Download Hemoglobin Learning Objective Hemoglobin

Molecular & Cell Biology
Hemoglobin
Hemoglobin
Hemoglobin (Hb) is an iron-containing metalloprotein that
functions to transport oxygen in the red blood cells of
vertebrates. The protein, which accounts for 97% of the RBC’s
dry content is responsible for delivering oxygen from the lungs to
all other tissues in the body where it is taken up for cell use.
Learning Objective
After interacting with this Learning Object, the learner will
be able to,
Explain the structure of Hemoglobin, oxygen binding & structural
changes.
Describe factors affecting oxygen binding & Hemoglobin disorders.
Compare the studies on Hemoglobin & Myoglobin.
Molecular & Cell Biology
Hemoglobin
Structure of hemoglobin
Hemoglobin is a heterotetramer
composed of two alpha and two beta
chains. The alpha globin gene locus
resides on chromosome 16 with each
gene contributing to the synthesis of
the alpha globin chain. The beta
globin
gene
locus
resides
on
chromosome 11 and consists of all
genes that are expressed from the
time of embryonic development of Hb
to that of the adult beta globin gene.
The globin chains are synthesized by
ribosomes in the cytosol.
Molecular & Cell Biology
Hemoglobin
Structure of hemoglobin
Every subunit of hemoglobin is bound
to a prosthetic group known as heme.
This consists of a central iron atom in
its ferrous state surrounded by a
complex organic ring structure known
as protoporphyrin. The heme group is
essential for the oxygen binding
property of hemoglobin. The iron
atom forms six coordination bonds,
four of which are to the nitrogen atom
of porphyrin rings, one to a His side
chain in the globin subunit and the
other being the binding site for
oxygen. Iron in its Fe3+ state does not
bind to oxygen.
Molecular & Cell Biology
Hemoglobin
Oxygen binding & structural changes
Hemoglobin binds four molecules of
oxygen per tetramer, one per heme, in a
cooperative manner. The first model for
cooperative binding, proposed by
Monod, Wyman and Changeux, also
known as the MWC model, assumes that
each subunit can exist in at least two
conformational states with all subunits
making the transition from one state to
the other simultaneously. Each subunit
of a cooperatively binding protein is
functionally identical and binding of
ligand can occur in either conformation
but with varying degrees of affinity for
each. Binding of ligand to a low affinity
state makes the transition to the high
affinity state more likely.
Molecular & Cell Biology
Hemoglobin
Oxygen binding & structural changes
The second model for cooperative
binding of oxygen to hemoglobin,
proposed by Daniel Koshland in 1966,
postulates that binding of a ligand to
one subunit can induce conformational
changes in the other subunits.
However, unlike the concerted model,
it does not state that all subunits must
transition from one state to the other
simultaneously. Any conformational
change brought about in one subunit
enhances the likelihood of a similar
change as well as binding of a ligand
in the neighboring subunit. A larger
number of intermediate states are
therefore more likely in the sequential
model.
Molecular & Cell Biology
Hemoglobin
Oxygen binding & structural changes
Two major conformations of Hb have
been deduced by X-ray analysis – the T
state which is stabilized in the absence
of oxygen and the R state which is
relatively more stable in the presence
of oxygen. The pair of identical 
dimers of Hb are linked by several ion
pairs at the interface that stabilize it in
its deoxy state. Many of these
interactions are disrupted upon binding
to oxygen and new ones are formed
instead. The porphyrin ring in the T
state is found to be slightly puckered,
thereby causing the iron atom to lie out
of plane and protrude onto the proximal
histidine.
Molecular & Cell Biology
Hemoglobin
Oxygen binding & structural changes
Upon binding to oxygen, one  pair
of subunits rotates with respect to the
other
by
around
15o.
Several
interactions at the  interaface are
therefore disrupted and new ones
formed.
As
Max
Perutz
rightly
postulated, there are changes in the
position of many key amino acid
residues upon oxygenation. Binding of
oxygen causes the iron atom to move
back into the plane of the porphyrin
ring thereby pulling the proximal
histidine along with it. Once the
transition from T state to R state takes
place in one subunit, the remaining
subunits also undergo the same
transition
more
readily,
thereby
favouring the cooperative binding to
oxygen.
