HEMOGLOBIN

&
MYOGLOBIN
Myoglobin Hemoglobin
 MYOGLOBIN
• Is a monomeric protein found in the muscles
• myoglobin is an example of a globular and tertiary
structure protein.
• Used in storage and transportation of oxygen within
the muscle.
• Binds a single oxygen molecule and it carries O2 from
capillaries to sites of usage in cells.
• It contains Heme group
• Myoglobin has eight α -helical regions.
• 75% of the residues are present in the 8 right handed helices.
• There about 7 – 20 amino acid residues in these α helices.
• The complete polypeptide of myoglobin is made up of 153
amino acids.
• Starting at the N-terminal, these are termed helices A – H.
• Hydrogen bonding in the polypeptide backbone stabilizes the
a-helical regions; amino acid side chains are also involved in
hydrogen bonds.
• Two polar histidine residues are found in the interior: they
interact with the heme and bound oxygen and thus play a role
in function of the protein.
• High concentrations of myoglobin in muscle cells allow
organisms to hold their breath for a longer period of time.
• Gives red colour to the muscles.
• The myoglobin polypeptide chain is folded to form a cradle
that nestles the heme prosthetic group.
• The polypeptide of myoglobin may be viewed as serving
three critical functions:
- it cradles the heme group,
- it protects the heme iron atom from oxidation, and
- it provides a pocket into which the O2 binds
 HEMOGLOBIN
• Tetrametric, two alpha chains and two beta chains.
• The overall structure of hemoglobin is α₂β₂
• Both the α- and β-chains of hemoglobin are very similar to
the myoglobin chain
• The α-chain is 141 residues long, and the β-chain is 146
residues long
• amino acids of α-chain and the β-chain are
homologous(same amino acid residues are in the same
positions).
• The heme group of myoglobin and hemoglobin is the same
• Found in red blood cells.
• Carries oxygen to tissues and transport carbon dioxide
and hydrogen ions back to lungs.
• Binds to a total of 4 oxygen molecules
• Both hemoglobin and myoglobin bind oxygen reversibly
but hemoglobin exhibits positive cooperativity.
• This means that binding of one Oxygen molecule, makes
it easier for the other Oxygen molecules to bind.
• This binding can be shown in an oxygen binding graph
for both myoglobin and hemoglobin
A comparison of the oxygen binding behavior of
myoglobin and hemoglobin.
• The oxygen binding curve for myoglobin is hyperbolic where as that
of hemoglobin is sigmoid.
• Myoglobin has a higher affinity for O2 than haemoglobin
• Myoglobin contains only a single globin chain: its dissociation curve
is a rectangular hyperbola.
• In the tetrameric form of hemoglobin, the binding of oxygen is, a
cooperative process.
• The oxygenation of each chain causes structural changes which
increase the affinity of the heme of the remaining chains for oxygen.
• This positive cooperative binding and increasing oxygen affinity as
oxygen loads is the cause of the sigmoid shape of the dissociation
curve
• The relationship between concentration, partial
pressure of O2 (PO2) and quantity of O2 bound to
hemoglobin or myoglobin is expressed as an O2
saturation curve.
• The binding of oxygen to heme increases as the
concentration of oxygen in the environment
increases and decreases as the oxygen
concentration decreases.
• The graph of hemoglobin indicates that the binding of
the first oxygen molecule facilitates binding of a second
oxygen molecule, which facilitates the binding of a third,
which in turn facilitates the binding of the fourth.
• This is precisely what is meant by the term cooperative
binding
• Hemoglobin binding curve is still lower than that of
myoglobin at any oxygen pressure. This shows that
myoglobin has a higher percentage of saturation than
hemoglobin at my given pressure
 HEME GROUP
• An example of a prosthetic group.
• It is made up of a metal iron (Fe2+) and an organic protoporphyrin
ring
• Porphyrin part consists of a cyclic tetrapyrrole consisting of four
molecules of pyrrole linked by 4 α-methylene bridges,2 propionate
groups and 2 vinyl groups.
• One atom of ferrous iron resides at the center of the planar
tetrapyrrole.
• The conjugated double bonds absorbs visible light and colors heme
deep red.
• The ability of myoglobin or haemoglobin to bind oxygen depends upon this
heme group (prosthetic group).
• Iron atom has 6 coordination positions
 4 to nitrogens that are part of the flat tetrapyrrole ring and 2
perpendicular to the ring
 Coordinated N atoms help prevent conversion of the heme iron to the
ferric (Fe3+) state.
 Iron in the Fe2+ state binds oxygen reversibly but not in the Fe3+ state.
 The coordination site is occupied by a side chain nitrogen of His F8
(proximal histidine) residue and the site is the binding site for molecular
oxygen.These 2 sites lie perpendicular to the ring on opposite sides.
 The other histidine in the interior of the molecule His E7 (distal histidine)
on the same side as oxygen acts as a gate that opens and closes as
oxygen enters the hydrophobic pocket to bind to the heme. It sterically
inhibits oxygen from binding perpendicularly to the heme plane
His F7

