Myoglobin & Haemoglobin

Biochemistry Objectives 4 7

Myoglobin monomeric protein of red muscle. Stores oxygen as a reserve against oxygen
deprivation.
Haemoglobin tetrameric protein of erythrocytes. Transports O2 to the tissues and returns
CO2 and protons to the lungs. Carbon monoxide disrupts the physiologic function of
haemoglobin.

Myoglobin
Oxygen stored in red muscle myoglobin is released during O2 deprivation (e.g. severe exercise) for
use in muscle mitochondria for aerobic synthesis of ATP.

Myoglobin consists of a single 153-residue polypeptide chain and has a MW of 17 000 kDa.

An unusually high proportion, almost 85% (about 75% according to Harpers and Nayak) of amino
acid residues, are present in eight right-handed helices. There are some random coils and no -
pleated sheets.

The surface of myoglobin is rich in amino acids bearing polar and charged side chains. The interior
contains only residues with non-polar R groups such as Leu, Val, Phe and Met, with two exceptions
(His E7 and His F8). These lie close to the heme iron, where they function in O2 binding.

The single heme of myoglobin lies in a crevice between helices E and F oriented with its polar
propionate groups facing the surface of the globin. The remainder resides in the non-polar interior.
The fifth coordination position of the iron is occupied by a nitrogen from the imidazole ring of the
proximal histidine, His F8. The distal histidine, His E7, lies on the side of the heme ring opposite to
His F8.

The iron moves toward the plane of the heme when oxygen is bound. The iron of unoxygenated
myoglobin lies 0.03 nm outside of the plane of the heme ring, toward His F8. When O2 occupies the
sixth coordination position, the iron moves to within 0.01 nm of the plane of the heme ring.
Oxygenation of myoglobin is thus accompanied by motion of the iron, of His F8, and of residues
linked to His F8.

Each molecule of myoglobin binds only one molecule of O2.

The iron of heme is always in the Fe2+ state. Oxidation of the Fe2+ of myoglobin or haemoglobin to
Fe3+ destroys their biologic activity.

The oxygen-binding curve of myoglobin is hyperbolic, following Michaelis Menten kinetics.

Myoglobin loads O2 readily at the PO2 of the lung capillaries (100 mm Hg), but releases only a small
fraction of its bound O2 at the PO2 values typically encountered in active muscle (20 mm Hg) or
other tissue (40 mm Hg). Thus, it is an ineffective vehicle for delivery of O2. When strenuous exercise
lowers the PO2 of muscle tissue to about 5 mm Hg, myoglobin releases O2 for mitochondrial
synthesis of ATP. Hence it functions more as a storage reservoir for O2 than a transport protein.
Figure 1: Oxygen-binding curves of both haemoglobin and myoglobin. Arterial oxygen tension is about 100 mm Hg; mixed
venous oxygen tension is about 40 mm Hg; capillary (active muscle) oxygen tension is about 20 mm Hg; and the minimum
oxygen tension required for cytochrome oxidase is about 5 mm Hg.
Haemoglobin
Composition
Haemoglobins are tetramers composed of pairs of two different polypeptide subunits.

HbA (normal adult haemoglobin): 22

HbF (fetal haemoglobin): 22
HbS (sickle cell haemoglobin): 2S2
HbA2 (minor adult haemoglobin): 22
Gower 1 (embryo): 22

The globin chain is 141 amino acids long while the globin chain is 146 amino acids long.

The subunit composition of haemoglobin tetramers undergoes changes during development. The
human embryo initially synthesizes a 22 tetramer. By the end of the first trimester, these have
been replaced by and subunits, forming HbF (22), the haemoglobin of late foetal life. While
synthesis of subunits begins in the third trimester, subunits do not completely replace subunits
to yield adult HbA (22) until some weeks postpartum.

Figure 2: Gene expression of haemoglobin before and after birth. Also identifies the types of cells and organs in which the
gene expression occurs.

The quantity P50 is the partial pressure of O2 that half-saturates a given haemoglobin. The P50
exceeds the PO2 of the peripheral tissues in all organisms.

The values of P50 for HbA and HbF are 26 and 20 mm Hg, respectively. In the placenta, this difference
allows HbF to extract oxygen from the HbA in the mothers blood. However, HbF is suboptimal
postpartum since its high affinity for O2 limits the quantity of O2 delivered to the tissues.
Oxygen binding
Haemoglobins bind four molecules of O2 per tetramer, one per heme. A molecule of O2 binds to a
haemoglobin tetramer more readily if other O2 molecules are already bound. Termed cooperative
binding, this phenomenon permits haemoglobin to maximize both the quantity of O2 loaded at the
PO2 of the lungs and the quantity of O2 released at the PO2 of the peripheral tissues.

The binding of the first O2 molecule to deoxyhaemoglobin shifts the heme iron toward the plane of
the heme ring from a position of about 0.04 nm beyond it. This motion is transmitted to the
proximal (F8) histidine and to the residues attached to this histidine. This causes the rupture of salt
bridges between the carboxyl terminal residues of all four subunits.

Figure 3: The iron atom moves into the plane of the heme on oxygenation. Histidine F8 and its associated residues are
pulled along with the iron atom.

