Download Heme group

Protein Classification
• Simple – composed only of amino acid residues
• Conjugated – contain prosthetic groups
(metal ions, co-factors, lipids, carbohydrates)
Example: Hemoglobin – Heme
Protein Classification
• One polypeptide chain - monomeric protein
• More than one - multimeric protein
• Homomultimer - one kind of chain
• Heteromultimer - two or more different chains
(e.g. Hemoglobin is a heterotetramer. It has two
alpha chains and two beta chains.)
Protein Classification
Fibrous –
1)
2)
3)
4)
polypeptides arranged in long strands or sheets
water insoluble (lots of hydrophobic AA’s)
strong but flexible
Structural (keratin, collagen)
Globular –
1)
2)
3)
4)
polypeptide chains folded into spherical or
globular form
water soluble
contain several types of secondary structure
diverse functions (enzymes, regulatory proteins)
Protein Function
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Catalysis – enzymes
Structural – keratin
Transport – hemoglobin
Trans-membrane transport – Na+/K+ ATPases
Toxins – rattle snake venom, ricin
Contractile function – actin, myosin
Hormones – insulin
Storage Proteins – seeds and eggs
Defensive proteins – antibodies
Globular Proteins
Myoglobin/Hemoglobin
Hemeproteins: group of specialized proteins that contain heme
group as a tightly bound prosthetic group.
prosthetic group: is a non-protein compound that is permanently
associated with protein
The role of heme group is dependent on the environment created by
the three-dimensional structure of the protein.
e.g. heme in cytochrome  electron carrier,
enzyme catalase  active site
Myoglobin and hemoglobin  Oxygen carrier
Myoglobin/Hemoglobin
 First protein structures
determined
 Oxygen carriers
 Hemoglobin transport O2
from lungs to tissues
 Myoglobin O2 storage
protein
Structure and function of Hemoglobin
Mb and Hb subunits structurally similar
•8 alpha-helices
•Contain heme group
•Mb monomeric protein
•Hb heterotetramer (α22)
- Hb found exclusively in red
blood cells
-transport O2 from lungs to
capillaries of tissues and
transfer CO2 from tissues to
lungs
-composed of 4 polypeptides
held together by non-covalent
interaction
-each subunit is similar to
myoglobin and contains a
heme group
myoglobin
hemoglobin
-Myoglobin is O2 binding protein found in almost all mammals mainly in
muscles and heart
 -its main function is to store O2 for periods where energy demands is high, it
also increases the rate of transport of oxygen within the muscle cells.
- Compact structure, 80% of its polypeptide chain is α-helix that labeled A to h
that terminated by Proline or by -bends
The interior of the
myoglobin is composed of
NON-polar a.a. they packed
together stabilized by
hydrophobic interaction.
Charged a.a are located at
the surface.
Structure of Heme
Heme is a complex of protoporphyrine IX (4 pyrrole rings linked by methene
bridges) and ferrous iron (Fe+2)
Ferrous ion has 6 coordination bonds: 4 with the N of pyrrole rings and 2 are
perpendicular one with N of histidine and the other is with O2
protoporphyrine IX
porphyrine
 Heme = Fe++ bound to
tertapyrrole ring
(protoporphyrin IX complex)
 Heme non-covalently bound
to globin proteins through His
residue
 O2 binds non-covalently to
heme Fe++, stabilized through
H-bonding with another His
residue
 Heme group in hydrophobic
crevice of globin protein
Heme group
 Heme = Fe++ bound to
tertapyrrole ring
(protoporphyrin IX complex)
Distal histidine: stabilizes the
binding of O2 to heme
 Heme non-covalently bound
to globin proteins through
His residue
 O2 binds non-covalently to
heme Fe++, stabilized through
H-bonding with another His
residue
 Heme group in hydrophobic
crevice of globin protein
Proximal histidine
Oxygen binding to
Heme group
Distal histidine: stabilizes the
binding ofO2 to heme
Heme
Oxygen
Ferrous ion
Proximal histidine
Hb tetramer can be described as two identical dimers, (α)1 and (α)2
The interaction between α and  subunits is strong (hydrophobic, ionic and
hydrogen interactions )
The interaction (α)1 and (α)2 is weak interaction (primary hydrophobic).
