Download Reading - Chapter 15 and the Appendix to Chapter 15

Biochemistry 462a – Hemoglobin Structure and Function
Reading - Chapter 15 and the Appendix to Chapter 15
Practice problems - Chapter 15 - 5,7; Proteins extra problems
Myoglobin and Hemoglobin







Oxygen is required for oxidative metabolism and energy production in most cells.
Oxygen is used to oxidize dietary substrates to carbon dioxide, yielding energy.
Vertebrates use hemoglobin (Hb) to transport O2 to tissues from lungs (or gills) and to
transport CO2 from tissues to lungs (or gills).
Mb is used in some tissues, notably muscle, as a storage reserve of O2.
Mb is a monomeric protein, while Hb is a tetramer.
However, each subunit of Hb has a tertiary structure very similar to the tertiary structure of
Mb.
In both Mb and Hb, the oxygen is carried bound to a iron-porphyrin complex called HEME.
The IRON (blue in figure below) is in the ferric state (Fe+2) state. OXYGEN (red in figure
below) binds reversibly to the Fe+2 without causing oxidation of the iron to Fe+3. Mb or Hb
in which the iron has been oxidized chemically to Fe+3 does not bind oxygen reversibly.
Deoxy Heme



Oxy Heme
The heme group is buried within a hydrophobic pocket in the protein.
The iron is coordinated to a histidine, THE PROXIMAL HISTIDINE.
The hydrophobic character of heme-binding pocket is responsible for the lack of oxidation of
Fe+2.
1
Oxygen Binding to Myoglobin

The binding of O2 to Mb follows a simple HYPERBOLIC saturation curve. The fractional
saturation, , is defined by these equations.

The partial pressure of O2, PO2, is the fraction of the gas phase that is O2 and P50 is the partial
pressure of O2 required to give 50% saturation. Note that [Mb] = [free myoglobin] and [MbO2] = [myoglobin that has bound O2].
Oxygen Binding to Hemoglobin






The O2 saturation curve of Hb is
SIGMODIAL.
Such a sigmodial saturation curve is
diagnostic
of
a
COOPERATIVE
INTERACTION between the ligand, O2,
and the protein.
Initially, Hb is in a low affinity T-STATE.
Binding of O2 causes a conformational
change in the Hb, which converts it to the
high affinity R-STATE.
Thus the sigmodial saturation curve is a
composite of a low affinity and a high
affinity curve.
The cooperative binding of O2 to Hb is an
example of an ALLOSTERIC EFFECT,
in which the binding of one ligand to a
protein influences the affinities of the
remaining unfilled binding sites.
2
The Hill Plot

The Hill equation describes the binding of n molecules of O2 to Hb by an equation similar
to the one shown above:
.

A linear form is given by

A plot according to this equation can be used to assess cooperativity in binding - the Hill
Coefficient, n.

For Mb, such a plot yields a straight line
with n = 1.0
For Hb, the plot is not a straight line, but
composed of three parts:
At low and high pO2 the plot has a slope of
1.
o These represent conditions where
the first (T state) or last (R state) O2
are being added.
o Under these conditions the Hb
behaves as through it had a single
binding site.
At intermediate values of pO2 there is a
slope > 1. This represents the region of
cooperative binding.
o The value of the slope pO2 = p50
gives n.
o For Hb this is about 3.5.
The maximal possible value for n would be
4, the number of subunits, but the Hill
coefficient is always less than this maximal
value.




3
The Significance of the Sigmodial Oxygen Binding Curve
The significance of the sigmodial O2
saturation curve of Hb can be appreciated
from the graph to the right.
While both Mb and Hb will be saturated
with O2 at the partial pressure of O2 in the
lungs, only hemoglobin will release
significant amounts of O2 at the partial
pressure of O2 present in the tissues.
In fact, the O2 released by Hb can then be
taken up by Mb for O2 storage in those
tissues, such as muscle, that have
significant amounts of Mb.
Hemoglobin Structure and Cooperative O2 Binding

