© amit saha roy

Metalloprotein-
Hemoglobin and Myoglobin
 The primary structure of a protein © amit saha roy

proteins are polymers of 20 different -amino acids, known as the common amino acids, which have a
specific codon in the DNA genetic code
2
Essential Amino Acids © amit saha roy

COO-

H3N+ C H

3
The four levels of protein structure © amit saha roy

4
 Metalloprotein © amit saha roy

A metalloprotein is formed when a protein incorporates one or more metal ions as

an integral part of its structure.

metalloprotein – metal ion = apoprotein

prosthetic group – tightly bound group (e.g., heme) to apoprotein

cofactors – small organic (e.g., vitamins, ATP, NAD, FAD) or inorganic molecules (particularly metal ions) that are
required for activity; can be loosely bound (coenzymes) or tightly bound (prosthetic groups)

holoprotein – active protein with cofactors and prosthetic groups attached

 © amit saha roy

Metalloprotein

Oxygen carrying protein Redox protein

i) Haemoglobin i) Ferredoxins
Fe containing heme protein
ii) Myoglobin ii) Cytochromes

iii) Hemocyanine Cu containing non-heme protein

iv) Hemerythrin Fe containing non-heme protein

6
 © amit saha roy
Nature has designed two oxygen carrying protein for transport and storage of oxygen in our
Biological system

i) Haemoglobin

ii) Myoglobin

Haemoglobin and Myoglobin were the first proteins to be completely successful X-rays analysis done by J.
C. Kendrew and Max Perutz (Nobel Prize for Chemistry 1962).

7
 What is Myoglobin? © amit saha roy
Myoglobin is the oxygen binding protein is found principally in the muscle tissues of the vertrabrata. It

contains only one heme unit. The Mb makes the oxygen available to the respiratory reaction of the cell.

It consists single polypeptide chain (globin) consisting 153 amino acids- made-up of 7 α-helical

and 6 non-helical segments. Fe atom coordinate to the imadazole ring of His –residue is the

oxygen binding prothesitic group is Fe(II)-protoporphyrin(ix)

8
What is Hemoglobin? © amit saha roy
Hemoglobin is a metaloprotein in red blood cells that binds to oxygen in the
lungs and carries the oxygen to the tissues.
• Hemoglobin has a tetrameric structure containing globin with prosthetic group heme; it is
made up of four subunits (2 α chains and 2 β chains) interlinked through hydrogen bonded
(COO- …. NH) interactions.
• Each subunit contains a heme group that can bind one molecule of oxygen (O2) reversibly.

Haemoglobin is also known as

tetrameric form of Myoglobin,
structurally and functionally
more complex than Myoglobin.

Haemoglobin Haem unit + globin unit

 Hemoglobin Hb © amit saha roy

 141 Amino acid

 146 Amino acid
Mb 153 Amino acid

Hb is not an exact tetramer of

Mb
Four units of Hb

3 major types of Hb
Hb A (Adult)
Hb F ( Fetal)
Hb S (Sickle cell)
Forms of Hemoglobin

Besides adult hemoglobin (HBA1) as described above other minor haemogobins such as HbA2 which are
found less than 5%,
Fetal Haemoglobin (HbF) are found during fetal development which are less then 2% and Glycosylated
haemoblogin (HbA1c) occupies less than 5% in a normal human body.

Forms of
Hb A Hb A2 Hb F HbA1C
Haemoglobin

Structure α2β2 α2δ2 α2γ2 α2β2-glucose

Normal % 96-98 % <5% <2 % <5 %

11
Structure of Haemoglobin © amit saha roy
Each subunit contains a
i. Non-protein part called a prosthetic group, which is called a heme, produced by the
combination of iron with a porphyrin ring

ii. Globin-the apoprotein part.