The two dimers can move with respect to each other two conformations
according to the presence or absence of O2
T-form (Taut or tense): the deoxy form of Hb. The two α dimers interact
through a network of ionic bonds and hydrogen bonds that constrain the
movement of the dimer. This form is the low O2 affinity form of Hb
R-form (relaxed form): the binding of O2 causes rupture of some ionic bonds and
H-bonds  the polypeptide chains have more freedom of movement. The R-form is
the high O2 affinity form of Hb.
Oxygen binding to Myoglobin
Myglobin has one heme group bind only one oxygen molecule.
Hemoglobin has 4 heme groups  bind to 4 oxygen molecules
O2 dissociation curve has hyperbolic shape
affinity than of
Hemoglobin
P50 of Mb is about 1
mmHg and for Hb is 26
P50 is O2 Partial pressure
needed to half saturation of
the Mb of Hb
Degree of saturation
Myglobin has higher O2
Oxygen dissociation curve
Concentration of Oxygen (Partial pressure)
Oxygen
transport
proteins
Efficient O2 transport
protein should bind to
O2 at high partial
pressure (loading in
lung) and release it
(low affinity) at low
Partial pressure of
(unloading in the
tissue)
Oxygen Binding Curves
•Mb has hyperbolic O2
binding curve
tissues
lungs
•Mb binds O2 tightly.
Releases at very low pO2
•Hb has sigmoidal O2
binding curve
•Hb high affinity for O2
at high pO2 (lungs)
Strong-binding
Transition from weak to
strong binding
•Hb low affinity for O2 at
low pO2 (tissues)
Weak-binding
O2 Binding to Hb shows positive cooperativity
O2 Binding to Hb shows sigmoidal shape,  low
binding affinity at low con of Oxygen and high affinity Hb
at higher con
cooperative binding by the four subunit of Hb
 The binding of one O2 molecule at one heme group
increases the oxygen affinity of the remaining heme
groups in the same hemoglobin molecule. The affinity
of hemoglobin for the last O2 bound is 300 times
greater than its affinity for the first O2
O2 affinity increases as each O2 molecule binds
Increased affinity due to conformation change
Deoxygenated form = T (tense) form = low affinity
Oxygenated form = R (relaxed) form = high affinity
Hemoglobin is efficient in delivering the O2 to the
tissues from lung, myglobin which has hyperbolic
O2-dissociation curve is unable to do that
Increasing
affinity for
O2
Cooperative O2 Binding to Hb
Myoglobin-Oxygen binding
Allosteric Interactions
• Allosteric interaction occur when specific molecules
bind a protein and modulates activity
• Allosteric modulators or allosteric effectors bind
reversibly to site separate from functional binding or
active site
• Modulation of activity occurs through change in
protein conformation
• 2,3 bisphosphoglycerate (BPG), CO2 and protons are
allosteric effectors of Hb binding of O2
How is CO2 Exported?
CO2 + H2O  H2CO3  HCO3 + H+
Most of the CO2 produced in metabolism is hydrated and transported as
bicarbonate ion. the hydration of CO2 by the zinc-dependent enzyme carbonic
anhydrase.
Binding of Hemoglobin to CO2
Some CO2 is carried as carbamate bound to the uncharged α-amino group
Carbon dioxide is transported in the form of a carbamate on the amino
terminal residues of each of the polypeptide subunits.
Direct binding of CO2 to Hb stabilizes the T- form (deoxy) of Hb  resulting
in a decrease in its affinity for oxygen
The formation of a carbamate also results in release of a proton into solution
 indirectly induces the Bohr effect
H+
O
C
O
H
H2 N
H
O
C
C
R
O
Protein
Amino Terminus
C
O
H
N
C
C
R
O
Protein
Carbamate on Amino Terminus
Bohr Effect
 Increased CO2 leads to decreased
pH
CO2 + H2O <-> HCO3- + H+
 At decreased pH several key a.a’s
protonated, causes Hb to be
converted to T-conformation
(low affinity)
HbO2 + H HbH + O2.
