BIOCHEMISTRY || Protein Function (Myoglobin & Hemoglobin)

Dr. Jose Oclarit

PROTEIN FUNCTION: Hemoglobin & Myoglobin The Backbone structure of MYOGLOBIN

Because of its red color, the blood pigment has been of
interest since antiquity.

- First protein to be crystallized- 1849

- First protein to have its mass accurately
measured.
- First protein to be studied by
ultracentrifugation.
- First protein to be associated with a
physiological condition.
- First protein to show that a point mutation can
cause problems.
- H is the last helix
- First proteins to have X-ray structures
- Amino acids that are interacting are indicated
determined.
in the helix and the position of the amino acid
- Theories of cooperativity and control explain
in which they are located are in what kind of
hemoglobin function.
helix.
- The center is known as the prosthetic group/
The structure of myoglobin and hemoglobin heme of the globin
- HEMOGLOBIN = heme + globin
- Andrew Kendrew and Max Perutz solved the
- Heme is the prosthetic group, non-protein and
structure of these molecules in 1959 to 1968.
holds the iron where oxygen is bound to.
- The questions asked are basic. What chemistry
is responsible for oxygen binding cooperativity,
BPG effects and what alterations in activity
does single mutations have on structure and
function.
- Myoglobin: 44 x 44 x 25 Å single subunit 153
amino acids residues.
 Nucleotides: 459 (1 amino acid is coded by
3 nucleotides)
 Alpha: 141 (423 AA residues)
 Beta: 146 (438 AA residues)
 Myoglobin: 153 (459 residues)
- 121 residues are in a helix. Helices are named
A, B, C,…F.
 363 nucleotides in alpha helix
- The heme pocket is surrounded by E and F but
not B, C, G, also H is near the heme.
- Amino acids are identified by the helix and
position in the helix or by the absolute
numbering of the residue.

***Homology- similarity of the structure/ sequence

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BIOCHEMISTRY || Protein Function (Myoglobin & Hemoglobin)

HEMOGLOBIN O2 Binding and Allosteric Properties of Hemoglobin

- Spherical 64 x 55 x 50 Å two-fold rotation of - Hemoglobin binds and transports H+, O2 and
symmetry α and β subunits are similar and are CO2 in an allosteric manner
placed on the vertices of a tetrahedron. There - Allosteric interaction:
is no D helix in the α chain of hemoglobin.  Of relating to, undergoing, or being a
Extensive interactions between unlike subunits change in the shape and activity of a
α2- β2 or α1- β1 interface has 35 residues protein (as an enzyme) that results from
while α1- β2 and α2- β1 have 19 residue combination with another substance at a
contact. point other than the chemically active site.
 ALPHA HELIX ONLY HAS 7 HELICES - A regulatory mechanism where a small
 OXYGENATION causes a considerable molecule (effector) binds and alters an
structural conformational change. enzyme’s activity.
 Alpha and beta globin are only found in  Effector molecule: Oxygen
hemoglobin not in myoglobin.  Enzyme activity: Globin activity

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
T- state R- state
(deoxygenated) (oxygenated)
O2
➢ Diving animals have large
concentration of myoglobin to keep
***Hemoglobin = 4 chains (2 alpha & 2 beta)
O2 supplied to muscles.
 Hemoglobin
➢ Found in red blood cells
Oxygenation Rotates α1β1 dimer in relation to α2β2
➢ Carries O2 from lungs to tissues and
dimer about 15°
removes CO2 and H+ from blood to
- The conformation of the deoxy state is called lungs
the T state. ➢ Lower affinity for O2 than myoglobin
- The conformation of the oxy state is called the ➢ Tetrameter- 2 sets of similar units (α2-
R state. β2)
- Individual subunits have a t or r in the deoxy or
oxy state.
- What causes the differences in the
conformation states?
 It is somehow associated with the binding
of oxygen.