Molecular & Cell Biology
Hemoglobin
Oxygen binding & structural changes
Cooperative binding of oxygen to
hemoglobin allows efficient transfer of
oxygen from lungs to tissues. The high
partial pressure of oxygen in lungs
allows Hb to be saturated upto 98%
due to the cooperative nature while
saturation level in tissues drops to 32%
due to lower partial pressure. This
allows 66% of the oxygen taken up to
be released. In case of proteins
showing no cooperativity, however,
the maximum amount of oxygen that
can be delivered under similar
conditions is only around 38% due to
less uptake and relatively less release.
This cooperative binding nature leads
to a sigmoidal binding curve for Hb
allowing it to deliver 1.7 times more
oxygen compared to non-cooperative
proteins.
Molecular & Cell Biology
Hemoglobin
Factors affecting oxygen binding
The effect of pH and CO2 concentration on the
binding and release of oxygen by hemoglobin is
called the Bohr effect. Lowering the pH and
raising the partial pressure of carbon dioxide
results in the release of O2 from
oxyhemoglobin. In peripheral tissues, CO2
released from Krebs cycle and other cellular
processes combines with water to form
carbonic acid, which dissociates into protons
and bicarbonate ions. Hb which has just
released its bound oxygen into the tissues acts
as a buffer by binding protons and delivering
them to the lungs. In the lungs, the uptake of
oxygen by hemoglobin releases protons that
combine with bicarbonate ion, forming
carbonic acid, which when dehydrated by
carbonic anhydrase becomes carbon dioxide,
which is then exhaled.
Molecular & Cell Biology
Hemoglobin
Factors affecting oxygen binding
The chemical basis for the effect of pH has
been found to lie in the interactions of the
various side chains in hemoglobin. In
deoxyHb, the -His 146 forms a salt bridge
with a lysine residue in the  subunit.
This interaction is however not possible
when the His residue is deprotonated,
which occurs at high pH. Upon lowering
the pH, protonation of the His residue
brings about salt bridge formation,
thereby favouring the deoxy form and
leading to release of bound O2. CO2 reacts
with the terminal amino groups forming
negatively charged carbamate groups,
thereby stabilizing the deoxyHb form.
Molecular & Cell Biology
Hemoglobin
Factors affecting oxygen binding
2,3-bisphosphoglycerate is a highly anionic
compound that is present in RBCs at around
the same concentration as Hb. 2,3-BPG
binds to a central pocket of the T form of
Hb tetramer and stabilizes it by interacting
with three positively charged amino acids
on each β-chain. Transition from T to R
state requires bonds to be broken between
Hb and 2,3-BPG. This enhances the oxygen
releasing capacity of Hb, without which it
would be an extremely inefficient
transporter molecule.
Molecular & Cell Biology
Hemoglobin
Hemoglobin disorders
A large number of mutations have been
described in the globin genes. The mutation
causing sickle cell anemia is a single
nucleotide substitution of A to T in the
codon for amino acid 6 of the  chain. This
change converts a glutamic acid residue to
valine in the corresponding amino acid
sequence. Replacement of the Glu residue
by Val creates a “sticky” hydrophobic
contact point at position 6 of the β chain.
These sticky spots cause deoxyHbS
molecules to associate abnormally with
each other leading to clumping of the cells.
Their oxygen carrying capacity is greatly
reduced and these patients require frequent
transfusions.
Molecular & Cell Biology
Hemoglobin
Hemoglobin disorders
Thalassemia are the result of abnormalities
in hemoglobin synthesis. Deficiencies in βglobin
synthesis
result
in
the βthalassemias . Mutation of a single base
from G to A in an intron of the β-globin gene
generates a new splice site. The resulting
mRNA contains a stop codon further
upstream
and
leads
to
premature
translation termination thereby producing
aberrant protein. Deficiencies in α-globin
synthesis due to inactivation of one or all
the four α-globin genes results in the αthalassemias.
Molecular & Cell Biology
Hemoglobin
Comparative study of hemoglobin
& myoglobin
X-ray crystallography is a very useful
visualization technique that facilitates the
determination
of
three-dimensional
coordinates of atoms in a protein. Myoglobin
was the first protein whose structure was
determined
by
X-ray
crystallography
studies. When a beam of X-ray was passed
through the crystals of myoglobin, some
part of the beam was found to pass straight
through while the others were scattered in
different directions. These scattered beams
were detected by means of an X-ray film
which, after several spot intensity
calculations, provided an electron density
map of the protein. The protein was found
to consist of a single polypeptide chain
having eight alpha helices along with a
heme group in the centre similar to
hemoglobin.
