Download Hemoglobin - Mercer University

Hemoglobin:
the “molecular lung”
by
Thomas A. Huber
Department of Biology
Mercer University
The red blood cells (erythrocytes) in our first story in lecture are about 33% by weight a
single protein: the tetrameric protein hemoglobin. Hemoglobin (Hb) is part of a
sophisticated O2 delivery system that transports O2 to the tissues of our bodies via the
vascular system and also transports CO2 away from those same tissues. The presence of
Hb increases the amount of O2 that can be transported per liter of blood about 100-fold.
Similarly to how we breathe into our lungs O2-rich air and breathe out CO2-enriched air,
the Hb protein alternates between an O2-enriched form and a CO2-enriched form.
Therefore, it has been called the “molecular lung”. Structurally, Hb is a heterotetramer;
it has four (tetra-) subunits (-mer) and they are not all identical (hetero-). More
specifically, Hb is a “homodimer of heterodimeric polypeptides” – its subunit
designation is best written as (αβ)2, but is often given as α2β2. The α and β subunits are
structurally and evolutionarily related to each other and to the monomeric protein
myoglobin (Mb). Your goal in this recitation is to improve your understanding of how
the structure of Hb is related to its functions of O2 and CO2 binding and transport.
Before coming to recitation, you should read pages 941-943 in your text and go to the
following two web site pages; this should all take about 20-25 minutes:
https://www.youtube.com/watch?v=LWtXthfG9_M
http://biochem.web.utah.edu/iwasa/projects/hemoglobin.html
You learned in BIO 211 that one way to analyze the interaction between an oxygenbinding protein – like hemoglobin or myoglobin – and O2 is by looking at oxygen
dissociation curves. Since myoglobin is a monomeric protein with a single heme group
it can only bind one O2 molecule at a time. The association reaction for this is given at
the top of the next page:
Mb  O2  Mb  O2
Ka
However, Hb is a tetrameric protein and can bind up to four O2 molecules at a time. The
association reaction (assuming that all four O2 bind or associate simultaneously and with
the same affinity) would be as follows:
Hb  4 O2  Hb  O2  4
Ka
Given the above reactions, how would we write the equations that express the value of
each association constant? Write your answers in the space below.
For myoglobin:
For hemoglobin:
Ka 
Ka 
Answer:
Answer:
The shapes of the oxygen dissociation curves of Mb and Hb result from the fact that the
ability of each protein to bind O2 depends on (1) the structure of the protein, (2) the
structure of O2, and (3) the partial pressure of O2. The oxygen dissociation curves for
Mb and Hb are shown in Figure 1.
Figure 1. Oxygen dissociation curves
for the proteins myoglobin and
hemoglobin. The curve for Mb is a
rectangular hyperbola; the curve for
Hb is sigmoidal. For Mb, P50 equals Kd
(= 1/Ka); for Hb, P50 would be equal to
the product of the four individual Kd
values, one associated with each
subunit. The figure is taken from
http://www.colorado.edu/intphys/Class
/IPHY3430-200/image/figure13g.jpg .
From the two oxygen dissociation curves in Figure 1, it is clear that at any one partial
pressure of O2, Mb is more or
less (circle one) saturated than Hb. This means that
myoglobin’s affinity for O2 is higher or lower (circle one) than hemoglobin’s
affinity for O2. We might conclude from this that when comparing two oxygen
dissociation curves, the one with the greater value of P50 indicates a protein or
circumstance of higher or lower (circle one) affinity for O2.
Not only is there a difference in the affinity for O2 between Mb and Hb, but the shapes
of their oxygen dissociation curves are quite different (see Figure 1). Myoglobin’s shape
is a rectangular hyperbola; hemoglobin’s shape is sigmoidal. This difference in shape is
a result of hemoglobin’s subunit positive cooperativity. That is, when one subunit of Hb
binds an O2 molecule, it makes it easier for the next subunit to bind an O2 molecule.
Similarly, when one subunit releases an O2 molecule, it makes it easier for the next
subunit to release an O2 molecule. Another way of stating this is to say that the affinity
of Hb for O2 increases as it binds O2 molecules or decreases as it releases O2 molecules.
Positive cooperativity is indicated by (1) increasing values of Ka (association
equilibrium constant) for the binding of subsequent O2 molecules, (2) increasingly
exergonic O2-binding reactions, and (3) sigmoidal dissociation curves.
