Myoglobin and Hemoglobin

O2 Binding and Allosteric Properties of Hemoglobin

Hemoglobin binds and
transports H+, O2 and CO2 in
an allosteric manner

Allosteric interaction of,

relating to, undergoing, or being a change
in the shape and activity of a protein (as
an enzyme) that results from
combination with another substance at a
point other than the chemically active
site

a regulatory mechanism
where a small molecule
(effector) binds and alters
O2 does not easily diffuse in muscle and O2 is
toxic to biological systems, so living systems
have developed a way around this.

Physiological roles of:

Myoglobin
Transports O2 in rapidly respiring
muscle
Monomer - single unit
Store of O2 in muscle high affinity for
O2
Diving animals have large
concentration of myoglobin to keep
O2 supplied to muscles
Hemoglobin
Found in red blood cells
Carries O2 from lungs to tissues and
removes CO2 and H+ from blood to
lungs
Lower affinity for O2 than myoglobin
Tetrameter - two sets of similar units
(22)
 Small protein found in muscle
x-ray crystallography
Made up of 153 residues of myoglobin
grouped into 8 helix A to H
(proline near end)

very small due to the folding

44 x 44 x 25

hydrophobic residues
oriented towards the interior
of the protein

only polar AAs inside are 2

histidines

Red indicates Heme group

 Hemoglobin and x-ray crystallography
myoglobin are only
slightly related in of myoglobin
primary sequence.

Although most amino

acids are different
between the two
sequences, the amino acid
changes between the two
proteins are generally
conservative.

More strikingly, the

secondary structures of
myoglobin and the
subunits of hemoglobin
are virtually identical.
 Synthesis of hemoglobin
Hemoglobin synthesis requires
the coordinated production of
heme and globin.

REMEMBER:
Heme is the prosthetic group
that mediates reversible
binding of oxygen by
hemoglobin.

Globin is the protein that

surrounds and protects the
heme molecule.
 Synthesis of heme
Heme is synthesized in a complex
series of steps involving enzymes in the
mitochondrion and in the cytosol of the
cell .

(1) the condensation of succinyl CoA

and glycine by ALA synthase to form
5-aminolevulic acid (ALA).
transported to the cytosol

(2) A series of reactions produce a

ring structure - co-proporphyrinogen
III.

returns to the mitochondrion

 Synthesis of heme
Back in the mitochohdrion:-

(3) an additional reaction produces

protoporhyrin IX

This step Primes the center of the ring

structure

(4) The enzyme ferrochelatase inserts

iron into the ring structure of
protoporphyrin IX to produce heme!!
 Structure of heme prosthetic group
Protoporphyrin ring w/ iron =
heme

Four Pyrrole groups [A to D]

linked by methane bridges

Fe+2 coordinated by prophyrin N

atoms and a N from Histidine
(blue)
This is known as His F8 (8th
residue of the F helix

Iron is out of plane due to His 8 bond

A molecule of O2 acts as 6th

ligand
 Synthesis of globin

Alpha gene cluster:

Each chromosome 16 has two alpha
globin genes that are aligned one after
the other on the chromosome. For
practical purposes, the two alph globin
genes (termed alpha1 and alpha2) are
identical.

The transiently expressed embryonic

genes that substitute for alpha very
early in development, designated zeta,
are also in the alpha globin locus.
 Synthesis of globin
Beta gene cluster:
The genes in the beta globin locus are
arranged sequentially from 5' to 3'
beginning with the gene expressed in
embryonic development (the first 12
weeks after conception; called epislon).

The beta globin locus ends with the

adult beta globin gene. The sequence of
the genes is: epsilon, gamma, delta, and
beta.
 Synthesis of globin
Beta gene cluster:
There are two copies of the gamma gene on
each chromosome 11.

The others are present in single copies.

These two beta globin genes express their

globin protein in a quantity that precisely
matches that of the four alpha globin genes.

The mechanism of this balanced expression

is still mostly unknown.
 Oxygenation
Oxygen binding to hemoglobin is due
to the effect of the ligand-binding
state of one heme group on the
ligand-binding affinity of another.

