1. No category

Molecules, Gene and disease
Session 2
Lecture 4
Oxygen transport proteins
Transport of O2 and CO2 by hemoglobin
Haemoglobin and Myoglobin
MYOGLOBIN
•
153 amino acids (18 KDa)
•
O2-carrying protein in muscle
•
Contains essential prosthetic group (haem: contains Fe
atom)
•
Compact protein, largely α-helices
•
Histidine involved in O2 –binding
•
Carbon monoxide can also bind
Heme consists of:
•an
organic part, protoporphyrin, made up of 4 pyrrole
rings,linked by methylene bridges (‘tetrapyrrole ring’) and
with 4 methyl, 2 vinyl and 2 propionyl side chains; and
•an
atom of Fe, which binds to the
N atoms of the protoporphyrin ring.
4
•The
Fe can form 2 additional bonds,
on either side of the plane.
ferrous (+2) state binds the oxygen.
one
The
Hemeprotein and Heme
Features of myoglobin structure
•
Compact (45 x 35 x 25 Å)
•
75% α-helical
•
Internal residues are non-polar, except for two His residues
which are involved in O2-binding
•
The heme is largely hidden, but the propionate
side-chains (-ve charge) are on the surface
•
Histidine F8 is directly linked to Fe
•
O2 is bound directly only to Fe
in heme, on the opposite
side to His F8
N
Position of Fe in myoglobin. The Fe lies slightly out of
the plane of the porphyrin ring, towards His F8.
Oxygen binding to heme of myoglobin.
O2-binding to Fe pulls the iron into the plane of the ring with
associated movement of His F8.
The binding of oxygen by myoglobin
k1
Mb + O2
The equilibrium constant: Kd =
MbO2
k-1
k-1
[Mb].[O2]
1
[MbO2]
=k
(1)
Define the degree of saturation of myoglobin with oxygen (Y) as the concentration of
oxymyoglobin as a proportion of the total myoglobin concentration:
[MbO2]
Y=
(2)
[Mb] + [MbO2]
Substituting equation (1) into equation (2) gives:
[O2]
pO2
Y = K + [O ]
= P + pO
d
2
50
2
(As oxygen is a gas, its concentration is usually expressed in terms of partial pressure
(pO2) and P50 is used to represent the equilibrium constant)
This equation gives a hyperbolic relationship between Y and pO2, reminiscent of the MichaelisMenten equation (will be discussed in session 3).
Binding of oxygen to myoglobin
1.0
0
0
pO2
5
(torr)
10
Haemoglobin
•
•
2 polypeptide chains: α (141 aa), β (146 aa) in α2β2
tetramer
Oxygen-carrying protein in red blood cells
•
Each chain contains an essential heme prosthetic
group
•
Conformation of each polypeptide chain is very
similar to that of myoglobin
•
Sigmoidal O2-binding curve (cooperativity)
Quaternary structure of Haemoglobin.
The α2β2 tetramer contains 4 heme groups.
Binding of Oxygen to hemoglobin
Deoxy
Oxy
Transition from T to R state in haemoglobin
On binding oxygen, one pair of αβ -subunits shifts with respect to the other by a
rotation of 15 degrees.
Binding of oxygen to one subunit ‘switches’ other subunits to a conformation
which favours oxygen binding - leading to ‘cooperative’ binding of oxygen.
Conformational changes in
hemoglobin
The movement of the iron atom on
oxygenation brings the iron-associated His
residue towards the porphyrin ring. The
associated movement of the His-containing a
helix alters the interface between the ab pairs
and initiates other structural changes
involving other subunits.
Cooperative’ binding of oxygen to
hemoglobin
‘Cooperative’ binding of oxygen to haemoglobin
The binding curve can be pictured as a composite of two simple
binding curves, of the kind seen for myoglobin - one corresponding
to the T state (low affinity)
and one to the R state (high
affinity).
As the concentration of
substrate increases, the
equilibrium shifts from
the T state to the R state,
resulting in a gradually
increasing slope
of the binding curve.
Fractional saturation changes
more steeply as a function of pO2.
pO2
Allosteric effects
• The ability o f hemoglobin t o reversibly bind oxygen is
affected by the following parameters:
• pH
• pO2
• pCO2
• the availability o f 2,3-bisphosphoglycerate .
These are collectively called allosteric ("other site")
effectors , because their interaction at one site on the
hemoglobin molecule affect s the binding of oxygen t o
heme groups at other locations on the molecule .
Regulation of oxygen binding
The highly anionic 2,3-BPG is present in red blood cells at
~ 2 mM. It binds to haemoglobin (one molecule per tetramer) and
decreases the affinity for O2, promoting release in the tissues.
The physiological adaptation to high altitude involves increased
tissue concentrations of BPG, leading to more efficient O2 release
to compensate for the reduced O2 tension.
Binding of 2,3-BPG to deoxyhaemoglobin
2,3-BPG binds in the central cavity of the tetramer, interacting with
three positively charged groups (2 His, 1 Lys) on each b chain.
The oxygenated haemoglobin has a smaller central gap and
excludes 2,3-BPG.
Differential oxygen affinity of foetal and maternal red blood cells
Foetal haemoglobin contains a variant of the β chain, called ɤ, which has a
HisSer substitution in the 2,3-BPG-binding site. The foetal
haemoglobin thus has a reduced affinity for 2,3-BPG, resulting in an
enhanced O2-binding affinity that allows transfer of O2 from the maternal
to the foetal red blood cells
The Bohr effect:
H+ ions and CO2 promote the release of O2
 Rapidly metabolising tissues, such as contracting muscle, have a high
need for O2 and generate large amounts of H+ and CO2. Both of these
species interact with haemoglobin to promote O2-release.
 The O2 affinity decreases as the pH
decreases from the pH 7.4 found
in the lungs.
 Increased CO2 concentrations also
lead to a decrease in O2-affinity.
 This regulation of O2-affinity by
pH and CO2 is called the Bohr
effect after its discoverer,
Christian Bohr (1904).
Chemical Basis of the Bohr effect
In deoxyhaemoglobin, three amino acid residues
form two salt bridges that stabilise the T state
(the low oxygen affinity state). The formation of
one of these depends on the presence of a proton on
His b146. Lowering of the pH by metabolic activity
favours the proton-addition and formation of the salt
bridge with Aspb94, thus stabilising the T structure
and increasing the tendency for O2 to be released.
The effect of CO2 on deoxyhaemoglobin
The CO2 reacts with the terminal aNH2 groups to form
carbamates, which are negatively charged. The N-termini lie at
the interface between the ab dimers, and the negatively charged
carbamates participate in salt-bridge interactions that favour the T
state, stabilising the deoxy-form and favouring the release of O2
Carbon monoxide and haem iron
 Carbon monoxide is a poison because it combines with
ferromyoglobin and ferrohaemoglobin and blocks oxygen
transport.
 Note that HisE7 sterically inhibits binding of CO, and
lowers its affinity for the haem Fe. This is then
sufficiently low that endogenous levels of CO can be
tolerated. High levels of CO from poorly ventilated gas
fires are highly toxic.
HisF8
N-Fe-O
(Plane of porphyrin ring)
O
HisF8
N-Fe-C
(Plane of porphyrin ring)
O
Summary