1

OXGEN TRANSPORT PROTEINS

Nature has designed four O -carrying proteins for transport and storage of oxygen in
2

biological systems.
1. Hemoglobin (denoted as Hb) and
2. Myoglobin (Mb) are dioxygen (O ) binding metalloproteins containing an iron
2

porphyrin system, Fe (II)-heme proteins. Hemoglobin is presentin red Blood Cells

(RBC) and helps in transport of dioxygen from lungs to tissues. Whereas,
myoglobin stores dioxygen and is present in muscles.
3. Hemerythrin, a non -hemeFe (II) proteins, occurring in several marine
invertebrates and lower forms of life. Its main function is O -storage.
2

4.Hemocyanine:Hemocyanine are copper containing oxygen transport proteins,

occuring in a number of invertibrate, viz., snail, quids, cattle, octopus etc.

HEMOGLOBIN & MYOGLOBIN

ACTIVE SITE STRUCTURE:

• Mb is a monomeric protein (MW = 17,100daltons, one Dalton = 1.6605402e-

24gm) having a single poly peptide chain that is not conductive of self
association.
• On the other hand, Hb is a tetrameric protein (MW= 64500daltons), consisting of
-
two and two  peptide chains interlinked trough hydrogen bonded (COO
+
………..NH ) interactions.
3

X-ray study showed that the disappearance of these salt bridge bonds on oxygenation
The active site of both Hb and Mb contains the heme group in which Fe(II) is
th
equatorially coordinated by the four pyrrole nitrogen atom of protoporphyrin IX.The 5
position is coordinated by the imidazole nitrogen atom of histidine of the chain (i.e., the
th
globin).The 6 position in deoxy-Hb or deoxy-Mb is vacant, but hdrophobically shielded
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by the protein chain.As a result, only non polarneutral molecules such as O CO, etc. can
2,

bind to the sixth position.

FUNCTION OF GLOBIN CHAIN

In the absence of the protein (globin), the 6th position is irreversibly oxidised by oxygen
of the air to Fe(III)- heme, hematin.The latter, because of its residual positive charge, is
reluctant to bind uncharged ligands such as O2 , but readily binds charged liands such as
CN-, S2- , OH- etc.; which inhibit the oxygenation.

(Globin) Fe(II) heme + O2 (Globin) Fe(II)-hem

me-O2

( Fe(II) heme + O2 Fe(III)-heme (i.e., hematin)

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Mechanism for the formation of hematin shown below-

The first step is the binding of di-oxygen molecule, as in hemoglobin

FeII + O2 FeII O The heme is here symbolised

The bound dioxygen can now O by circle:
coordinate to a second heme,
forming a  -peroxo complex

FeII O + FeII FeIII O

O O FeIII

Cleavage of the peroxo complex results in two molecules of a ferryl

complex with the iron in the +4 formmal oxidation state:

FeIII O
FeIV O
O FeIII

Finally, attack of the ferryl complex on another heme results in the

formation of hematin:

FeIV O + FeII FeIII O

FeIII

peroxo dimer known as hematin
i.e. during formation of one mole hematin three mole heme is destroyed

Role of Distal (E7) andProximal (F8) Histidine Residues in Hb& Mb

In deoxy-hemoglobin, four of the coordinated sites of iron are occupied by nitrogens of

porphyrin ring. The fifth site is occupied by Histidine residue (called proximal histidine)
of globin. The sixth position is occupied by weakly bonded water molecule. Hence some
authors tend to report Fe (II) ion in deoxy form as pentacoordinated. Deoxy-hemoglobin
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is said to be in T-state (tense).On the opposite side of the proximal histidine, there is one
more histidine group (called distal histidine) placed near the iron ion. It forces the
binding of dioxygen in "end on bent" confirmation.

