities for their substrate than single
proteins and seen by the higher P50 of Hb than Mb. This is what makes them good carriers.

The graph above shows the difference in the pO2 from the lungs to the tissue is 66% for a
sigmoidal curve and only 38% for a non cooperative curve. This means that picking up oxygen
in the lung and dropping it off in the periphery would be done more efficiently by a protein that
exhibits sigmoidicity, like hemoglobin, vs. one that doesn’t.
PBIO-534 Myoglobin & Hemoglobin CELL & MOLECULAR PHYSIOLOGY

The two forms of hemoglobin

There are two forms of hemoglobin. The T
(taut) form AKA deoxyhemoglobin and the R
(relaxed) form AKA oxyhemoglobin. The T-form
represents the inactive, low-affinity, off state and the
R form represents the high-affinity, active, on state.
Now how do these structures come in to play when
talking about sigmoidicity? In the high affinity R-
state, it shows a hyperbolic curve and the P50 is very
low. In the low affinity T-state we see a very shallow
curve and the P50 is at a much higher value. When you
combine the two curves, we see that the transition
between these two structures is what gives us the
sigmoidicity that we see with Hemoglobin. Even more
so, the shape forces the function and is not just a
consequence of function.

Now let’s look at someone who is anemic. When someone has anemia that means that they only
have 50% of the hemoglobin that a normal person would have. Now, let’s compare them to
someone has 50% of the heme occupied by CO.

So why is it that an anemic individual will survive but

someone has 50% CO/Hb will die? The answer lies in the
graph. With anemia, we see that there is still some sigmoidal
character for their hemoglobin to bind to Oxygen. It is
obviously not as high as a normal person but it is still
sigmoidal. Whereas someone who has 50% COHb is almost
completely hyperbolic and the ability for hemoglobin to be a
carrier is no longer there. The Hemoglobin is in a high affinity
binding state due to the presence of CO and they can not let
go and will not drop off Oxygen in the periphery.

Models of Cooperativity
PBIO-534 Myoglobin & Hemoglobin CELL & MOLECULAR PHYSIOLOGY

The Concerted Model is also called the Equilibrium Model. This model states that once a T form
binds substrate, all subunits will switch to R. This is the model that hemoglobin would most
likely follow.

The Sequential Model is also called the Induced Fit Model. This model states that in the absence
of substrate, only T exists. In the presence of substrate, T is induced to the R form. Unlike the
concerted model, the subunits remain in T form but are more
easily changed.

Allosteric Regulation
1. 2,3-Bisphosphoglycerate
2,3 BPG is a negatively charged molecule that raises the
affinity of hemoglobin. It raises the P50 for binding oxygen from
1 to 26 torr. It interacts with Hemoglobin at its + charged
center. This allows communication between the subunits so 2,3
BPG helps to relay the message that Oxygen is bound.
Application: We can look at hemoglobin at high
altitudes and see how essential 2,3BPG is. At high altitudes and
no BPG, the curve turns hyperbolic and there would be no drop off of oxygen because it would
be permanently bound. However, when you have 2,3 BPG in the system, we see that at higher
concentrations (8mM vs. 5mM), we reestablish a Delta (0.37 at 4500m) that is similar to one that
we would have at sea level (0.38).

2. The Bohr Effect

The Bohr effect is shown by hemoglobin’s role in CO2 transport and changes in pH. Carbon
dioxide lowers the affinity for hemoglobin for Oxygen by lowering the pH of the environment
through its interaction with water.

CO2 + H2O → HCO3- + H+

Looking at the chart, in an environment that is higher in pH and no CO2, like 7.4 (lung), Hb has a
higher affinity for oxygen. But in environments that are lower in pH and have CO2 present
(tissue), Hb has a lower affinity for oxygen and will drop it which is what we want. Also, the
protonation of Hb stabilizes its T state and deprotonation of Hb stabilizes its R state.
Pregnancy
Fetal Hb has a higher affinity for oxygen which makes sense because the fetus must be
able to pull blood from the mom’s blood through the placental interface. It is slightly
different than a mother’s Hb and has a lower affinity for 2,3 BPG because it has a more
positive, less negative pocket. This too makes sense because it also gives fetal Hb a
higher affinity for Oxygen than Mommy’s Hb allowing transfer from mother to fetus.