As a result of rupture of the salt bridges, one pair of / subunits rotates 15 with respect to the
other, compacting the tetramer. The haemoglobin thus transitions from the low-affinity T (taut)
state to the high-affinity R (relaxed) state.
 Figure 4: Conformational changes in the structure of deoxygenated and oxygenated haemoglobin.

In deoxygenated haemoglobin, In oxygenated haemoglobin, Asp-94 of

Tyr-42 of 1 binds to Asp-99 of 2. 1 binds to Asn-102 of 2.
Asp-94 of 1 and Asn-102 of 2 Tyr-42 of 1 and Asp-99 of 2 are free.
are free.

1. At P1, both myoglobin and haemoglobin are 100% saturated.

2. At P2 (lower partial pressure), the myoglobin is still about 90% saturated but the
haemoglobin is 20% saturated.
3. At P3, even though haemoglobin has lost all its oxygen (5% saturated), myoglobin still has a
high percentage saturation of oxygen (55%).
Transport of CO2 and protons by haemoglobin
Haemoglobin transports CO2 and protons from peripheral tissues to the lungs. Haemoglobin carries
CO2 as carbamates formed with the amino terminal nitrogens of the polypeptide chains.

Haemoglobin carbamates account for about 15% of the CO2 in venous blood. Most of the remaining
CO2 is carried as bicarbonate, which is formed in erythrocytes by the hydration of CO2 to carbonic
acid (H2CO3), a process catalyzed by carbonic anhydrase. At the pH of venous blood, H2CO3
dissociates into bicarbonate and a proton.

Bohr effect and proton transport

Deoxyhaemoglobin binds one proton for every two O2 molecules released, contributing significantly
to the buffering capacity of blood. Each haemoglobin therefore picks up 2 protons.

The somewhat lower pH of peripheral tissues, aided by carbamation, stabilizes the T (taut) state and
thus enhances the delivery of O2. Put another way, haemoglobin acts as a buffer since a decrease in
pH (increase in H+) results in a decreased affinity for O2. The oxygen-haemoglobin dissociation curve
is thus shifted to the right by a decrease in pH. Since an increase in CO2 concentration decreases
pH, increased CO2 concentrations also shift the curve to the right. This causes O2 to be released to
the tissues.

In lungs, the process reverses. As O2 binds to deoxyhaemoglobin, protons are released and combine
with bicarbonate (HCO3-) to form carbonic acid (H2CO3). Dehydration of H2CO3, catalysed by carbonic
anhydrase, forms CO2 which is exhaled. Binding of oxygen thus drives the exhalation of CO2. This
reciprocal coupling of proton and O2 binding is termed the Bohr effect. The Bohr effect is dependent
upon cooperative interactions between the hemes of the hemoglobin tetramer. Myoglobin, a
monomer, exhibits no Bohr effect.
Figure 5: The Bohr effect. CO2 generated in peripheral tissues combines with water to form carbonic acid, which dissociates
into protons and bicarbonate ions. Deoxyhaemoglobin acts as a buffer by binding protons and delivering them to the
lungs. In the lungs, the uptake of oxygen releases protons, which combine with the bicarbonate ion, forming carbonic acid,
which when dehydrated by carbonic anhydrase becomes carbon dioxide which is then exhaled.
Allosteric regulation by 2,3-Bisphosphoglycerate
A low PO2 in peripheral tissues promotes the synthesis of 2,3-BPG in erythrocytes from the glycolytic
intermediate 1,3-BPG:

Figure 6: 2,3-BPG. The highlighted part of the molecule is electronegative and so will be stabilised by an amino acid that is
electropositive (e.g. lysine, histidine or arginine).

The haemoglobin tetramer binds one molecule of 2,3-BPG in the central cavity formed by its four
subunits. However, the space between the H helices of the chains lining the cavity is sufficiently
wide to accommodate 2,3-BPG only when haemoglobin is in the T (taut)/deoxygenated state.

2,3-BPG forms salt bridges with 6 positive terminal amino groups (3 per chain): 2 histidines and 1
lysine per chain. (1 each of valine, histidine and lysine according to Harpers.)

2,3-BPG therefore stabilizes deoxygenated (T-state) haemoglobin by forming additional salt

bridges that must be broken prior to conversion to the R state. This means that the affinity of
haemoglobin for O2 is decreased by 2,3-BPG, thus the oxygen-haemoglobin binding curve is shifted
to the right and haemoglobin gives O2 more readily to the tissues.

Fetal haemoglobin and 2,3-BPG

In the subunit of fetal haemoglobin, the histidine residue H21 that usually facilitates binding to 2,3-
BPG by the subunit of adult haemoglobin is replaced by serine. Serine has 2 negative charges as
opposed to the positive charge of histidine and cannot form a salt bridge, hence 2,3-BPG binds more
weakly to HbF than to HbA. The stabilization afforded to the T (deoxygenated) state is thus lower,
accounting for the higher affinity of HbF for O2 than HbA.

Figure 7: The oxygen saturation curve for fetal hemoglobin (blue) appears left-shifted when compared to adult hemoglobin
(red) since fetal hemoglobin has a greater affinity for oxygen.
Adaptation to high altitude
At high altitudes there is a relative deficiency of oxygen. Prolonged exposure to high altitude induce
an increase in the number of erythrocytes and in their concentrations of haemoglobin and of 2,3-
BPG. Elevated 2,3-BPG lowers the affinity of HbA for O2, enhancing the release of O2 at peripheral
tissues.