Allosteric regulators of hemoglobin: DPG
Figure 2 shows the effect of increasing concentrations of 2,3-diphosphoglycerate (DPG
or BPG) on hemoglobin’s oxygen dissociation curve. This allosteric regulator was
discussed on a web site page you reviewed before completing this recitation.
Figure 2. The effect that an
increasing concentration of 2,3DPG has on the oxygen
dissociation curve of hemoglobin.
One molecule of 2,3-DPG can
bind to one Hb tetramer, but only
tightly if Hb is in the T state.
Therefore, 2,3-DPG stabilizes the
T state of Hb and makes it easier
to unload oxygen gas. The figure
is from
http://www.coheadquarters.com/P
ennLibr/MyPhysiology/lect5p/lec
t5.04.htm .
At lower concentrations of DPG, the oxygen dissociation curve is more hyperbolic; at
higher concentrations of DPG, the oxygen dissociation curve is more sigmoidal. From
the data summarized in Figure 2, we could conclude that increasing concentrations of
DPG increases or decreases (circle one) the affinity of Hb for O2, and this
makes it easier or harder (circle one) to unload the O2 in the tissues.
The concentration of DPG stays relatively constant at 4.7 mM (about the same
concentration as hemoglobin’s) in the blood plasma unless one goes to a place of higher
elevation where the air is “thinner” and the partial pressure of oxygen gas is lower. At
first you might experience shortness of breath at the higher elevation, but after a day or
two you start feeling more “normal.” This is because your body acclimates to the higher
elevation (in part) by increasing the concentration of DPG in the plasma so that it’s
easier to deliver the appropriate amount of oxygen gas to the tissues. Having the genetic
information that codes for the components responsible for regulating DPG concentration
at varying altitudes is an adaptation that we have that influences our fitness if our
environment changes. There are other responses that allow us to acclimate to higher
elevations (such as producing more erythrocytes) that take longer – usually several
weeks – to complete. For this reason, if athletes train at high elevation for several weeks
before competing at a lower elevation, they might have an advantage before these
physiological changes reverse.
Allosteric regulators of hemoglobin: CO2, H+, Cl- and
temperature
Although the concentration of DPG only changes slowly (in days), several other
allosteric regulators of Hb change in concentration or value on much shorter time scales
and are consequently more important to understanding how hemoglobin functions as a
“molecular lung”. Chief among these are the concentrations of CO2, H+ and Cl-. The
effects of two of these regulators (and the effect of temperature) are shown in Figure 3
on the next page and are sometimes called the Bohr effect, after Christian Bohr, the
father of the physicist Niels Bohr, who first reported them in 1904.
From Figure 3 we can conclude that increasing the concentration of CO2 or H+ – or
increasing the temperature – will increase or decrease (circle one) the affinity
of hemoglobin for O2, making it easier or harder (circle one) to unload O2 in the
tissues. The effects of increasing Cl- concentrations would be similar to those of
increasing CO2 or H+ concentrations. That is, the curve would shift to the right.
Figure 3. The effect that increasing
or decreasing the concentration of H+
or CO2 – or the temperature – on the
oxygen dissociation curve of Hb. The
figure is from
http://www.octc.kctcs.edu/GCaplan/anat2/no
tes/FG28_12.jpg .
Hemoglobin’s two stable conformations
As you saw in the web page animation, Hb has two possible stable conformations.
These are referred to as the T state (T for “tense”) and the R state (R for “relaxed”). In
Figure 4 below, the T state is on the left and the R state is on the right. We will not
concern ourselves with all of the differences between the two states, but notice (It will
help if you “zoom” this picture up to 200% before looking …) that amino acid 97 in β1
in the T state “fits” between amino acids 44 and 41 of α2, but in the R state it has
“ratcheted” down (in the picture) to between amino acids 41 and 38. Likewise, β2 has
“ratcheted” up (in the picture) on α1 when moving from the T state to the R state.
Figure 4. T state (left) and R state (right) conformers of hemoglobin. The T state
conformation is stabilized by increasing the plasma concentrations of CO2, H+ and Cl- or by
decreasing the concentration of O2. The R state conformation is stabilized by increasing the
plasma concentration of O2 or by decreasing the concentrations of CO2, H+ and Cl-. The T
state is more common in the tissue capillary beds and the R state is more common in the
capillary beds surround the alveoli of the lungs. These pictures are from the chapter on
hemoglobin in the text by Voet, Donald and Judith Voet. 2004. Biochemistry, 3rd edition.