Molecular & Cell Biology
Hemoglobin
Comparative study of hemoglobin
& myoglobin
Myoglobin, found largely in muscle tissues,
has been found to be structurally similar to
the alpha subunit of hemoglobin. The alpha
helix arrangement of both proteins has been
found to be the same with the recurring
structure being known as the globin fold.
The hemoglobin chain having 141 amino
acids and myoglobin having 153 residues
have also been found to have very high
sequence
homology.
Despite
the
similarities, myoglobin functions largely as
an oxygen binding protein that stores a
reserve supply of oxygen in muscle tissues
while hemoglobin serves to transport
oxygen.
Molecular & Cell Biology
Hemoglobin
Comparative study of hemoglobin
& myoglobin
Myoglobin binds strongly to oxygen and acts
as an oxygen storage protein rather than a
transporter. It shows 50% saturation at a
pressure as low as 2 torr and gets saturated
even under low oxygen pressure conditions
that prevail in the muscle. Myoglobin can
use only 7% of the oxygen carrying capacity
as opposed to hemoglobin which can utilize
nearly 90% of the carrying capacity. Unlike
hemoglobin, which has a sigmoidal oxygen
binding curve, myoglobin has a hyperbolic
curve indicating that it binds to oxygen
irrespective of the surrounding partial
pressure of oxygen in the tissues. It is this
property that allows sea mammals such as
whales that have very high amounts of
myoglobin in their muscle to remain
underwater for long periods of time.
Molecular & Cell Biology
Hemoglobin
Structure of Hemoglobin
1.Hemoglobin: Hemoglobin isthe predominat metalloprotein in the red blood cells that
carries oxygen. Each hemoglobin molecule is
made up of four heme groups surrounding a
globin group.
2. ,  subunits: Subunits that occur in the
functional organization of macromolecules,
usually proteins.
3. Heme: Heme is a prosthetic group
containing carbon, nitrogen and hydrogen
atoms, with a single Fe2+ ion at the center.
4. Prosthetic group: A tightly bound non –
protein chemical compound that is required for
the activity of an enzyme.
5. Protoporphyrin IX: A metal-free porphyrin
that combines with iron to form the heme of
iron-containing proteins. It is made up of four
pyrrole rings linked by methene bridges to form
a tetrapyrrole ring. Four methyl groups, two
vinyl groups, and two propionate side chains are
attached.
Molecular & Cell Biology
Hemoglobin
Oxygen binding and structural changes
6. Cooperative binding: It is a form of
allosteric binding in which ligand binding to
macromolecules having more than one binding
site is carried out in a cooperative manner
such that binding of ligand at one site
increases the affinity of another site for the
ligand. In tetrameric hemoglobin, binding of
first oxygen molecule to one subunit brings
about structural changes which in turn
positively influences the binding of the
remaining subunits to oxygen.
7. Oxyhemoglobin: When all subunits of
hemoglobin are bound to oxygen, it is known
as oxyhemoglobin and it transports oxygen to
the various tissues of the body.
8. Deoxyhemoglobin: Hemoglobin in oxygen
unloaded form is called deoxyhemoglobin.
9. T state: T stands for the tense state. It is one of
the two quaternary forms of hemoglobin that
predominates in absence of oxygen. It has a lower
affinity for substrates and, hence, lower catalytic
activity.
10. Models for oxygen binding to Hb: Two models
have been proposed by different groups of
scientists to explain the binding of oxygen to
hemoglobin. These are the concerted and
sequential models:
Molecular & Cell Biology
Hemoglobin
Oxygen binding and structural changes
a) Concerted model: The first model proposed
by Monod, Wyman and Changeux,
also
known as the MWC model, assumes that each
subunit can exist in at least two conformational
states and hypothesizes that all subunits make
the transition from one state to the other
simultaneously. According to this model, each
subunit of a cooperatively binding protein is
functionally identical. Binding of ligand can
occur with either conformation but with varying
degrees of affinity for each. Binding of ligand to
a low affinity state makes the transition to the
high affinity state more likely.
b) Sequential model: The second model,
proposed by Daniel Koshland in 1966,
postulates that binding of a ligand to one
subunit can induce conformational changes in
the other subunits. Unlike the concerted
model,
it does not state that all subunits must exhibit
transition from one state to the other
simultaneously, thereby making a larger number of
intermediate states more likely.