binding site of
oxygen

His F8
• Isolated heme binds carbon monoxide (CO) 250 times more strongly
than oxygen.
• CO is present in small quantities in the atmosphere and arises in
cells from the catabolism of heme.
CO does not completely displace O2 from heme iron?
• The accepted explanation is that the apoproteins of myoglobin and
hemoglobin create a hindered environment.
• In myoglobin and hemoglobin the distal histidine sterically prevents
the orientation of Fe, C and O perpendicular to the plane of heme.
• Binding at a less favored angle reduces the strength of the heme-CO
bond.
• Great excess of O2 over CO normally present, dominates heme-O2
bond.
 Structural changes that occur on binding of oxygen to
haemoglobin
• In deoxyhemoglobin, the Fe atom lies out of the
porphyrin plane by about 0.04nm or about 0.06 nm.
• As the Fe atom moves, it drags histidine F8 and the helix F
along with it.
• These shifts are transmitted to the subunits where they
trigger conformational re-adjustments that lead to the
rupture of interchain salt links.
• Deoxyhemoglobin resists oxygenation because the deoxy
form is stabilized by specific hydrogen bonds and salt
bridges.
• In deoxyhemoglobin, with all of these interactions
intact, the C-termini of the four subunits are restrained,
and this conformational state is termed T (the tense or
taut) form.
• The binding of the first O2 molecule to deoxyHb shifts
the heme iron towards the plane of the heme ring.
• This motion is transmitted to the proximal histidine F8
and to the residues attached to it.
• The shift in helix F upon oxygenation leads to rupture of
salt bridges between the carboxyl terminal residues of
all four subunits.
• In oxyhemoglobin, C-termini almost has complete freedom of
rotation, and the molecule is now in its R (relaxed) form.
• Oxygen is accessible only to the heme groups of the α-chains
when hemoglobin is in the T conformational state and the
heme of β-chains in the T state is virtually inaccessible because
of steric hindrance by valine residue in the E helix.
• This hindrance disappears when the hemoglobin molecule
undergoes transition to the R state.
• The conformational changes significantly increase the affinity
of the remaining unoxygenated hemes for O2.
• The terms T and R also are used to refer to the low affinity and
high-affinity conformations, respectively.
• The binding of oxygen to hemoglobin is loose and
reversible.
• It is governed by the following factors:
1.Partial pressure of oxygen (PO2)
2.Partial pressure of carbon dioxide (PCO2) .
3.pH (acidosis) – the effect of H⁺ is also known as
the Bohr effect
4.Temperature
• Protons, carbon dioxide, and the metabolite 2,3-
bisphosphoglycerate (or 2,3 BPG), all affect the binding of O2 to
haemoglobin.
• Low pH favors T form, which has low oxygen affinity and releases
oxygen to the tissues.
• Thus, as the pH decreases, dissociation of O2 from hemoglobin is
enhanced.
• Protons arise from rupture of salt bridges during the binding of O2
to T state.
• When a tissue's metabolic rate increases, its carbon dioxide
production increases.CO2 affect O2 binding to Hb similar to that of
H+, partly because it produces H+ when it dissolves in the blood:
 carbonic
anhydrase
−
+¿ + 𝐻𝐶𝑂 3 ¿
𝐶𝑂 2 + 𝐻 2 𝑂 ⇋ 𝐻 2 𝐶𝑂 3 ⇌ 𝐻
carbonic acid bicarbonate

• HCO3- causes the pH of tissues to decrease, and so,

promotes the dissociation of oxygen from hemoglobin to
the tissue, allowing the tissue to obtain enough oxygen
to meet its demands.
How the oxygen Saturation curve of hemoglobin is
affected by different factors
 Effect of Temperature
• Increase in metabolic activity increases temperature.
• Increase in temperature decreases Hb affinity for O2 by
weakening and denaturing the bond between O2 and Hb.
• Decreasing affinity increases O2 unloading to the tissues.
Effect of pH
• When O2 binds to Hb protons are released due to conformational
changes in the Hb.
Hb + O2 HbO2 + H+
• Active tissue produces protons.
• Low pH, the H+ protonates terminal amino acids driving it into the
T-state. This increases oxygen unloading to the tissues.
 Effect of CO2
• Increased metabolic activity increases CO2. Most of the CO2 content
(80 –90%) is transported as bicarbonate ions. The formation of a
bicarbonate ion will release a proton into the plasma.
• Lowering the pH.
• Increased O2 unloading to tissues.
Effect of 2,3-bisphosphoglycerate (2,3-BPG)
• Produced in red cells
• Production increased under conditions of low O2 such as anaemia
and high altitude.
• In tissues 2,3-BPG decreases affinity of Hb for O2 enhancing O2
unloading
Subunit Composition of different types of Hemoglobin

• The subunit composition of the principal

hemoglobin’s are:
• α2β2 (Hb A; normal adult hemoglobin),
• α2γ2 (Hb F; fetal hemoglobin),
• α2S2 (Hb S; sickle cell hemoglobin),
• α2δ2 (Hb A2; a minor adult hemoglobin).
 Fetal Haemoglobin
• Fetal Hb differs from adult Hb in that the β-chains are
replaced by very similar, 146-residue subunits called γ-
chains - fetal Hb is α2γ2.
• 2,3 BPG binds less effectively with the γ-chains of fetal Hb
(Hb F).
• Hence fetal Hb has higher affinity for oxygen.
• This enables the foetus to extract oxygen from maternal
circulation.
• Fetal γ-chains have Ser instead of His at position 143 (H21),
and thus lack two of the positive charges required for 2,3
BPG binding.