Chem*3560
Lecture 4:
Factors that control the allosteric behaviour of hemoglobin
Story so far
Hemoglobin exists in two structural states, T (tense) which has low O2 affinity (high P50),
and R (relaxed) which has high O2 affinity (low P50).
These two states are distinguished on the global scale by the relative orientation of the α1β1
pair of globins relative to α2β2. R-state is rotated 15o relative to the T-state orientation.
On the microscopic level, there are no changes in the α1β1 interface (within a pair), but
significant effects are seen in the interactions between α1 and β2 (between opposites).
In T state, β2 globin His HC3 is held in position by α1 globin Lys C3 so that the His side chain
can forms an ion pair with Asp FG1. This pulls helix F8 away from heme-Fe, which in turn
pulls Fe out of the heme plane, making it less available to bind O2. There is tension between
His HC3 and heme Fe, hence the term tense state.
When O2 binds to T-state hemoglobin (which can happen if pO2 is high enough), the bound O2
exerts an opposing force on the heme Fe. The presence of a single O2 may be insufficient to
bring about change, but as more O2 molecules occupy a single hemoglobin, the collective force
may be sufficient to break the ion pairs that stabilize the T-state.
When this happens, the whole hemoglobin molecule switches to R state by rotating the globin
pairs. (R for relaxed, due to absence of tension between His HC3 and heme-Fe). This means
that the remaining vacant sites in the hemoglobin molecule are now in the high affinity R state.
pO2 decreases as blood circulates to peripheral tissues, so some O2 will be released. As more
sites become vacant, electrostatic forces tend to pull hemoglobin back into T-state, and this
will promote the release of the remaining O2 in the molecule.
Any factor that selectively favours T or R state of hemoglobin will affect the
allosteric behaviour.
Active metabolism releases CO2 which acidifies the blood
e.g. TCA cycle oxidation of acetyl CoA derived from fats or sugars
CO2 + H2O ! H+ + HCO3–
increases [H+] so pH is lower
If tissues are O2 deficient, anaerobic glycolysis may also occur:
glucose → 2 lactate– + 2 H+
Hemoglobin responds to lower pH and the presence of CO2 by decreasing O2 affinity (shifts
the binding curve to the right, so P50 increases; affinity is inversely related to P50). This allows
more O2 to be released.
These responses are known as the Bohr effect, from Christian Bohr (father of Nils Bohr, the
atomic physicist). (Lehninger pp.216-217).
Structural basis of the Bohr pH effect
H+ is taken up by His HC3 of the β-globin.
His HC3 ion pairs with Asp FG1 in the T-state of hemoglobin.
Proximity to Asp FG1 gives His HC3 a higher pKa than normal, so it is protonated at pH 7.2
His HC3 and Asp FG1 move apart when hemoglobin switches to the R-state so His becomes
closer to the usual pKa = 6.5, and will tend to be deprotonated.
There are two His HC3 in hemoglobin (one in each β-globin) so the H+ stoichiometry is 2 H+
per Hb tetramer
"
this direction is promoted by high O2
⇒
Hb.(H+)2 + 4 O2 ! Hb.(O2)4 + 2 H+
T-state
R state
⇐
this direction is promoted by lower pH (higher [H+]
Le Chatelier's Principle in action!
The Bohr pH effect shifts the sigmoidal curve to the right at lower pH
pH in the lung tends to be higher, around
7.4, but this makes little difference at the
higher pO2 because hemoglobin is already
near saturation
pH in actively metabolizing tissues drops
to 7.2, causing the binding curve to shift
rightwards. For typical pO2 in the
peripheral tissues, this resultings in
release of 10-15% more O2.
Hemoglobin also acts as a carrier for CO2
CO2 binds to the globin, not to the heme (unlike CO).
CO2 reacts reversibly with the α-globin N-terminal amino group to form a carbamate derivative.
""""# carbamate (carboxylate bonded directly to NH)
The N-terminus of one α-globin is close to the C-terminus of the opposite α-globin. Lys HC1
and Arg HC3 are found at the α-globin C-terminus, and give this region a positive charge.
(His HC3 is at the β-globin C-terminus).
Proximity of the positive charge to the N-terminus causes the N-terminal amino group to have a
lower pKa (normally around 8). This allows deprotonation, and promotes the reaction with CO2.