12
 Function of globin chain
© amit saha roy
From the study of the model system Fe(tetraphenylporphyrin)(N-methylimidazole)

1. It creates hydrophobic non-polar environment.

2. In absence of the globin protein, the 6th position is readily coordinated by polar water molecule.

3. In absence of the globin protein, Fe(II)-heme is irreversibly oxidised by oxygen of the air to
µ-oxoFe(III)-heme, called haematin

II
II III P-Fe III III
O2
P—Fe P—Fe—O2 - P—Fe—O – O – Fe - P

Fe(III)-superoxo species Fe(III)-peroxo species

Fe2+ + O2 Fe2+ O
O III III IV
P-Fe
Free Heme P—Fe—O – Fe - P 2 P—Fe—O2 -

4+
Fe2+ O + Fe2+ 2 Fe O
haematin
O P = porphyrin ring
µ-oxoFe(III)-dimmer species
Fe4+ O + Fe2+ Fe3+ O
Fe3+

13
 © amit saha roy
Model System of Hb

These type of the model system has been developed

to increases the steric crowding so dimerisation not possible.

In order to avoid the dimerisation of two Fe centre the steric

factor is utilised.
This type of porphyrin ring is called picket-fence porphyrin
Or capped porphyrin.

14
Porphyrin: © amit saha roy
Porphyrin: Porphyrins are cyclic compounds formed by the fusion of 4 pyrrole rings linked by methenyl
bridges (=CH-). Since an atom of iron is present heme is a ferroprotoporphyrin. These rings are names as
I,II,III, IV and the bridges are names as Alpha, beta, gamma and delta. Porphyrins contain side chains
attached to each of the other four pyrrole rings. Different Porphyrins vary in the nature of the side chains
that are attached to each of the pyrrole rings.

15
 Active site structure of both Hb /Mb
Structure of Heme (prosthetic group):
© amit saha roy
Heme is a derivative of porphyrin.
Here Fe(II) is coordinated by the four pyrrole nitrogen atoms of protoporphyrin (ix).

The 5th position is coordinate by the imidazole nitrogen atom of a histidine of the protein chain.

The 6th position is vacant, but hydrophobically shielded by the goblin in protein chain. As a result
Only non-polar neutral molecules such as O2, CO etc can bind to this position.

Fe

16
 © amit saha roy
Since iron atom has six coordination
sites, four coordinate to nitrogen
atoms that are part of the flat
porphyrin ring system and two are
perpendiculars to the porphyrin.
One of these two other sites forms a
coordinate covalent bond to a nitrogen
atom in histidine F8 (proximal). Proximal
Another histidine (E7, distal) is close histidine
to the sixth coordination position.

Distal
histidine
The coordinated nitrogen atoms (which have an electron-donating
character) help prevent conversion of the heme iron to the ferric
(Fe3+) state. One of these two coordination bonds is occupied by a
side-chain nitrogen of a His residue. The other is the binding site for
molecular oxygen (O2). Therefore, the oxidation of the heme iron is
prevented by the presence of the distal histidine side chain
preventing easy release of oxygen. This is an important mechanism to
understand the regulation of haemoglobin's affinity for oxygen as
well.
17
 Changes at the active site during oxygenation © amit saha roy
of Myoglobin/Hb

In the deoxy-Hb or Mb, the iron atom is slightly displaced towards the proximal side of
the ‘heme’ i.e. (above) the plane of the protoporphyrin cavity.

In the deoxy-Hb Fe(II) is (HS), so comparatively larger in size thus it does not fit in the hole and
is raised by about 0.42 A° above the macrocyclic N4 basal plane created by the four sp2 nitrogen.

Structural study reveal that in the

deoxy-Hb : Fe(HS)(t2g4)(eg2), radius : 92 pm
oxy-Hb : Fe(LS)(t2g6)(eg0), radius : 75 pm

DEOXYMYOGLOBIN OXYMYOGLOBIN
Distal
N histidine
N
N Protein
H N H H N Protein
N H
N N
N Fe
N Fe O
N N O
Protein Protein
Proximal
histidine

Fe2+ t2g4eg2, HIgh spin, radius 92 pm Fe3+ t2g5eg0, Low spin, radius 75 pm

Paramagnetic Diamagnetic

Fe 42 pm outside porphyrin plane Fe fits inside the porphyrin plane

Proximal
histidine 18
 Changes the electronic structure of Oxy-Hb
© amit saha roy
On oxidation
Changes the conformation of the globin protein

As oxygen binds to the distal side of the ring, it pulls the iron atom about 0.2 angstrom closer to the
plane of the ring. Although this distance is small, the movement is amplified, causing significant shifts
throughout the tertiary structure of the protein.