The differences in O2 affinity between T-State (deoxy) and R-State (oxy) Hb can be
understood in terms of the changes in quaternary structure that accompany the
conversion of deoxy Hb to oxy Hb.
o The shift from the deoxy to oxy conformation arises from the fact that in deoxy Hb
the iron lies out of the plane of the heme ring, but when O2 binding occurs, the iron
moves into the plane of the heme ring.
o Because the proximal His is bound to the Fe, it moves also, causing the helix in which
it is found to move.
o This movement alters the structure of each subunit, which in turn alters the interaction
between subunits at the interfaces.
o This ultimately leads to the shift from the deoxy to the oxy conformation.
o Because of the way in which the subunits are packed together, one subunit cannot
shift from the deoxy to oxy conformation without causing the others to change also,
even though the other subunits have not bound O2.
o Once the other subunits have shifted to the oxy conformation, the affinity of the
unoccupied sites for O2 increases because there are no steric blocks to O2 binding,
i.e., the conformational changes have already taken place.
4
The Bohr Effect








In the tissues, the pH is lower due to the
presence of CO2.
.
The lowered pH causes Hb to lose O2.
This is known as the BOHR EFFECT and
increases the delivery of O2 to the tissues.
The origin of the Bohr effect lies in the fact
that deoxy Hb is a weaker acid than oxy
Hb:
A major contribution to the Bohr effect
involves the C-terminal His of each subunit.
In the deoxy state, this His forms a salt bridge
to Asp 94, if the His ring is protonated.
The salt bridge stabilizes the protonated form
of the His, causing a high pKa, in the deoxy
state.
The salt bridge does not form in the oxy state.
Thus protonation of His-146 favors the deoxy
conformation.
Carbon Dioxide Transport

The presence of CO2 in the tissues alters the affinity of Hb for O2 in two ways:
1. Through lowering the pH - the Bohr effect
2. Through the formation of carbamates by the amino terminal amino groups of
Hb:

The carbamation reaction releases H+, which favors the Bohr effect and the negatively charge
introduced allows formation of additional salt bridges, but only in the deoxy state. Thus,
carbamation favors the deoxy state.
5
Bisphosphoglycerate



The red blood cell contains high concentrations of bisphosphoglycerate (BPG),
which is an ALLOSTERIC EFFECTOR of Hb's affinity for O2.
BPG binds strongly to the deoxy form of Hb, but only weakly to the oxy form.
Thus, BPG favors the deoxy conformation.
The importance of bisphosphoglycerate (BPG) as an allosteric regulator of hemoglobin's
O2 affinity
High altitude adaptation
Adaptation to high altitude is a complex
physiological process that involves many
events.
One event that occurs within 24
hours is an increase in the content of
BPG in the erythrocyte.
The effect of the increased
concentration of BPG is to reduce
the affinity of hemoglobin for O2,
which increases the efficiency of O2
delivery to tissues.
6
Fetal respiration
The fetus obtains O2 by diffusion across the
placenta.
This diffusion is aided by the fact that fetal
blood has a higher affinity for O2 than does
maternal blood.
Fetal hemoglobin (HbF) is a tetramer that
contains two and two  chains.
The  chains in HbF, which replace the
chains, have a low affinity for BPG.
Thus, HbF has a higher O2 affinity than does
maternal HbA.
The Physiological Transport of O2 and CO2

How do all these allosteric effects combine to allow Hb to carry O2 from the lungs (gills)
to the tissues and to carry CO2 from the tissues to the lungs (gills)?
o As shown below, Hb stripped of all its allosteric effectors has too high an affinity for
O2 to allow effective transport of O2 to tissues.
o However, the presence of both CO2 in the tissues and BPG in the red blood cell create
a situation in which O2 is efficiently transported from lung to tissue.
7
We can summarize these events as follows:
1. In the lungs the partial pressure of O2 is high, which overcomes any negative allosteric
effects and causes complete oxygenation of Hb.
2. As Hb-O2 enters the tissues the presence of CO2 and a lowered pH combine to favor the
deoxy conformation and release of O2.
3. The presence of BPG aids the delivery of O2 by favoring the deoxy conformation.
4. Deoxy Hb binds CO2.
5. The deoxy Hb returns to the lungs where the pH is higher, the O2 content higher and the CO2
content is lower. All these factors favor reverse of the carbamation reaction, deprotonation
of His 146 and the formation of oxy Hb.
Mutant Hemoglobins









Several hundred mutant Hemoglobins are known to exist.
In most, a single amino acid replacement occurs in either the or  chain of normal Hb A.
Many of these changes cause no known effect, but several lead to pathologies associated with
abnormal O2 transport.
First, lets look at HbS or sickle cell hemoglobin.
In HbS there is a single amino acid replacement of a Val for Glu at position 6 of the chain.
This seemingly innocuous change places a hydrophobic sidechain on the surface of the
protein.
In the deoxy conformation the Val sidechain of a  chain in one HbS binds to the  chain of
another HbS.
This leads to polymer formation and precipitation of the deoxy HbS.
This leads to red cell lysis and anemia.
Mutant Hemoglobins - what can be learned from experiments of nature?