•
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Function of proximal HistidineResidues (F8)

The proximal histidine (F8) residue acts as a good -donor to facilitate the central metal
to act as a better -donor towards the -acid ligand(e.g.O )at the trans-position.This
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largely helps O to acts as a better -acid ligand(i.e. -acceptance) to induce the spin at
2

iron, i.e. O acts as a relatively strong field ligand. If the base (B) is itself a good -acid
2

ligand, then formation of the O -adduct will be disfavored. CO is a powerful poison to

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Hb and Mb,as the heme group has very high affinity for the -acid ligand like CO.

Function of Distal HistidineResidues (E7)

Presence of distal histidine residue in the region of sixth coordination site, it does not
allow CO to form the linear Fe-CO bond and CO is forced to make a bent bond.The bent
conformation discourages the binding of CO to heme iron. Otherwise, CO may have even
more affinity with the iron ion. It is observed that CO binds to hemoglobin 200X stronger
than dioxygen but binds 20,000X stronger with unprotected heme.Thus the distal histdine
(protein) 1. Weakens the interaction with CO and optmises the binding of O2 in Hb and
Mb. 2.Additionally the imidazole moity of distal histidine stabilizes the oxygenated
compound through H-bonding.

BIOLOGICAL IMPORTANCE OF Hb& Mb AND ROLE OF METAL ION

Hemoglobin(Hb) and Myglobin(Mb) are responsible for the transport and storage of
oxygen in higher animals. Viz., mammals.Hb transports oxygen from its source(vis.,
lungs, skin and gills) to the site of its use inside the muscle cells, where oxygen is
transferred to Mb for use in mictrochondrialoxidation,i.e., respiration.
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n
c hai
lo bin
G
i n
cha
bin
Glo
H
N

His
H
N
N

Fe His
N
0
0.8A N
N
+ O2 Fe
N N

N N
N N

O=O

deoxy-Hb/Mb oxy-Hb/Mb
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Conformational Change, role of metal ion

Being 5-coordinated, Fe(II) in deoxy –Hb and deoxy-Mb, is present in high spin
configuration.Fe (II)-N bond lengths in high spin Fe(II)-N compounds are  2.18A0 ,
which is much greater than the mean radius ( 2.05A0) of porphyrin cavity.Penta
coordinated Fe(II) in deoxy –Hb and deoxy-Mb, has a square pyramidal geometry and it
is situated about 0.8A0 out of the pophyrin plane, being shifted towards the epically
coordinated histidine.Oxygen binds to the Fe(II) –heme at the vacant 6th position and
resulting octahedral field is sufficiently strong to transform high spin Fe(II) (radius 0.92)
to low spin Fe(II) (radius 0.75).As a result of that, Fe (II) radius is contracted by about
0.17A0 (0.92A0 - 0.75A0) and Fe (II) in the active site of oxy –Hb and oxy-Mb, moves
towards the pophyrin plane and ultimately sits in the porphyrin cavity. This movement of
Fe(II) causes the coordinated histidin to move towards the pophyrinplane. This brings
about a conformational change throughout the peptide chain amounting to rupture of
some or all the COO-…….NH3 salt bridge interactions. The constrained hemoglobin
tetramer then relaxes by exposing the 6th positions of the remaining heme groups to
oxygenation. This phenomenon is known as comparative interaction. Oxygenation of Hb
is autocatalytic due to this cooparativeinteraction. But such effect is absent in Mb due to
its monomeric nature.