 Deoxy form of Hb has higher
affinity of H than O2
 Protonation of some amino acids
stabilizes the deoxyhemeglobin
(T-form)
 HCO3- combines with N-terminal
α-amino group to form carbamate
group.
 Carbamation stabilizes Tconformation
Bisphosphoglycerate (BPG)
 2,3-Bisphosphoglycerate is an
important allosteric effecter of
hemoglobin Binding of BPG to Hb
causes low O2 affinity
 One molecule binds at the
interface of all four subunits, and
makes contacts with the β subunits. BPG binds in the cavity
between β-Hb subunits
 Its binding stabilizes the
deoxyhemoglobin state so
stabilizes T-conformation. This
promotes oxygen dissociation
from oxyhemoglobin.
2,3-Bisphosphoglycerate
 2,3-BPG concentration increases in response to chronic hypoxia as
in pulmonary obstruction or to high altitude or chronic anemia.
 2,3-BPG is present in erythrocytes at about 5 mM (at sea level), At
high altitudes it is present at 8 mM. Shifts the curve to the right 
increase the O2 delivery to the tissue.

At high altitudes, wherein the partial pressure of oxygen is low,
one would want hemoglobin to give up more of its bound oxygen
to the tissues
 Fetal Hb (α22) has low affinity for BPG, allows fetus to compete
for O2 with mother’s Hb (α2 β2) in placenta

Role of 2,3-BPG in transfused blood
Carbon monoxide binding to Hb
• CO binds tightly to one or more of heme iron forming carbon
monoxyhemoglobin (HbCO) and hemoglobin is shifted to R-form 
causing the remaining heme with high O2 affinity  shifts the O2binding curve to the hyperbolic (left)  inability of affected hemoglobin
to deliver O2 to the tissue
Formation of methemoglobin:
• oxidation of the heme component of Mb and Hb into ferric (Fe+3) state
form metmyglobin and methmoglobin
• The oxidized heme can't bind the O2
• This oxidation can result from drugs or toxins or from inherited defects
• Occasional oxidation of heme is corrected by the enzyme NADHcytochrome b5 reductase that found in the red blood cell.
• Methemoglobin binds strongly to CN (poison that inhibits the cytochromal
electron transport), so in the case of the cyanide poisoning amyle nitrite is
taken which able to oxidize the heme group  sequestering the CN
Types of Hemoglobins
There are 4 different types of hemoglobins known:
The most common is Hb A that form 90% of total Hb and consists of α2 β2
Hb F (α22) less than 2%
Hb A2 (α22) 2-5%
Hb A1C (α2 β2-glucose) 3-9 %
Fetal hemoglobin (Hb F): tetramer α22
• Hb F is major Hb in the fetus and newborn. During the last month of pregnancy , it
accounts for 60 % of the total Hb.
• In the first few weeks of pregnancy embryonic Hb is synthesized Hb Gower1 (ζ2ε2)
after that the liver starts HbF synthesis. After the development of the bone marrow
the Hb A is synthesized at about the eighth month of the pregnancy and gradually
replaces the Hb F
Binding of the 2,3-BPG to HbF (α22)
• HbF has higher affinity for O2 than dose HbA, bec it has lower binding affinity to
2,3-BPG and this facilitates the transfer of O2 from maternal circulation across the
placenta to the red blood cells of the fetus
• 2 globin chains (HbF) lack some positively charged amino acids found that found in
the β globin (Hb A) reduce the 2,3BPG binding  higher affinity to O2
Hemoglobin A2 (Hb A2 (α22))
• Hb A2 is a minor component of normal
adult hemoglobin, appear firstly about 12
week after the birth and can form about 2%
of the total Hb
Hemoglobin A1c
• Under physiologic conditions HbA is slowly
and non-enzymatically glycosylated
• The extent of glycosylation is dependent on
the plasma level of particular hexoses
• The most abundant glycosylated Hb is
HbA1c which has glucose unit that
covalently linked to amino group of Nterminal valines of the beta chain
• In the case of Diabetes mellitus, the amount
of HbA1c will increase
Hemoglobinopathies
Defined as a family of disorders caused either by production of structurally
abnormal hemoglobin molecule, synthesis of insufficient quantities of normal
hemoglobin or rarely both
 Sickle- cell anemia (HbS)
 Hemoglobin C disease (HbC)
 Thalassemia
Sickle- cell anemia (Hemoglobin S disease “HbS”)
• a glutamate residue is replaced by valine residue in the β-chains. This results
in two fewer negative charges for the tetrameric structure.