11. R state: R stands for relaxed state. One of the
two quaternary forms of hemoglobin that
predominates when oxygen is bound. . It has a
higher affinity for substrates and, hence, higher
catalytic activity.
12. Distal histidine: Distal histidine in vertebrate
hemoglobins plays an important role in oxygen
binding and has been strongly conserved during
evolution. It is considered to be important in finetuning the ligand affinities of hemoglobin.
Molecular & Cell Biology
Hemoglobin
Oxygen binding and structural changes
13. Proximal histidine: The heme in
hemoglobin is held in the cleft both by
hydrophobic interactions and by a covalent
bond between the iron and a nitrogen atom of a
nearby histidine side chain. This histidine is
referred to as the proximal histidine.
14. Sigmoidal plot: The binding of oxygen to
hemoglobin
displays
marked
sigmoidal
behaviour, which is indicative of cooperation
between subunits. The binding of first oxygen
causes a conformational change in the other
binding sites that makes it easier for oxygen to
bind there. This explains the initial up-swing in
the sigmoidal curve. The "leveling out" at the
top of the curve is caused by saturation of the
hemoglobin-oxygen binding sites.
Molecular & Cell Biology
Hemoglobin
Factors effecting oxygen binding
15. Bohr Effect: The effect of pH and CO2
concentration on the binding and release of
oxygen by hemoglobin is called the Bohr effect.
Lowering the pH and raising the partial pressure
of carbon dioxide results in the release of O2
from oxyhemoglobin.
16. Effect of 2,3-bisphosphoglycerate: 2,3bisphosphoglycerate is a highly anionic
compound that is present in RBCs at around the
same concentration as Hb. 2,3-BPG binds to a
central pocket of the T form of Hb tetramer
and stabilizes it by interacting with three
positively charged amino acids on each β-chain.
17. Fractional saturation: It is the fraction of
active sites that are bound to the substrate and
is directly proportional to reaction velocity.
18. Oxygen partial pressure: It is the partial
pressure of oxygen in the gas phase above the
solution.
Molecular & Cell Biology
Hemoglobin
Hemoglobin disorders
19. Sickle cell anemia: Sickle-cell anemia is a
genetic disease in which an individual has
inherited the allele for sickle-cell hemoglobin
from both parents characterized by abnormal,
rigid, sickle shape (HbS) as compared to the
normal flexible biconcave disk shaped red blood
cells (HbA). It results from a single amino acid
substitution, a Val instead of a Glu residue at
position 6 in the two β chains. As a result of this
change, deoxyhemoglobin S has a hydrophobic
patch on its surface, which causes the
molecules to aggregate into strands that align
to form insoluble fibres. Sickle-cell disease may
lead
to
various
acute
and
chronic
complications, several of which are potentially
lethal.
20. Thalassemia: This is another inherited genetic
disorder due to deletions or mutations in the globin
genes which results in abnormalities and
in α-globin synthesis. Signs and
symptoms of thalassemias are due to lack of
oxygen in the bloodstream. This occurs because
the body doesn't make enough healthy red blood
cells and hemoglobin. The severity of symptoms
depends on the severity of the disorder.
deficiencies
21. Point mutation: A type of mutation resulting
from substitution of a single nucleotide base with
another.
Molecular & Cell Biology
Hemoglobin
Comparative study of Hemoglobin and Myoglobin
22. Myoglobin (Mb): Myoglobin is a globular
protein having a single polypeptide chain
consisting of eight alpha helices linked by short
polypeptide segments, with a total of 153
amino acid residues. It is found in muscle
tissues of most mammals and plays a role in
oxygen binding. Myoglobin resembles the alpha
subunit of hemoglobin and like hemoglobin, it
consists of a central heme group which enables
it to bind oxygen. Myoglobin was the first
protein to have its X-ray crystallography
structure determined in 1959 by John Kendrew.