Lecture Proteins 12 Oxygen
Binding Proteins
Suggested reading: Lehninger, 5th
edition, Chapter 5.1
Hb subunits are structurally similar
to Mb
Recall that hemoglobin is a tetramer containing 4
heme groups (one heme associated with each
polypeptide chain).
It is also called a dimer of protomers.
Why ? When Hb dissociates in urea, the
closest and strongest contacts are between and
chains rather than - or -.
Fewer than the aa residues of and subunits
are identical, but their 3D structures are very
similar.
Hb Subunits
Furthermore, when comparing Mb to Hb subunits (Fig
5-6), their structures are very similar even though the
amino acid sequences of Mb, Hb and Hb are
identical at only 27 positions.
When considering Hb quaternary structure, most of
the interactions between chains are hydrophobic.
There are a few H bonds and salt bonds between
chains.
Because Hb is a tetramer, each Hb molecule can bind 4
oxygens.
These sites exhibit cooperativity in binding.
Hb undergoes a structural change
on binding oxygen
There are two major conformations of
hemoglobin which have been confirmed by X-ray
crystal structures: the R state and the T state.
T = tense and R = relaxed
R state:
Higher oxygen affinity for Hb is observed in the R
state.
Oxygen binding stabilizes R state.
The R state is predominant conformation for
oxyHb.
T state
The absence of oxygen stabilizes the T state.
T state is the predominant conformation of deoxyHb.
T state is stabilized by a greater number of ion pairs,
many at the interfaces (Fig 5-9).
The binding of O2 to Hb in the T state promotes a
change in conformation to the R state. The subunit
pairs slide past each other, rotate and narrow the
pocket between the subunits. (Fig 5-10)
Ion pairs are broken and formed. This is an example
where ion pairs are important to structure/function.
What happens on the molecular level to trigger the
switch from the T state to the R state with O2 binding?
Changes in the positions of key amino acid side chains
surrounding heme may cause the change from T to R.
In the T state, the porphyrin ring of heme is slightly
puckered, and the heme iron protrudes to some extent
on the proximal His side as shown in Fig 7-13.
When O2 binds, it pulls the Fe2+a short distance down
into the heme, and a more planar heme conformation
results.
Molecular rearrangement must occur to relieve the
strain, where the proximal his (His F8) shifts its position
to the perpendicular, also shifting the attached F helix
and the FG corner.
Molecular level: Switch from T to R
state (contd)
Val FG5 is pushed to the right and its H-bond with
Tyr HC2 is broken. This change weakens the H
bonds and salt bonds that connect FG corners of
one subunit with C helices of another subunit.
As small changes occur in the structures of the
other subunits, O2 binding increases.
In summary, the binding of O2 pulls the Fe a
fraction of a nanometer into the heme and
causes a larger shift in the surrounding structure,
particularly at the interfaces. This facilitates
oxygen binding.
Hb Binds Oxygen Cooperatively
As stated earlier, Mb with its hyperbolic binding curve,
could bind O2with high affinity in the lungs (where pO2=
13.3 kPa or about 100 mm Hg), but it would not release
much of it in the tissues (where pO2= 4 kPa or about 30 mm
Hg).
Alternatively, a protein with a hyperbolic curve could also
bind O2at low enough affinity to release it in the tissue, but
it would not pick up enough O2in the lungs.(Fig 5-12).
Hb solves the problem by undergoing a transition from the
low affinity (T-state) to the high affinity state (R-state) as
more oxygen molecules are bound.
The binding curve for Hb is sigmoidal (Fig 5-12) because O2
binding is cooperative.
Hb Binds Oxygen Cooperatively
By cooperative, we mean that the binding of O2 to the
heme Fe in one subunit increases the binding affinity (Ka)
of oxygen in the other subunits.
The first O2 molecule interacting with deoxyHb binds
weakly (T state).
Conformational changes occur to make it easier to bind
oxygen.
By the time the last (4th) O2 molecule binds, it binds to a
heme in a subunit already in the R state.