• The substitution of a hydrophobic amino acid for a hydrophilic one makes the
resulting molecule “sticky.” This is because a hydrophobic patch has been
created, which causes molecules to stick together at this point. This causes
aggregation to occur in deoxyhemoglobin.
• Subsequent to strand formation, several strands can assemble to form an
insoluble fiber, which is what gives sickled cells there shape.
• People with sickle cell anemia suffer from repeated crises brought on by
physical exertion.
Hemoglobinopathies
Sickle- cell anemia (Hemoglobin S disease “HbS”)
Hemoglobin C disease
 HbC is a hemoglobin variant having a single substitution in the sixth position
of the β-globin chain. In this case lysine is substituted.
 Patients have a relatively mild chronic hemolytic anemia and they don't suffer
from infractive crises
Hemoglobin SC disease
in this disease some β-globin chains have sickle-cell mutation and other βglobin chains carry mutation found in HbC
Thalassemias
 Thalassemia is a hereditary hemolytic disease in which an imbalance in the
synthesis of globin chains occurs
 Normally the synthesis of α-chains and β-chains are coordinated so that each
α-globin has its β-globin
 in the thalassemia the synthesis of either α- or β-globin chain is defective
α-thalassemia: defect in the synthesis of the α-globin and there are 4 different
levels of this type
β-thalassemia: β-globin is decreased or absent, there are 2 different level of this
type
The End
2,3-bisphosphoglycerate Binding to Hemoglobin
The negative charges on 2,3bisphosphoglycerate interact with
positive charges on hemoglobin (shown
in blue)
2,3-bisphosphoglycerate
binding to hemoglobin
stabilizes the T state.
Shown here is the R state of
hemoglobin, to which oxygen
has a greater affinity. Notice
how the binding site for BPG
collapses.
Sickle Cell Anemia
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Sickle cell anemia was the first condition for which a genetic
mutation was correlated with a physiological response. This is a
homozygous recessive condition, in which offspring must inherit
both of the mutated genes in order to develop the disease fully.
There are more than 300 different genetic variants of hemoglobin
that are known.
In the case of sickle cell disease, a valine residue is substituted for a
glutamate residue in the b chains. This results in two fewer
negative charges for the tetrameric structure.
The substitution of a hydrophobic amino acid for a hydrophilic one
makes the resulting molecule “sticky.” This is because a
hydrophobic patch has been created, which causes molecules to
stick together at this point. This causes aggregation to occur in
deoxyhemoglobin.
Subsequent to strand formation, several strands can assemble to
form an insoluble fiber, which is what gives sickled cells there
shape.
People with sickle cell anemia suffer from repeated crises brought
on by physical exertion. The hemoglobin content of their blood is
about 1/2 of normal erythrocytes, and the sickled cells can block
capillaries, causing severe pain.
Mutations in a- or b-globin genes can
cause disease state
• Sickle cell anemia – E6 to V6
• Causes V6 to bind to
hydrophobic pocket in deoxyHb
• Polymerizes to form long
filaments
• Cause sickling of cells
• Sickle cell trait offers
advantage against malaria
• Fragile sickle cells can not
support parasite
Structure and function of Hemoglobin
-found exclusively in red blood cells
-transport O2 from lungs to capillaries of tissues and transfer CO2
from tissues to lungs
-composed of 4 polypeptides held together by non-covalent interaction
-each subunit is similar to myoglobin and contains a heme group
Oxygen Binding Curve
Oxygen Binding Curve