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BIOCHEMISTRY || Protein Function (Myoglobin & Hemoglobin)
3D Structure of Hemoglobin and Myoglobin

x-ray crystallography of myoglobin - α1- β1 units have 35

MYOGLOBIN interactions
- α1- β2 units have 19
- Small protein interaction sites
found in muscle - similar units have few
- made up of 153 polar contacts
residues grouped - the 2 α and 2 β subunits
into 8 α helix A to face each other through
H (proline near aqueous channels.
end)  Aqueous channel: presence of water, polar
- Very small due to 44 x 44 x 25 Å interaction.
- Hydrophobic residues oriented towards the
interior of the protein SYNTHESIS OF HEMOGLOBIN
 Hydrophobic residues: AAs that are afraid
of water. Positioned at the interior of the
globin
- Only polar AAs inside are 2 histidine
- Red indicates Heme group (prosthetic group)
- 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. - Hemoglobin synthesis requires the
 Between Mb and Hgb, they have a region coordinated production of heme and globin.
called conserve region. - REMEMBER:
- Most strikingly, the secondary structures of  Heme
myoglobin and the subunits of hemoglobin are ➢ Is the prosthetic group that mediates
virtually identical. reversible binding of oxygen by
 They both have 8 helices. hemoglobin.
 Globin
MYOGLOBIN and HEMOGLOBIN ➢ Is the protein that surrounds and
protects the heme molecule.
- Hemoglobin is structurally related to
- Synthesis of heme begins with glycine and
myoglobin
succinyl- CoA (Krebs Cycle) catalyzed by ALA-
- Very different primary sequence about an 18%
synthase -> 5-aminolevulinic acid then
homology in the primary sequence
transported outside the mitochondria and
- 2 alpha subunits and 2 beta subunits
with the action of ALA-dehydrase in the
- In adults, there are very small amount of alpha
cytoplasm will then be formed into
2 delta 2 hemoglobin.
porphobilinogen -> undergoes series of
- Significance of conserved amino acids between
multiple steps -> porphobilinogen through the
myoglobin and hemoglobin
enzymatic reaction in the cytoplasm is then
 These are the important AAs which keeps
converted into Coproporphyrinogen III ->
hemes in contact with the protein
absorbed into the membrane ->
 Stabilizes helical arrangement
Protoporphobilin III -> Protoporphyrin IX and
 Interacts with heme/iron
when combined with iron (Fe2+) through the
enzyme ferrochelatase -> HEME

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BIOCHEMISTRY || Protein Function (Myoglobin & Hemoglobin)
FREE vs. BOUND heme—role of Apoprotein
SYNTHESIS OF HEME - Apoprotein
- Heme is synthesized in a complex series of  The protein moiety of a molecule or
steps involving enzymes in the mitochondrion complex
and in the cytosol of the cell. - Restricts heme dimers
- (1) the condensation of succinyl CoA and - Keeps iron reduced
glycine by ALA synthase to form 5-aminolevulic - Stabilizes transition state (O2 binding)
acid (ALA) - CO, NO, and H2S binding—greater affinity
 Transported to the cytosol than O2
- (2) a series of reactions produce a ring - His E7 decreases affinity of ligands (CO and O2)
structure—co-proporphyrinogen III for Fe2+
 Returns to the mitochondrion
- (3) an additional reaction produces
protoporphyrin IX SYNTHESIS OF GLOBIN
 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 THE HEME PROSTHETIC GROUP

- Protoporphyrin ring w/ iron

= HEME
- Four Pyrrole groups (A to D)
linked by methane bridges - Alpha gene cluster:
- Fe2+ coordinated by  Each chromosome 16 has 2 alpha globin
porphyrin N atoms and a N genes that are aligned one after the other
from Histidine (blue) on the chromosome. For practical
 This is known as His F8 purposes, the two alpha globin genes
(8th residue of the F α (termed alpha1 and alpha2) are identical
helix) ➢ Alpha globin is found in chromosome
- Iron is out of plane due to 16
His 8 bond ➢ Beta globin is found in chromosome
- A molecule of O2 acts as 6th 11
ligand. ➢ Chromosome 11- epsilon, 2 gammas
- Two hydrophobic sidechains on O2 binding site (gamma G, gamma A = Hb F), delta (Hb
of the heme help hold it in place A2), beta (Hb A)
 Valine E11 and phenylalanine CD1 ➢ Chromosome 16- Zeta 2, Zeta 1
- Oxygenation changes state of Fe (embryonic hgb), Alpha 2, Alpha 2
 Purple to red color of blood, Fe3+- brown  The transiently expressed embryonic
 Ferrous state is very important because genes that substitute for alpha very early
this is the state where iron will bind with in development, designated zeta, are also
oxygen, ferrous state is the most active in the alpha globin locus.
part
- Oxidation of Fe2+ destroys biological activity of
myoglobin.
- Physical barrier of proteins is to maintain
oxidation state of Fe2+.