Too far apart to interact! (25 to 37

apart)

Mechanically transmitted between

heme groups by motions of the
proteins

This means the molecule changes

shape!
 The two states
There are two general structural states - the deoxy or T
form and the oxy or R form.
One type of interactions shift is the polar bonds between
the alpha 1 and the beta 2 subunits.
oxy and deoxy quaternary structures are
different
change takes place between 1 - 2 and 2 - 1
amino acids between 1 and 2 help to stabilize each forms
Oxygen binding shifts
quaternary structure at long
distances

binding of O2 ligand at 6th

coordinate position pulls Fe into
heme

moves proximal histadine (F8) and

the alpha helix it is attached to.

shift in the helix is transmitted

throughout of molecule
How Do Myoglobin (Hemoglobin) Bind
to Oxygen

Move down
upon O2
binding

N His E7
NH
How Do Myoglobin (Hemoglobin) Bind
to Oxygen

Move down
upon O2
binding

N His E7
NH
So what also
allows this to
change shape?
Functional Structure of Hemoglobin -
allosteric regulations

Allosteric interaction - the binding of one ligand at

one site in a protein that affects the binding of
other ligands at other sites in the protein.

This can affect binding and can be cooperative (pos

or neg).
 diphosphoglycerate BPG
present in human red
blood cells at
approximately 5 mmol/L.

It binds with greater

affinity to deoxygenated
hemoglobin

In bonding to partially
deoxygenated hemoglobin it
allosterically upregulates the
release of the remaining
oxygen molecules bound to
the hemoglobin, thus
enhancing the ability of
RBCs to release oxygen
near tissues that need it
most
2,3 bisphosphoglycerate (BPG)

Purified Hb has a different O2

affinity than it does in blood

26 fold decrease change in
affinity is due to 2,-3
diphosphoglycerate BPG
(BPG replaced by nucleotides
IHP and ATP in fish and birds)
- 1 BPG per Hb - binds in
central cavity of Hb
- binds preferentially to deoxy
Hb
- hydrophobic bonds with Lys
and salt bridge with His
- O2 binding changes
conformation and kicks out BPG
change in altitude increases
concentration of BPG
Fetal F Hb has replaced His 143
with Ser - What might the
consequences be?
Cooperative interactions between subunits
Both models do not fully account for the effects of allosteric
effectors
sequential model (D Koshland)
binding of one O2 induces T-R conformation change
1st change is most difficult due to influence by 3 other
subunits
binding of next three subunits happens sequentially, with
higher affinity (easier T-R changes)
kinetics increase to the fully oxy Hb state as more O2 is bound
Concerted model (J Monod)
All R or all T no in between as in the
Koshland model
concerted model means as more O2
binds, the R conformation is favored
until all units are in the R conformation
regardless of the total units bound to
O2
Affinities do NOT change until
conformation changes
1 O2 - all T; 2 O2 - nearly even
equilibrium; 3 O2 mostly R; 4 O2 -
mostly R form
energy from O2 binding causes the
change in equilibrium
this model best fits O2 dissociation
curve but with limits.
Simple definition of the concept of
cooperativity. In its simplest meaning,
cooperativity is a measure of communication
between binding sites. Positive cooperativity
describes a relationship in which the binding
to one site facilitates the binding to the
second, third, ect.
No cooperativity puts all sites on equal footing
and indicates no site to site influence.
Negative cooperativity implies binding to one
site hinders binding to subsequent sites.
 Bohr Effect
The Bohr effect is the reversible shift in Hb affinity for O2 with
changes in pH.

H+ Transport (effect) - O2 binding to Hb releases H+ due to

conformational changes in Hb
- deoxyform (T form) brings Asp 94 close to His 146
- the proximity of an acidic amino acid increases the pK of histidine
(pKa is now above the pH) and results in H+ binding to deoxyHb
- in other words the His becomes protonated where it normally would
be ionized
- increasing pH stimulates Hb to bind to O2

- Bottom line - when O2 binds Hb, H+ is released from several

amino acid's functional groups. When O2 is released, the amino
acids become protonized and then "picks" up a H+.

Sowhen the H+ is high (acidic conditions) the H+ is driven onto the

terminal amino acids driving it into the T conformation
 Oxygen Dissociation Curve
Right-Shift
Hgb has less attraction
for O2
Hgb willing to release O2
to tissue
Examples: anemia,
acidosis
Even though there may
be less RBCs, they act
more efficiently to
deliver O2 to target
 Oxygen Dissociation Curve
Left shift
Hgb has more attraction
for O2
Hgb less willing to
release O2 to tissue
Examples: presence of
abnormal Hgbs,
alkalosis
Types of Hb:

Hb A or HbA1: is the normal Hb in adults represents about 97%

of total Hb. it is composed of 2 and 2 chains.