Mutant hemoglobins provide unique opportunities to probe structure-function relations in
a protein.
There are nearly 500 know mutant hemoglobins and >95% represent single amino acid
substitutions.
About 5% of the population carries a variant hemoglobin.
Some mutant hemoglobins cause serious illness.
The structure of hemoglobin is so delicately balanced that small changes can render the
mutant protein nonfunctional.
The following images were generated using hemoglobin A (2hhb.pdb, C. Fronticelli,
unpublished and 1 hho.pdb, B. Shaana, J. Mol. Biol. 17 31 (1984)), and the SwissPdbViewer to introduce the various mutations. Thus, the images are not derived from
actual crystal structures but represent approximations to what those structures would
probably look like.
8
Mutations that lead to loss of heme.






The heme group can be dislodged by mutations in
the heme-binding pocket.
In Hb Hammersmith [Phe(42)Ser] the Phe in
Hb A at position 42 in the  chain is converted to
Ser.
In Hb A the Phe blocks access of water to the
heme pocket.
But the smaller polar Ser allows water to enter
the heme pocket.
This causes the heme to be dislodged from the
pocket.
This produces a nonfunctional molecule.
Mutations that disrupt the tertiary structure of a subunit.




In the  chain there are two helices (Red and Green below) that pass so close together that
there is only room for the H sidechain of Gly between them.
In Hb Savannah [Gly(24)Val] the presence of the Val sidechain forces the two helices
apart.
This leads to disruption of the tertiary structure of the  chain.
In turn, this leads to an unstable hemoglobin molecule.
This is Hb A with Gly
This is Hb Savannah with Val
9
Mutations that stabilize Methemoglobin





In order for hemoglobin to
reversibly transport O2 the iron
must remain in the ferric (Fe+2)
state.
Oxidizing the iron to Fe+3 produces
methemoglobin, which does not
transport O2.
All known methemoglobins arise
from mutations which provide a
negatively charged oxygen atom as
a ligand for the iron.
The negatively charged
oxygen stabilizes iron in the
Fe+3 state.
This is Hb Milwaukee
[His(87)Glu] in which the
distal His, which normally
protects the iron from
becoming oxidized has been
replaced by Glu, which
stabilizes the Fe+3 state.
Mutations that change the stability of the R State or the T State.



Mutations at the 1-2 interface often interfere with the quaternary structure of
hemoglobin.
Such mutations can change the relative stabilities of hemoglobin's R and T states, thereby
affecting the O2 affinity of the mutant hemoglobin.
Hb A has a p50 = 26 mm Hg.
10










In Hb Kansas [Asn(102)Thr] a
critical hydrogen bond in the 1-2
interface between 2-Asn(102) and
1-Asp(94) that stabilizes the R state
is lost.
This means that hemoglobin remains
in the T state.
This mutant hemoglobin has a low O2
affinity (p50 = 70 mm Hg).
There is little cooperativity in O2
binding (Hill coefficient = 1.3).
The 1subnit is red and 2 subunit is
green.
In Hb Yakima [Asp(99)His] a
critical hydrogen bond in the 1-2
interface between 2-Asp(99) and 1Tyr(42) that stabilizes the T state is
lost.
This means that hemoglobin remains
in the R state.
This mutant hemoglobin has a high O2
affinity (p50 = 12 mm Hg).
There is no cooperativity in O2
binding (Hill coefficient = 1.0).
The 1subnit is red and 2 subunit is
green.
Crocodile Hemoglobin
Crocodiles can remain under water for up to one hour and usually kill their prey by drowning
them.





How do the tissues of the crocodile get O2 while submerged?
Unlike deep diving whales, which use myoglobin to store O2 in muscle, crocodiles rely
on a unique allosteric effector of Hb to ensure delivery of O2 to tissues.
While submerged, metabolism produces CO2, which is converted to HCO3- and it is the
HCO3- that acts as the allosteric effector by binding to residues at the 12 interface in
deoxy Hb and stabilizing the deoxy Hb.
This unique allosteric effect is only found in crocodile Hb.
Crocodile Hb does not bind BPG due to mutations in the BPG-binding pocket.
11