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BIOCHEMISTRY || Protein Function (Myoglobin & Hemoglobin)

SYNTHESIS OF GLOBIN (cont’) What about the other genes? (cont’)

- Beta gene cluster - With the exception of the first 10 to 12 weeks
 The genes in the beta globin locus are after conception, fetal hemoglobin is the
arranged sequentially from 5’ to 3’ primary hemoglobin in the developing fetus. (2
beginning with the gene expressed in alpha, 2 gamma)
embryonic development (the first 12 - The combination of 2 alpha chains and 2 beta
weeks after conception; called epsilon). chains form “adult” hemoglobin, also called
 The beta globin locus ends with the adult “hemoglobin A”.
beta globin gene. The sequence of the - Although hemoglobin A is called “adult”, it
genes is: epsilon, gamma, delta, and beta. becomes the predominate hemoglobin within
 There are 2 copies of the gamma gene on about 18 to 24 weeks of birth.
each chromosome 11.
 The others are present in single copies. DEVELOPMENT OF GENE EXPRESSION
 Therefore, each cell has 2 beta globin
genes, one on each of the two
chromosomes 11 in the cell.
 These 2 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?

- Embryonic hemoglobin
- Two distinct globin chains (each with its  Zeta (2), Epsilon (2)
individual heme molecule) combine to form  Alpha (2), Epsilon (2)
hemoglobin. One of the chains is designated  Seta (2), Gamma (2)
alpha. - Fetal hemoglobin
- The second chain is called “non-alpha”. With  Hemoglobin F: alpha (2), gamma (2)
the exception of the very first weeks of - Adult hemoglobin
embryogenesis, one of the globin chains is  Hemoglobin A- alpha (2), beta (2)
always alpha.  Hemoglobin A2- alpha (2), delta (2)
- A number of variables influence the nature of - The globin genes are activated in sequence
the non-alpha chain in the hemoglobin during development, moving from 5’ to 3’ on
molecule. the chromosome.
- The fetus has a distinct non-alpha chain called - The zeta gene of the alpha globin gene cluster
gamma. is expressed only during the first few weeks of
- After birth, a different non-alpha globin chain, embryogenesis.
called beta, pairs with the alpha chain. The - Thereafter, the alpha globin genes take over.
combination of two alpha chains and two non- - For the beta globin gene cluster, the epsilon
alpha chains produces a complete hemoglobin gene is expressed initially during
molecule. embryogenesis.
- The combination of 2 alpha chains and 2 - The gamma gene is expressed during fetal
gamma chains form “fetal” hemoglobin, development.
termed “hemoglobin F” (Hb F)  The combination of 2 alpha genes and 2
gamma genes form fetal hgb, or Hb F.

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BIOCHEMISTRY || Protein Function (Myoglobin & Hemoglobin)

Development of gene expression (cont’) The two states (cont’)

- Around the time of birth, the production of - The T form finds the terminals in several
gamma globin declines in concert with the rise important H bonds and salt bridges.
of beta globin synthesis. A significant amount - In the T form, the C terminus of each subunit
of fetal hgb persists for 7 or 8 months after are “locked” into position through several
birth. hydrogen and ionic bonds.
- Most people have only trace amounts, if any,  If there is no Oxygen, there is presence of
of fetal hgb after infancy. H bonds and ionic bonds but during the
- 2 alpha genes and 2 beta genes comprises the binding of O2 to hgb the H bond and ionic
normal adult hgb, Hb A. bonds are broken -> O2 bind to the iron
- The delta gene, which is located between the part of the heme.
gamma and beta genes on chromosome 11 - Shifts the R state break these and allow an
produces a small amount of delta globin in increased movement throughout the molecule.
children and adults. The product of the delta - Note that binding of one or more oxygen can
globin gene is called hemoglobin A2, and have a dramatic effect on the other subunits
normally comprises less than 3% of that have not yet bound an O2.
hemoglobin in adults, is composed of 2 alpha - Binding of O2 dramatically alters the
chains and 2 delta chains. interactions and brings about a twisting of the
OXYGENATION two halves (alpha beta pairs)
- Much of the quaternary changes takes place in
- Oxygen binding to hgb is due to the effect of the salt bonds between the C terminals of all
the ligand binding state of one heme group on four chains.
the ligand-binding affinity of another
- Too far apart to interact (25 to 37 Å apart)
- Mechanically transmitted between heme Oxy and Deoxy Quaternary structures are
groups by motions of the proteins different:
- This means the molecule changes shape.
- Changes takes place between α1- β2 and α2-
β1.
The two states: - Amino acids between α1 and β2 help to
stabilize each form.