Nature of bonding
The bonding mechanism of oxy-Hb and oxy-Mb can be explain by considering simplest
possibilities:Coordination of singlet O2 to low spin Fe (II); O2 as a one electron acceptor
leading to low-spin Fe(III) and O2- (Superoxide).In the de-oxy form, if square pyramidal
geometry is consider the electronic configuration of high spin Fe(II) is,
(dxzdyz)3((dxy)1(dz2)1(dx2-y2)1.In the oxy-condition, if FeIII –O2-interaction is considered
then the electronic configuration of low-spin FeIII is t2g5(assuming octahedral
geometry).Oxy Hb/Mb is diamagnetic to to anti ferromagnetic coupling between the
unpaired electron of Fe3+ and superoxide ion(O2-). In the model system, FeIII –O2- the
unpaired electron (t2g5 ) of FeIII undergoes anti-ferromagnetic coupling with the unpaired
electron in g* of O2-.Thus both the models explain the diamagnetic character of
oxygenated heme unit.
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Experimental evidences:
• X-ray photoelectron spectroscopy suggests iron has an oxidation state of
approximately 3.2
• Infrared vibrational frequencies of the O-O bond suggest a bond length fitting
with superoxide (a bond order of about 1.6, with superoxide being 1.5).
• X-ray Absorption Near Edge Structures strongly suggests an actual local charge
closer to Fe3+ than Fe2+.
• Thus, the nearest formal oxidation state of iron in Hb-O2 is the +3 state, with
oxygen in the −1 state (as superoxide .O2−). The diamagnetism in this
configuration arises from the single unpaired electron on superoxide aligning
antiferromagnetically from the single unpaired electron on iron, to give no net
spin to the entire configuration, in accordance with diamagnetic oxyhemoglobin
from experiment.

Question: 1. Give the active site structure of Hemoglobin and Myoglobin. Explain
the biological importance of Hb&Mb. Mention the role of metal ion involved.
2. Oxyhmoglobin is diamagnetic comment.
3. Explain the roe of i) globin chain ii) distal histidine iii) proximal histidine in
Hb/Mb.

Cooperative effect:
As one subunit of tetrameric Hb is oxygenated, cooperative interaction predisposes
another subunits to take up oxygenation.

K1
Hb + O2 Hb(O2)

K2
Hb(O2) + O2 Hb(O2)2

K3
Hb(O2)2 + O2 Hb(O2)3

K4
Hb(O2)3 + O2 Hb(O2)4
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As a result, the successive oxygen binding constants of Hb gradually increases (K1K2

K3K4) instead of a statistically expected steady decrease (K1K2K3K4) (the
phenomena are commonly known as cooperative effect). The constant K4 corresponds to
oxygenation of the relaxed Hb tetramer, and it is quite close to the oxygen binding
constant of Mb, in which cooperative interaction is absent
The Bohr Effect:

• The Bohr effect is a physiological phenomenon first described in 1904 by the

Danish physiologist Christian Bohr:
• Hemoglobin's oxygen binding affinity is inversely related both to acidity and to
the concentration of carbon dioxide.
• That is, the Bohr effect refers to the shift in the oxygen dissociation curve caused
by changes in the concentration of carbon dioxide or the pH of the
environment.Since carbon dioxide reacts with water to form carbonic acid, an
increase in CO2 results in a decrease in blood pH, resulting in hemoglobin
proteins releasing their load of oxygen. Conversely, a decrease in carbon dioxide
provokes an increase in pH, which results in hemoglobin picking up more oxyge
• Due to absent of cooperative interaction, Mb oxygenation does not show Bohr
Effect. Oxygenation is favoured in basic condition due to elimination of COO-
…NH3+ salt bridge bonds. Deoxygenation is favoured in acidic condition.

Hemoglobin

Myglobin
logp1/2

pH
pH dependent of oxygenation of hemoglobin (Bohr effect),
p1/2 = oxygen pressure required to half saturate hemoglobin/
myoglobin
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• The maxima between pH 6.0-6.5 corresponds to the pH range of lowest O2-

affinity of Hb. Under such weakly acidic (lactic acid) condition as prevails in
working tissues, the transfer of O2 from oxy–Hb to Mb is greatly favoured.

Posing of Hb and Mb

Different -acid ligands like CO, NO,PF3 which are electrically neutral and not much
bulky can competitively replace O2 from the sixth octahedral site of
Hb&Mb.Consequently the O2 transport mechanism gets arrested and toxic effect arises.
CN- may also bind the site but due to presence of heme pocket surrounded by
hydrophobic environment does not welcome CN- easily.CO affinity of Hb and Mb is
drastically diminished due to the steric hindrance by the distal histidine (E7).But in the
industrial pollution and automobile exhaust produce a large amount of CO which on
inhalation produces carboxyhemoglobin(HBCO).This is why, it is now almost mandatory
to use catalytic converters into the exhaust system to convert CO and NO into CO2 and
N2 respectively.
Recent report demands that ZnO + CuO catalyst can effectively convert CO into CO2 .
It is very interesting that the poisoning effect of CO is not cumulative, as it can be
replaced by O2 in fresh air.

