Download Protein Function – Myoglobin and hemoglobin

Protein Function
Myoglobin and Hemoglobin
Chapter 7
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 an
enzymes activity
Protein Function
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 (22)
– Small protein found in muscle
– Made up of 153 residues
grouped into 8  helix A to H
(proline near end)
– very small due to the folding
 44 x 44 x 25 Å
– hydrophobic residues
oriented towards the interior
of the protein
– only polar AAs inside are 2
histidines
– Red indicates Heme group
x-ray crystallography
of myoglobin
– Hemoglobin and
myoglobin are only
slightly related in
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.
x-ray crystallography
of myoglobin
Myoglobin and Hemoglobin




Hemoglobin is structurally related to myoglobin
very different primary sequence about an 18%
homology in the primary sequence
2 alpha subunits and 2 beta subunits
in adults there are very small amount of alpha
2delta 2 hemoglobin
- significance of conserved amino acids
between myoglobin and hemoglobin
• these are the important aas which
keep hemes in contact with the
protein
• Stabilizes helical arraignment
• Interacts with heme/iron
3D structure of hemoglobin and myoglobin




1-  1 units have 35
interactions
 1-  2 units have 19
interaction sites
similar units have few
polar contacts
the two  and two 
subunits face each other
through aqueous channels
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
Structure of heme prosthetic group

Two hydrophobic sidechains on
O2 binding site of heme elp hold it
in place
– Valine E11 and phenylalanine
CD1

oxygenation changes state of Fe
– Purple to red color of blood,
Fe+3 – brown

Oxidation of Fe+2 destroys
biological activity of myoglobin

Physical barrier of protein is to
maintain oxidation state of Fe+2
free vs. bound heme - role
of apoprotein

Apoprotein -
the protein moiety of a
molecule or complex

restricts heme dimers

keeps iron reduced


stabilizes transition state (O2
binding)
CO, NO and H2S binding greater affinity than O2
His E 7 decreases affinity of
ligands (CO and O2 ) for Fe+2
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.
Therefore, each cell has two beta globin
genes, one on each of the two chromosomes
11 in the cell.
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.
What about the other genes?





Two distinct globin chains (each with its
individual heme molecule) combine to
form hemoglobin. One of the chains is
designated alpha.
The second chain is called "non-alpha".
With the exception of the very first
weeks of embryogenesis, one of the
globin chains is always alpha.
A number of variables influence the
nature of the non-alpha chain in the
hemoglobin molecule.
The fetus has a distinct non-alpha chain
called gamma.
After birth, a different non-alpha
globin chain, called beta, pairs with the
alpha chain. The combination of two
alpha chains and two non-alpha chains
produces a complete hemoglobin
molecule.
What about the other genes?




The combination of two alpha chains and
two gamma chains form "fetal"
hemoglobin, termed "hemoglobin F".
With the exception of the first 10 to
12 weeks after conception, fetal
hemoglobin is the primary hemoglobin in
the developing fetus.
The combination of two alpha chains and
two beta chains form "adult"
hemoglobin, also called "hemoglobin A".
Although hemoglobin A is called "adult",
it becomes the predominate hemoglobin
within about 18 to 24 weeks of birth.
Developmental gene expression!!!!!


Embryonic hemoglobins
zeta(2), epsilon(2)
alpha(2), epsilon (2)
zeta(2), gamma (2)

Fetal hemoglobin

hemoglobin F- alpha(2), gamma(2)

Adult hemoglobins

hemoglobin A- alpha(2), beta(2)
hemoglobin A2- alpha(2), delta(2)
Developmental gene expression!!!!!





The globin genes are activated in sequence
during development, moving from 5' to 3'
on the chromosome.
The zeta gene of the alpha globin gene
cluster is expressed only during the first
few weeks of embryogensis.
Thereafter, the alpha globin genes take
over.
For the beta globin gene cluster, the
epsilon gene is expressed initially during
embryogensis.
The gamma gene is expressed during fetal
development.
– The combination of two alpha genes
and two gamma genes forms fetal
hemoglobin, or hemoglobin F.
Developmental gene expression!!!!!