Oxygen binding shifts quaternary structures at

long distances

- Building of O2 ligands at 6th

coordinate position pulls
Fe into heme.
- There are 2 general structural states—the - Moves proximal histidine
deoxy or T form and the oxy or R form. (F8) and the alpha helix it is
- One type of interaction shift is the polar bonds attached to.
between the alpha 1 and the beta 2 subunits. - Shift in the helix is
- T- form (deoxy) interacting sites transmitted throughout of
 Asparagine G1 & Tyrosine C7 molecule.
- R-form (oxy) interacting sites
 Asparagine G4 & Aspartic acid G1

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BIOCHEMISTRY || Protein Function (Myoglobin & Hemoglobin)

T and R State of Hemoglobin

- How many hgb molecule in 1 rbc?

 270 million hgb molecules
- 1 hgb molecule can carry 4 oxygen molecules.
- 1 rbc = 1,080,000 O2 molecules

- 2 major conformations of hemoglobin as

predicted by the models for allosteric
activation.
- Oxygen will bind to hgb in either state;
however, it has a significantly higher affinity
for hgb in the R state.
 Salt bridges and H bonds are broken during
the R-state. (and vice versa w/ T-state)
- In the absence of O2, hgb is more stable in the Functional Structure of Hgb—Allosteric
T-state, and is therefore the predominant form regulations
of deoxyhgb. R stands for relaxed, while T
- Allosteric interaction
stands for tense. Since this is stabilized by a
 The binding of 1 ligand at one site in CHON
greater number of ion pairs.
that affects the binding of other ligands at
- Upon conformational change from the T state
other sites in the CHON.
to the R state, ion pairs are broken mainly
 This can affect binding and can be
between the a1b2 subunits.
cooperative (+/-)
- The T form finds the terminals in several
 Allosterism is typically seen when
important H bonds and salt bridges.
sigmoidal binding/activity curves
- In the T form the C terminus of each subunit
are “locked” into position through several Diphosphoglycerate BPG
hydrogen and ionic bonds.
- Shifts into the R state break these and allow an - Present in human rbc at approx. 5 mmol/L
increased movement throughout the molecule. - It binds with greater affinity to deoxyhgb.
- NOTE that binding of 1 or more O2 can have a  In bonding to partially deoxyhgb it
dramatic effect on the other subunits that allosterically upregulates the release of
have not yet bound an O2. the remaining O2 molecules bound to the
hgb, thus enhancing the ability of RBCs to
release O2 near tissues that need it most.

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BIOCHEMISTRY || Protein Function (Myoglobin & Hemoglobin)

Why is it important to lessen the affinity? 2,3 biphosphoglycerate (BPG)

- The Hill plot and the plot of the O2(?) binding - Purified Hb has a different O2
vs pO2. Think of when hgb should be mostly - Affinity than it does in blood
saturated and when it would be best if it had a  26-fold decrease change in affinity is due
low saturation/ affinity and thus “give up” its to 2,3-diphosphoglycerate BPG
O2. ➢ (BPG replaced by nucleotides IHP and
O2 pressure in various fluids ATP in fish and birds)
➢ 1 BPG per Hb—binds in central cavity
of Hb
➢ Binds prefentially to the deoxyhgb
➢ Hydrophobic salt bridge with His
➢ O2 binding changes conformation and
“kicks out” BPG
➢ Change in altitude increases conc of
BPG
 Fetal F Hb has replaced His 143 with Ser

Cooperative Interactions between subunits

Region or fluid pO2 (Torr)

Inspired Air 158

Alveolar Air 100

- Both models do not fully account for the
Arterial Blood 90
effects of allosteric effectors sequential model
Capillary 40 (D Koshland)
 Binding of one O2 induces T-R
Interstitial fluid 30
conformation change
Cell Cytosol 10  1st change is most difficult due to influence
by 3 other subunits
 Binding of next 3 subunits happens
- The lesser the Torr, the greater is the affinity of sequentially, with higher affinity (easier T-
BPG to the hgb thus favoring the delivery of O2. R changes)
As the rbc moves from the lungs to the tss,  Kinetics increase to the fully oxyhgb state
there is a decreasing alveolar pressure (pO2). as more O2 is bound
Decreased pO2 favors the delivery of O2 to the
tss, strong binding of BPG to hgb as it moves
from lungs to tss.

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