Hb(CO) + O2 Hb(O2) + CO

ALLOWABLE CONCENTRATION OF CO

• The maximum allowable concentration of CO in air is 50 ppm for working 8

hours.
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• Working of 4 hors in the atmosphere of 300ppm CO, leads to blocking of 35% of

Hb.

• Working of 4 hors in the atmosphere of 800ppm CO, leads to blocking of 60% of

Hb.
• During cigarette smoking, CO concentration may go upto 200-400
200 400 ppm and in a
heavy smoker 5-15%
15% of Hb remain as HbCO.

Why CN- is deadly toxic?

CN- actually blocks the cytochrome C oxidase involved in the respiratory chain.
• Treatment:
 To remove the bound CN- from cytochrome c oxidase, some methemoglobins
(Met-Hb)
Hb) are to be generated either by inhalation of amyl nitritevapour
vapour or by
III) can bind CN- more
injection of NaNO2 solution. (Met-Hb) bearing Fe (III)
strongly than the cytochrome c oxidase. Consequently, CN- is removed from the
respiratory chain to regenerate the electron tunneling path.
 Presently Co(II)-edtacomlpex
edtacomlpex is also therapeutic use.

Hemerythrin, a non -hemeFe(II) proteins

• Occurring in several marine invertebrates and lower forms of life.
• Its main function is O2-storage.

Active site of hemerythrin before and after oxygenation.

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Its O2 binding is very rapid (close to diffusion controlled rate) and shows no pH
dependence.Autoxidation to Fe(III)-methemerythrin results in a loss of the ability to bind
O2, while the affinity for binding with small anionic such as OH-, CN-, N3- etc. is greatly
increased.Deoxyhemerythrin is paramagnetic due to high spin Fe (II).Oxyhemerythrin
has lower magnetic moment at room temperature and it becomes diamagnetic at 1.4-
4.2K.Each Fe(II) of the binuclear active site transfers one electron to O2 to reduce it to a
peroxide (O22-) ion. Antiferromagnetic coupling of the two resulting Fe(III) centres gives
rise to the diamagnetism of oxyhemerythrin at low temperature.

Hemocyanin
• Hemocyanine are copper containing oxygen transport proteins, occurring in a
number of invertebrate, viz., snail, quids, cuttle, octopus etc.

• Hemocyanin are mainly two types-

• 1. molluscanHemocyanin2. ArthropodanHemocyanin
• MolluscanHemocyanine contain 10-20 subunits, each with 7-8 domains. Whereas
ArthropodanHemocyanin are hexamers or oligomers.
• Deoxy Hemocyanine is diamagnetic and colourless, in which both copper atoms
are in Cu(I) state. It takes up oxygen in the ratio 2Cu:O2
• Oxy-hemocyanin is blue but diamagnetic.
• The oxy form may have the following resonating structures.
• Cu+O2Cu+↔ Cu2+O2-Cu+↔ Cu+O2-Cu2+↔ Cu2+O22-Cu2+
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• Resonance and Raman spectrum of oxy Hemocyanin however, indicates a