Because of cooperative binding, Hb will be almost fully
saturated at the pO2 of the lungs but only partially
saturated at the pO2 in tissues. Most of the O2 bound by Hb
in the lungs will be released by Hb in the tissues.
Hemoglobin : An Allosteric Protein
When the binding of a small molecule (ligand) to one site on a
protein affects the binding at another site on the same
protein, the protein is called allosteric.
The ligands which induce different conformations of allosteric
proteins when they bind to the protein are called
modulators. The modulators may be either inhibitors or
activators.
Homotropic interaction= when the normal ligand and
modulator are identical.
Heterotropic interaction = when the modulator is a molecule
other than the normal ligand.
Cooperative binding of O2 to hemoglobin is an example of
allosteric binding.
O2 is both a ligand and an activating homotropic modulator.
The binding sites of an allosteric protein contain stable
segments in close proximity to unstable segments.
Quantitating Cooperative Ligand
Binding: Hill Plot
For a protein with n binding sites:
P + nL PLn
and Ka=[PLn]/ [P][L]n
= [L]n/ ([L]n+ Kd)
This equation can be rearranged as follows:
{ / (1-)} =[L]n/Kd
Taking the log of both sides gives us the Hill equation:
Log(/ (1-)) = n log [L] log Kd where Kd= [L]n0.5
A plot of log [/ (1-)] vs. log [L] is a Hill plot.
Hill Plots (contd)
Based on the equation, the Hill plot should have a slope= n.
But the experimentally determined slope reflects the
degree of interaction between binding sites, not their
number.
The slope of a Hill plot is nH, the Hill coefficient.
nH is a measure of the degree of cooperativity.
If nH= 1, ligand binding is not cooperative.
If nH> 1, there is positive cooperativity in ligand binding .
Hb is an example.
If nH< 1, negative cooperativity . This situation, where the
binding of one ligand impedes the binding of others, is rare.
Hill Plots (contd)
Hill The equation can be adapted to the binding
of oxygen to Hb:
log [/(1-)] = n log pO2n log P50
Hill plots for Mb and Hb are shown in Fig 5-14.
For Mb, the Hill plot is a straight line with a slope
of 1.
For Hb, the Hill plot is not linear, but the
maximum slope is a measure of the cooperativity
of binding (the greater the slope, the more
cooperative the binding).
Two major models: Mechanisms for
cooperative binding (Fig 5-15)
Two major models attempt to explain the cooperative
binding of ligands to multisubunit proteins (e.g. Hb):
MWC model (or concerted model) and Sequential
model
MWC model (Fig 5-15 a)
Assumes the subunits of a cooperatively binding
protein are functionally identical, that each subunit can
exist in two conformations, and that all subunits
undergo simultaneous transition from one
conformation to the other.
No protein has subunits in different conformations.
The two conformations are in equilibrium.
Mechanisms for Cooperative Binding
(contd)
Sequential Model (Fig 5-15b)
Ligand binding can induce a change of
conformation in an individual subunit.
A conformational change in one subunit
makes a similar change in an adjacent subunit,
and the binding of a second ligand is more
likely.
More potential intermediate states are in this
model.
Hemoglobin transports H+ and CO2
Hb carries H+ and CO2 (two end products from cellular
respiration) from the tissues to the lungs and kidneys.
The CO2 is hydrated to form bicarbonate:
CO2 + H2O H+ + HCO3
Bubbles of relatively insoluble CO2 would form in the
tissues and blood if not converted to bicarbonate.
Hydration of CO2 results in an increase in the H+ conc.
(decrease in pH)
Binding of oxygen by Hb is influenced by
pH and CO2 concentration
Hb transports ~40 % of the total H+ and 15 -20 % of CO2
formed in tissues to lungs and kidneys.
Binding of H+ and CO2 is inversely related to the binding of O2.
In peripheral tissues (low pH and high CO2), affinity of Hb for
oxygen decreases as H+ and CO2 are bound; O2 is released to
the tissues.
In lungs, CO2 is excreted, blood pH rises, and affinity of Hb for
oxygen increases. Hb binds more O2 for transport to tissues.