John Wiley & Sons, Inc.
Conformations “in between” these two
stable conformations are not sterically
possible. T state hemoglobin has a low
affinity for O2 and is often called deoxyHb;
R state hemoglobin has a high affinity for
O2 and is often called oxyHb. But what
usually causes the shift in conformation if
hemoglobin is in the T state? In the lungs,
O2 molecules are plentiful and one binds to
the Fe2+ ion of an α subunit’s heme group of
a deoxygenated T state hemoglobin
tetramer (the heme groups of the β subunits
are sterically blocked by a valine residue in T
Figure 5. This figure shows the effect of O2
state hemoglobin). When this happens, the
2+
binding to the heme group. The T state is
electronic properties of the Fe change, and
shown in blue; the R state is shown in red.
the iron ion becomes smaller in volume and
When O2 binds to Fe2+, the heme group
can now be accommodated into the center of
becomes less “domed” and the Fe2+ moves
the heme group (see Figure 5). But the
into the center of the heme. This pulls His F8
proximal histidine (His F8 in Figure 5) is
towards the heme. In turn, His F8 pulls Helix
F, which triggers a general T state to R state
bonded to the iron, and when the iron ion
conformational change. This picture is from
moves so does the helix (Helix F in Figure 5)
the chapter on hemoglobin in the text by Voet,
of which it is a part. This change in the
Donald and Judith Voet. 2004. Biochemistry,
position and orientation of Helix F eventually
3rd edition. John Wiley & Sons, Inc.
results in this subunit shifting from the t state
(a lower case “t” is used when referring to a
single subunit instead of the whole hemoglobin molecule) to the r state. But because this
α subunit interfaces with other subunits (see Figure 4), it tends to convert them from
their t states to their r states, too. The R state hemes of β subunits are not sterically
blocked by valine because it has moved aside. This is the essence of cooperativity!
What happens in one subunit affects the other subunits. In this case, when one α subunit
converts to the r state (which has a higher affinity for O2 gas) the entire tetramer tends to
convert to the R state. Now each of the other subunits has a higher affinity (higher value
of Ka) for O2.
Relating structure to function … finally!
So, what is “tense” in T state hemoglobin and what is “relaxed” in R state hemoglobin?
Oddly enough, this language seems to refer to the C-terminal amino acid residues of the
four subunits. When X-ray crystallography is used to obtain electron density maps of T
state hemoglobin, the C-terminal amino acid residue of each subunit appears to form
definite bonds with other parts of the protein. But in R state hemoglobin, the C-terminal
amino acid residues appear as “blurs” and do not seem to form definite bonds.
Apparently, the bonds that stabilize T state Hb are broken in order to make the
conformational shift to R state Hb. Figure 6 below shows the bonds that occur near the
C-terminal amino acid residue – Arg 141 – of an α subunit and the C-terminal amino
acid residue – His 146 – of a β subunit when Hb is in the T state.
Figure 6. This figure shows the bonds that stabilize the C-terminal amino acid residue (Arg
141) of the α1 subunit of T state hemoglobin (left) and those that stabilize the C-terminal amino
acid residue (His 146) of the β2 subunit (right). This picture is from the chapter on hemoglobin
in the text by Voet, Donald and Judith Voet. 2004. Biochemistry, 3rd edition. John Wiley &
Sons, Inc.
Looking at Figure 6 (left), identify the type of bond between the following functional
groups or ions:
____________________ N-terminal amino group of α2 and Cl____________________ phenolic hydroxyl group of Tyr 140 of α1 and carbonyl oxygen
next to Val 93 of α1
____________________ carbonyl oxygen of β2 and R-group of Arg 141 of α1
____________________ C-terminal carboxyl group of α1 and R-group of Lys 127 of α2
If each hydrogen bond represents 30 kJ/mol and
each ionic bond (“salt bridge”) represents 20
kJ/mol, how much energy is involved in the bonds
shown in 6a?
Answer:
When these bonds form, energy is released or required
for bond
formation (circle one). When these bonds form, the free energy of the hemoglobin
decreases or increases (circle one) and this decreases or increases the
stability of the Hb.
In Figure 6b you can see that the R-group of His 146 can form an ionic bond (“salt
bridge”) with the R-group of Asp 94 in T state hemoglobin if His 146 is in the His1+
form (that is, if it is protonated).