HbA2: minor adult Hb, comprised 3% of normal adult Hb.

Composed of 2 and 2 chains

HbF(fetal Hb): is the main Hb during fetal life and about 60% of
normal Hb at birth then disappear gradually. It is composed of 2
and 2 chains.
Hb F has greater affinity for O2 than HbA so ensure O2 transfer
from maternal circulation to fetus RBCs through placenta.

Note: The overall hemoglobin composition in a normal adult

is approximately 97.5% HbA1, 2% HbA2 and 0.5% HbF.
 A quick look at what
happens to blood when it
all goes wrong
 Sickle-Cell Anemia, a Molecular Disease
One of the first molecular diseases found - sickle cell anemia
sickle cell - blood cell is elongated , mis-shaped (sickle)
occurs at low O2 concentration
caused by hemoglobin aggregates
inflammation in capillaries and pain
red blood cells break down - anemia
between 10% of American blacks and 25% of African
blacks are heterozygous for sickle cell anemia
homozygous usually do not survive into adult hood
heterozygous individuals usually have no problem except
when in severe oxygen deprivation
Single amino acid (point mutation) HbS vs. HbA changes
structure
sickle cell b chains have a valine in place of glutamate
leads to more Hb S (sickle cell) has 2 more + charges
than normal hemoglobin
Glu -Val occurs on exterior of protein - does not
change O2 dissociation/allosteric properties of
protein
 Rh Factor
There are four blood groups but eight blood types.

The Rh-factor!!

85% Positive
15% Negative
Genetic factor
Can cause Hemolytic Disease and death of infants.
 The genetics of the Rh factor
Another blood grouping system independent of ABo the Rh-
factor
three genes: located very close together on the same chromosome.
First C & c, second D & d, third E & e

Unlike the ABo system there is no co-dominance, c, d, and e are

recessive to C, D, and E.

ccddee is known as Rh-negative. All others Rh-positive.

 Hemolytic disease
If a child is Rh+, a Rh- Mother can begin to produce
antibodies Rh+ red blood cells
Rh factor crosses placenta and mother makes antibodies
In subsequent pregnancies these antibodies can cross the
placenta and cause hemolysis of a Rh+ Childs red blood
cells.
Can lead to mental retardation or death
Prevented by giving Rh- women a Rh immunoglobulin
injection no later than 72 hours after birth. Attacks
any of the babies Abs in mother before her own
antibodies are produced
Hemolytic disease
Figure 7.5 (1)
Hemolytic disease
Figure 7.5 (2)
 Hemolytic disease
Figure 7.5 (3)

Prevented by giving Rh- women a Rh

immunoglobulin injection no later than
72 hours after birth.

Attacks any of the babies Abs in

mother before her own antibodies are
produced.
 Thalassemia
A group of genetic diseases in which a defect
occur in the rate of synthesis of one or more of
Hb chains, but the chains are structurally normal.
This due to defect or absence of one or more of
genes responsible for synthesis of or chains
leading to premature death of RBCs.
 Types

-thalassemia: When synthesis of chains is decreased or absent.

There are two copies of the gene responsible for synthesis of chains.
Individuals with globin gene defects have either :
- -thalassemia minor ( thalassemia trait) : when the synthesis of
only one globin gene is defective or absent. Those individuals make
some chains and usually not need specific treatment.
- -thalassemia major ( Cooley anemia): if both genes are defective.
-Babies will be severely anemic during the first or second year of life and
so require regular blood transfusion. Bone marrow replacement is more
safe treatment.

Fig. 10.11
--thalassemia: in which synthesis of globin chain is defective
or absent. There are four copies of gene responsible for
synthesis of globin chains so patients may have:
i - Silent carrier of -thalassemia with no symptoms:
if one gene is defective
ii- -thalassemia trait: if two genes are defective.
iii- Hb H disease: if 3 globin genes are defective, with mild to
moderate anemia. The produced Hb will be 4 which is called
HB H. Oxygen delivery to tissues will be blocked because Hb H
(4 ) has high affinity to O2 and not deliver it to tissues.
iv- Hydrops fetalis: when all 4 globin genes are defective. It
causes fetal death because globin chains are required for
synthesis of Hb F.
The End, at last!