The effect of pH and CO2 on oxygen binding and release by Hb
is called the Bohr effect.
HHb+ + O2 HbO2 + H+
Effects of H+ and CO2(contd)
From this equation, we know the binding of oyxgen to Hb is
influenced by H+ (and thus the pH). See Fig. 5-16
H+ binds to any of several amino acid residues in the protein.
A major contribution to Bohr effect is made by His 146 (HC3)
of the subunits.
When protonated, His 146 residue forms one of the ion pairs
to Asp 94 (FG1) that stabilizes deoxyHb in the T state; oxyHb
releases O2.
Carbon dioxide binds as a carbamate group to the -amino
group at the amino-terminal end of each globin chain to form
carbaminoHb (See Figure packet for this reaction).
This reaction produces H+ which contributes to the Bohr
Effect.
Bound carbamates also form additional salt bridges that
stabilize the T state (deoxy state) and promote O2 release.
H+ and CO2 are allosteric effector molecules for Hb.
Oxygen Binding to Hb is Regulated by
2,3-bisphosphoglycerate (BPG)
BPG is another allosteric effector molecule for Hb.
BPG is present in relatively high conc. in erythrocytes.
It greatly reduces the affinity of Hb for oxygen by binding
to groups that stabilize the deoxy (T) state.
HbBPG+ O2 HbO2+ BPG
Site of BPG binding to Hb is the cavity between the
subunits in the T state (positively charged residues interact
with negatively charged groups of BPG).
At high altitudes, BPG conc. in the blood rises, leading to a
decrease in the affinity of Hb for oxygen; O2is released to
the tissues. (Fig 5-17).
At sea level, the situation is reversed. (Fig 5-17).
BPG and Fetal development
Since a fetus must get oxygen from its
mothers blood, fetal hemoglobin must have
greater oxygen affinity than maternal
hemoglobin.
Fetal Hb synthesizes subunits rather than ,
forming 22 Hb.
This tetramer has a lower affinity for BPG than
adult Hb and a correspondingly higher affinity
for O2.
Sickle-Cell Anemia is a Molecular
Disease of Hb
Sickle cell anemia is a genetic disease in which an
individual inherits the allele (variant gene) for sicklecell Hb from both parents.
Characterized by a large number of immature cells with
many long, thin, crescent shaped erythrocytes that
look like a sickle blade (Fig 5-19 in Lehninger).
When Sickle Hb (HbS) is deoxygenated, it becomes
insoluble and forms polymers which aggregate into
tubular fibers. Normal Hb (HbA) remains soluble on
deoxygenation.
Sickle Cell Anemia (contd)
Altered properties of HbS result from a single
aa substitution:
Val instead of glu at position 6 in the two
chains.
HbS has two fewer negative charges. A sticky
hydrophobic contact at position 6 is created,
causing aggregation into bundles of fibers.
This condition results in serious medical
complications.
Carbon Monoxide (CO) Binds Hb
CO (carbon monoxide) binds to hemoglobin 250 times better than
oxygen to form COHb.
The tight binding of CO to Hb means COHb can accumulate over
time as people are exposed to a constant low-level source of CO.
Smokers have increased levels of COHb.
Symptoms of CO poisoning are rarely observed at levels less than
10 % total Hb. At 15 %, headaches begin. Death occurs when COHb
levels rise above 60 %.
As CO binds to one or two subunits of Hb, the affinity of the
remaining Hb subunits for O2 is increased.
Hb tetramer with 2 bound CO molecules can efficiently bind O2 in
the lungs-but it releases very little O2 to the tissues (See Fig 2).
Oxygen deprivation results.
Note that in an anemic individual, the amount of O2 bound is
similar to 50 % CoHb, but unlike the CoHb situation, the
hemoglobin from the individual with anemia can release oxygen to
the tissues.
This concludes our work in chapter 5.
You should be able to do problems 1-8.
I also put a few oxygen binding protein
problems on blackboard. You can go over
these in discussion with your TAs.
Please read Chapter 7.1 and 2 (Carbohydrates)
for Mondays class.