Formation of the carbamate gives the N-terminal a negative charge instead of the usual positive;
the negative carbamate can attract the C-terminal Lys-139 and Arg 141 from the opposite
α-globin.
Carbamate formation favours the T-state
The two α-globins move apart in R state, but move closer in T state.
A positive N-terminal amino group therefore is favoured by R state, because of the increased
distance from Lys HC1 and Arg HC3 on the opposite α-globin.
Hence when O2 binds in the lungs, this favours CO2 release.
R state
T-state
carbamate
When carbamate forms in peripheral tissues, it drives the equilibrium towards the T state side,
promoting O2 release.
The elusive missing factor X
The value P50= 4 kPa was measured for
hemoglobin present in whole blood.
However, when hemoglobin was
purified, O2 affinity increased
dramatically, giving P50= 0.25 kPa, similar
to the value for myoglobin, although the
sigmoidal shape is still present. (Lehninger
pp.218-219)
What has changed?
A missing factor accounts for difference,
however its identity as
2,3-bisphosphoglycerate (BPG) was not
discovered until 1967.
2,3-bisphosphoglycerate5– (not the same as
1,3-bisphosphoglycerate produced in
glycolysis) is formed by phosphorylation of
3-phosphoglycerate. Note the high negative
charge.
When 5 mM BPG is added back to pure
hemoglobin, P50 is restored to 4 kPa, the level found for hemoglobin in whole blood.
BPG is an example of an allosteric effector, a ligand that binds to a protein such as hemoglobin
and increases or decreases the allosteric effect.
BPG binds selectively to
T-state hemoglobin
The highly negative 2,3-BPG5–
binds in a recess between β1
and β2 globins, near a cluster
of positive amino acids His 2,
Lys EF6 and His H21.
In T-state, the β-globins
move apart so there is room
for BPG to fit in bound by
the electrostatic attraction.
The β-globins are shown
cyan and yellow; α-globins
in the background are
shown blue and green.
The two β-globins move
closer together in
R-state, so BPG can’t
bind.
Hb.(O2)4 ! Hb ! Hb.BPG
R state
T-state
BPG binding helps shift the equilibrium to the right, away from R-state, hence the low
affinity T state persists until much higher pO2 levels are reached.
BPG provides an adaptation to low O2 levels at altitude
At sea level, [BPG] = 4.7 mM in blood,
lung pO2 = 12 kPa.
At 4,500 m, lung pO2 drops to 7 kPa.
Within 24 hours, [BPG] rises to 8 mM.
The result is counterintuitive!
O2 affinity of hemoglobin is decreased,
because BPG favours the T state!
Decreased affinity (increasing P50)
makes little difference to the amount of
O2 bound, because hemoglobin is already near saturation.
However decreased affinity allows much more O2 to be released at tissue pO2
At sea level, with normal BPG levels, occupancy goes from 0.97 in lungs to 0.50 in tissues
At 4500 m, with 8 mM BPG, occupancy goes from 0.75 in lungs to 0.35 in tissues.
Different hemoglobin genes are expressed at embryonic, fetal and adult stages
of development
Globin genes are distributed in two groups:
Chromosome 16: α-like globins ζ (zeta) α α (two identical copies of a)
Chromosome 11: β-like globins ε (epsilon) γG γA δ β
Embryonic hemoglobin ζζεε is expressed during weeks 4-6 after conception.
Fetal hemoglobin (HbF) ααγγ is expressed until birth; γG γA are identical except for amino acid
136, Gly in one case and Ala in the other. There is no effect on function.
Adult hemoglobin (HbA) ααββ
ααββ is expressed post-birth (plus about 2% ααδδ
ααδδ which is
functionally similar).
His H21 of β-globin is replaced by Ser in ε- and γ-globins. His H21 is one of the positive amino
acids in the BPG binding recess, so there is less positive charge with neutral Ser.
Less positive charge means that BPG does not bind as well, so the R ! T equilibrium shifts in
favour of R state and higher O2 affinity. Less positive charge also means less electrostatic
repulsion between neighbouring γ-globins in R state. The higher O2 affinity of fetal hemoglobin
allows O2 to transfer from maternal to fetal blood in the placenta.