Around the time of birth, the production of
gamma globin declines in concert with a rise
in beta globin synthesis. A significant amount
of fetal hemoglobin persists for seven or
eight months after birth.
Most people have only trace amounts, if any,
of fetal hemoglobin after infancy.
two alpha genes and two beta genes
comprises the normal adult hemoglobin,
hemoglobin A.
The delta gene, which is located between
the gamma and beta genes on chromosome
11 produces a small amount of delta globin in
children and adults. The product of the
delta globin gene is called hemoglobin A2,
and normally comprises less than 3% of
hemoglobin in adults, is composed of two
alpha chains and two delta chains.
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
Å 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.
– The T form finds the
terminals in several important
H bonds and salt bridges.
– In the T form the C terminus
of each subunit are "locked"
into position through several
hydrogen and ionic bonds.
– Shifts into the R state break
these and allow an increased
movement throughout the
molecule.

Note that binding of one or
more oxygen can have a
dramatic affect on the other
subunits that have not yet bound
an O2.
Binding of oxygen dramatically alters the interactions and brings about a
twisting of the two halves (alpha beta pairs)
Much of the quaternary changes takes place in the salt bonds between the
C terminals of all four chains
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



T and R State of Hemoglobin
Below are the two major conformations of
hemoglobin as predicted by the models for
allosteric activation.
Oxygen will bind to hemoglobin in either state;
however, it has a signficantly higher affinity
for hemoglobin in the R state.

T and R State of Hemoglobin
In the absence of oxygen, hemoglobin is more stable in
the T state, and is therefore the predominant form of
deoxyhemoglobin. R stands for relaxed, while T stands
for tense, since this is stabilized by a greater number
of ion pairs.


Upon a conformational change from the T state to the
R state, ion pairs are broken mainly between the a1b2
subunits.

– The T form finds the terminals in several important H
bonds and salt bridges.
– In the T form the C terminus of each subunit are
"locked" into position through several hydrogen and
ionic bonds.
– Shifts into the R state break these and allow an
increased movement throughout the molecule.
Note that binding of one or more oxygen can have a
dramatic affect on the other subunits that have not yet
bound an O2.
How Do Myoglobin (Hemoglobin) Bind
to Oxygen
Move down
upon O2
binding
His E7
N
NH
How Do Myoglobin (Hemoglobin) Bind
to Oxygen
Move down
upon O2
binding
His E7
N
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).
Allosterism is typically seen when sigmoidal binding
/ activity curves.
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
So why is this important.
Look at the Hill plot and the plot of
the of binding vs pO2 (figs 7-8 and 7-7 respectively). Think of when
hemoglobin should be mostly saturated and when it would be best if it
had a low saturation / affinity and thus "give up" its oxygen. Use the
table below to help your thoughts.
Oxygen pressure in various fluids
Region or fluid pO2 (Torr)
Inspired Air
158
Aveolar Air
100
Arterial Blood
90
Capilary
40
Insterstitual Fluid
30
Cell Cytosol
10
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.
How is this cooperation actually forced on
hemoglobin?

The structures in one subunit effects the others
• Structural differences of bound and unbound Hb
– Two structures - T and R form depending on the
oxygenation of Hb
– T is the deoxy form, R is oxy form
– allosteric modifiers shift or stabilize one particular
conformation over the other
– Monomer interactions, most chain interaction occurs
between  and  chains
» through mostly hydrophobic residues
»  and interactions are few and polar
»  and  contact act as a switch
CO2 effects - In the red blood cells the picture is even more
complicated. CO2 is removed by converting CO2 to bicarbonate
CO2 + H2O <-> HCO3- + H+
bicarbonate (HCO3-) formed by the enzyme carbonic anhydrase
–H+ produced by this enzyme is removed by the Hb as described
above
–This allows more CO2 to be removed in the form of bicarbonate
CO2 binding aids in reducing O2 affinity by
changing conformation by the production of
more H+ (R to T change)
In
the lungs the O2 binds Hb and forces
the R conformation
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 (fig 7-11 (b))
- 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+.
 So
when the H+ is high (acidic conditions) the H+ is driven onto the
terminal amino acids driving it into the T conformation