Case -1

In the oxy-Hb, Fe(II) is (LS) and due to the smaller radius it fit into the N4 basal plane.

Case - 2

On oxygenation, Fe(II) is converted to Fe(III), electron loosed during this process is gain by dioxygen
to form superoxide.
19
 © amit saha roy

The superoxo-oxygen is hydrogen bonded to the distal histidine residue and in fact the
Fe-O-O bond angle is 115° (end on bent fashion).
Due to oxygenation change in electronic structure:
Π*

Δ1
π
Fe2+  Fe3+
O2  O2 -
Fe(III)(LS) (t2g5eg0) MO diagram of O2 (-) 20
Oxymyoglobin and oxyhemoglobin: Evidence for Fe3+ & O2

IR -study
O2
 O-O of oxyhemoglobin, 1107 cm-1 is closer to the
 O-O O2 value of 1145 cm-1 than
 O-O O2 value of 1550 cm-1

N N
Fe3+ Magnetic study:

Unpaired electron of O2 combine with low spin Fe3+

N which is also have spin ½ and these two spins can pair
by antiferromagnetic coupling and will be
diamagnetic
N

21
 © amit saha roy
Globin and the quaternary structure of haemoglobin:

The haemoglobin comprises of the globin polypeptide chains of two dimers. Thus, hetero-
tetrameric is composed of two identical dimers,(αβ)1 and (αβ)2.The two polypeptide chains
within each dimer are held tightly together, primarily by hydrophobic interactions as well as
the Ionic and hydrogen bonds. Though they are held together by polar bonds, the two dimers
are able to move with respect to each other.

Change of Conformation of Hb
It should be noted that after oxygenation the terminal Hb is presumed to have two quaternary structure
R ( relaxed) and T (Tense).

This is a reversible reaction between two conformations of hemoglobin, the T (tense) state and the R
(relaxed) state. The two states have different affinities for oxygen.

T form: The deoxy form of hemoglobin is called the ―T,‖ or taut(tense) form. In the T form, there are
two αβ dimers interact through a network of ionic bonds and hydrogen bonds. These bonds restrict
the movement of the polypeptide chains. The T form is the low oxygen-affinity form of haemoglobin.

22
R form: In Oxy-Hb, After two oxygen molecule is bound to a Hb molecule. The quaternary structure
of protein are switches to a structure called the ―R,‖ or relaxed form, in which the polypeptide chains
have more freedom of movement . The R form is the high oxygen-affinity form of haemoglobin.

This is due to the movement of the Fe(III) into porphyrin cavity along with the distal Histidine.The
binding of oxygen to haemoglobin causes the rupture of some of the ionic bonds and hydrogen
bonds between the αβ dimers which in turn may lead to some changes in the protein conformation
and breakdown salt-bridge interaction.

Now the constrained Hb tetramer relaxes by exposing the 6th position of the remaining heme groups
to oxygenation. This phenomenon is known as Co-operative effect.

The R structure have high oxygen affinity while this is diminished in the T state. The T state prevailed,
when all the 4 subunits are unligated to oxygen and in this condition the inter-subunit interaction are
believed to constrain proximal Histidine to reverts to its movement in the plane of porphyrin ring.

23
 © amit saha roy
K1
(Hb)4 + O2  (Hb)4 (O2)
K2
(Hb)4 (O2) + O2  (Hb)4 (O2)2 K1 <K <K <
2 3 K4

K3
(Hb)4 (O2)2 + O2  (Hb)4 (O2)3 Experimentally proved

K4
(Hb)4 (O2)3 + O2  (Hb)4 (O2)4

24
 Hemoglobin: Tense (T) and Relaxed (R) States;
Deoxy versus Oxy: The cooperative effect

Binding of O2 to Hb is cooperative. The presence of bound oxygen favor addition of more O2. The Hb
molecule goes from a tense to a relaxed state. Pockets of heme gets more easy for the following O2 units to
access due to breaking of some weak interactions.
This happens like chain and pulley. The pulling of the proximal histidine along with the activity of Fe getting
into the plane of the porphyrin triggers this activity.

Removing the first stamp Deoxy Hb Oxy Hb

requires more effort

© amit saha roy