If the pKa value for the R-group of His 146 is 7.10, then what fraction of the His is
protonated at pH = 7.40?
At pH = 7.20?
Show work!
The following equation will be useful:
Show work!
[ His 0 ]
pH  pKa  log
[ His1 ]
The pH of the blood entering active muscle capillary beds is about 7.40, and as it moves
through the capillary beds the pH is lowered to about 7.20. And, as you have calculated
above, at a blood pH of 7.20 a greater or lesser (circle one) fraction of the His
146 will be protonated and this means more or fewer bonds can form with the Rgroups of Asp 94 and this will stabilize or destabilize the T state of
hemoglobin.
The pH of the blood decreases because active muscle produces CO2 gas, and the action
of the enzyme carbonic anhydrase (located inside of erythrocytes) allows this CO2 gas
to react quickly with water to produce carbonic acid (see Figure 42.30 in your text).The
carbonic acid dissociates spontaneously to form H+ and bicarbonate anions. This is
shown in the equation below.
CO2  H2 O  H2 CO3  H   HCO3
carbonic anhydrase
The protons produced are the reason the pH decreases in the blood. The bicarbonate
ions produced in the erythrocytes leave via Band III in exchange for Cl- anions. This is
the reason [Cl-] increases in the erythrocytes. Therefore, in the capillary beds of active
muscle, the concentrations of CO2, H+ and Cl- all rise inside of erythrocytes. And, as we
saw in Figures 3 and 6 above, this means that hemoglobin will tend to covert to the T
state and release or bind (circle one) O2.
A Brief Look at the Thermodynamics of O2 binding
The table to the right provides data from a paper in which researchers measured the
changes in free energy, enthalpy and entropy when hemoglobin binds one, two, or four
O2 molecules (apparently, in 1987 it
was very hard to get these values for
when Hb binds three O2 molecules).
Note that numbers in the brackets
provide the average value for each O2
that Hb binds. Is the binding of O2 by
Hb thermodynamically favorable?
Yes or No (circle one).
How do you know?
Answer:
How is positive cooperativity expressed by
comparing the average values (in the
brackets) of ΔGº for each O2 bound?
Answer:
This table is from Parody-Morreale, Antonio,
Charles H. Robert, Gary A. Bishop, and Stanley
J. Gill. 1987. Calorimetric Studies of Oxygen
and Carbon Monoxide Binding to Human
Hemoglobin. The Journal of Biological
Chemistry 262: 10994-10999.
Is the reaction enthalpically or entropically driven (circle one)?
Is cooperativity primarily a result of differences in entropy changes or enthalpy
changes (circle one)? For your choice, explain the source of the difference that appears
to contribute to cooperativity. (Hint: Remember what happens in the T state to R state
change!)
Answer:
Since 1987, the value of ΔGº for the last O2 bound (on the average value for each O2
bound basis) has been measured and is about -8.52
kcal/mol. Using this value and the average value of -5.8
 G o   RT ln Ka
kcal/ mol for the first O2 bound (from the table on the last
 G o
page), quantify the increased affinity for the last O2 bound
or Ka  e RT
compared to the first O2 bound. (Hint: The affinity in each
case is given by the Keq constant, in this case an
association constant (Ka). Using the equation to the right,
you should be able to calculate each Ka. Since we are working with association
constants, next determine the ratio of Ka for the last O2 bound divided by the Ka for the
first O2 bound. This will give you the value of the “-fold increase”. Don’t worry about
units, since (in this case) they will cancel each other out. Some constants you will need
are R = 1.9872 × 10-3 kcal/mol·K and T = 25º C = 298.15 K.
Show work!
The End of the Story … Back to the Lungs
When the deoxygenated blood is pumped to the capillary beds located in the alveoli of
the lungs, it is then in an O2-rich, but CO2-poor, environment. So, you start exhaling
CO2 gas and the reactions shown in the box at the top of the last page reverse. This
means that as you exhale CO2 gas carbonic acid concentrations decrease and
bicarbonate reenters the erythrocytes (in exchange for Cl- ions that now exit) and
combine with protons to form more carbonic acid (which continues to form more CO2
and H2O, etc.). Therefore, the concentrations of CO2, H+ and Cl- in the erythrocytes are
all decreasing at the same time that O2 concentrations are now high. The result of this is
that Hb molecules start converting to the R state and quickly bind four oxygen gas
molecules each. The formation of the bond between each O2 and its heme iron
releases or requires (circle one) energy.