This all aids the function of Hb.
In active
tissues respiration, (glycolysis) results in lactic acid formation.
These tissues need more O2. Without the H+ effect Hb would hold
on to more of the O2. The H+ induces Hb to dump 10% more of
it's O2.
– CO2 reversibly binds to N term (carbamate) to remove
remaining CO2
R - NH2 + CO2 <-> R - NH - COO- + H+
R is the Hb N term amide

The carbamide increases the T formation - deoxy form.
The reverse occurs in the lungs. This results in 1/2 of CO2 removal
from tissues.
H+
O
C
O
H
H2N
H
O
C
C
R
O
Protein
Amino Terminus
C
O
H
N
C
C
R
O
Protein
Carbamate on Amino Terminus
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
Deoxy HbS precipitates
– oxyHb phenylalanine b85 and
leucine b88 interior
– phe and leu shift to exterior
– create a sticky patch with
valine (hydrophobic bonding)
– nucleation (cluster of
aggregate) occurs
logarithmically
– homozygous - 1000 times
faster than heterozygous
– that means mixed genes can
re-oxygenate faster than
polymerization can occur
Malaria – an agent of natural Selection




New traits are produced by mutation and are then
subject to natural selection.
The traits that survive are adaptations.
Malaria causes 110 million cases of illness each year
– Close to 2 million deaths each year.
Rare before the invention of agriculture
– Did much to change the selective pressure on human
populations
A small change in a gene
can have many phenotypic Figure 7.10
consequences.
Malaria – an agent of natural Selection



Most victims of malaria are young children
Where malaria occurrence is high, so is the HBs allele
– Odd, as Sickle Cell Anemia is nearly always fatal before
reproductive age
– HBs allele confers resistance to malaria
So in areas of high occurrence to malaria, the HBs allele
may cause a genetic disorder, but increases the overall
fitness of a population where malaria occurs.
Malaria – an agent of natural Selection
Rh Factor

There are four blood groups but eight blood types.

The Rh-factor!!




85% Positive (US population)
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 Rhfactor
– 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.
O2 affinity




pH affects the binding of
oxygen to Hb
In the lungs
(pO2 = 100 mm Hg), Hb is 98%
saturated at both pH 7.4 and
7.2.
binding of oxygen to Hb in the
lungs is not affected by
changing the pH and oxygen will
load normally.
O2 affinity



at the tissues
The change in the pH results is
a lower % saturation of Hb even
though the pO2 in the tissues
has remained at 40 mm Hg (for
this example).
the % saturation at pH 7.4 with
a pO2 of 40 mm Hg is about
70%
– meaning 30% of the
oxygen coming to the
tissue is released.
O2 affinity




at the tissues
At a pH of 7.2, the value is
close to 60% (meaning 40% of
the oxygen coming to the tissue
is released).
Thus more oxygen is delievered
to tissues at a lower pH even
when the pO2 remains
unchanged.
Lowering the pH appears to
shift the entire red curve to
the right.
Alteration in oxygen binding by:
-
H+, CO2 2,3 bisphosphoglycerate (BPG)
In deoxyhemoglobin, the N-terminal amino groups of the α-subunits and
the C-terminal Histidine of the β-subunits participate in ion pairs. The
formation of ion pairs causes them to decrease in acidity. Thus,
deoxyhemoglobin binds one proton for every two O2 released.
In oxyhemoglobin, these ion pairings are absent and these groups increase
in acidity. Consequentially, a proton is released for every two O2 bound.
Specifically, this reciprocal coupling of protons and oxygen is the Bohr
effect.
Alteration in oxygen binding by:
-
H+, CO2 2,3 bisphosphoglycerate
(BPG)
Simple rule of thumb-
– High H+ conc and higher
CO2 levels decrease O2
affinity for Hb
– High O2 concentration
decrease H+ and CO2
affinity for Hb

Hb acts as a H+ buffer in
respiring tissue
The End, at last!