Cu2+O22-Cu2+ structure and diamagnetic due to Antiferromagnetic interaction
between the two Cu(II) ions.
Why is hemoglobin red
Each hemoglobin protein is made up subunits called hemes, which are what give
blood its red color. More specifically, the hemes can bind iron molecules, and these
iron molecules bind oxygen. The blood cells are red because of the interaction
between iron and oxygen. (Even more specifically, it looks red because of how the
chemical bonds between the iron and the oxygen reflect light.)
When the iron is oxygenated (Fe+3), it becomes red. When the iron is deoxygenated
(Fe+2), it becomes blue. This is why your veins are blue. When hemoglobin is carrying
a lot of oxygen (like when just leaving the lungs), blood is bright red. When most of
the oxygen has been released to the body, blood is dark red. Therefore, contrary to
popular belief, blood is never blue. Veins under light colored skin only look blue
because the skin changes the optical properties of the light that passes through the
skin.
Function of Hb in Acid-base balance and CO2 transport
• Both the deoxy and oxy-forms of hemoglobin can acts as weak Bronsted acids.
Hence in the presence of corresponding conjugate base, it can acts as a buffer to
restore the biological pH as the corresponding pKa values lies close to 7.0.
• Besides this, hemoglobin plays an active role in transporting carbon dioxide from
the tissues(i.e. sites of generation of CO2) to lungs. There are two establish
pathways of CO2 transport.
First pathway
 Both the de-oxy and oxy- form of Hb are Bronsted acid having pKa values-

deoxy-Hb.H+ deoxyHb +H+, (pKa = 7.9)

oxy-Hb.H+ oxy-Hb +H+, (pKa = 6.7)

 The CO2 produced in Krebs cycle gets dissolved in the water of blood to produce
COH22CO
carbonic acid, + 3H 2O = 6.1). This processH
( pKa CO3 , (pKa
is 2controlled by a Zn –=containing
6.1)
metalloenzyme, carbonic anhydrase (CA).
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 Now the conjugate base of the deoxygenated form ( i.e. deoxy-Hb) which is a
large excess in tissues, acts a proton acceptor and H2CO3 with lower pKa values
acts as a proton donor.

H2CO3 + deoxy-Hb H2CO3- + deoxy-Hb.H+

• In this way, CO2 gets converted into HCO3- with the help of carbonic
anhydrase(AC) and deoxygenated hemoglobin. The HCO3- with ions is
transported through the plasma of blood stream to lungs.
• In the lungs, oxygenated Hb is largely available and it participates as follows.

oxy-Hb.H+ + H2CO3- oxy-Hb + H2CO3H

• The produced H2CO3 immediately breaks down by the Carbonic anhydrase(AC)

and CO2 is removed during exhalation.
CA
H2CO3 CO2 + H2O

• This process accounts for the 60-70% of the total CO2 transport.
Second pathway:
 In this process, Hb acts as the main carrier of CO2 transport. CO2 can combine in
a rather loose covalent structure with the α-amino groups (present in the globin
protein chain) of Hb molecule. The Carbamino compounds are of the following
nature.
RNH2 + CO2 RNHCO2- + H+
 The process is significantly dependent on the degree of oxygenation of Hb. The
Halden effect suggests that the de-oxy-Hb has got more thermodynamic affinity
to form carbamino compounds than the oxy-Hb. In fact the bound carbamates
form salt-bridge to stabilise the T-form. The released proton also stabilises the
deoxy-form through the formation of salt-bridge interaction.
 The controbution of the second pathway in transportation of CO2 is ˜ 10% in an
adult at rest.
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O2 is not toxic but Cl2 is toxic to the biological world

 From the knowledge of reduction potential of dioxygen(O2),
O2 + 4H+ + 4e = 2H2O, Eo = 1.23V, (pH = 0)
 At biologial pH (ca. 7.0), the formal potential of 1/2O2/H2O = 0.81V and ΔG is
associated with -305kj/mole.
305kj/mole. That is the thermodynamic potential indicates the
spontaneous oxidation of organic molecule having reduction potential less than
0.81V by O2 to produce CO2, H2O,and N2.
 But surprisingly, the organic molecules (i.e. biological kingdom) are not burnt
away with O2 in spite of the thermodynamic favour.
 It is due to the kinetic stability of the biological system. O2 is fairly powerful
oxidising agent, but it is not toxic to us.

MO diagram of O2
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• The kinetic inertness of O2 towards the oxidation of organic molecules arises from
the fact that the ground stste of oxygen molecule is triplet (2S+1 =3, S =1), i.e. it
contains two unpaired parallel electrons.
• On the other hand the organic molecules (i.e. the biological system) are singlet(
2S+1 =1, S= 0, i.e. no unpaired electron) in ground state and the reaction products
originating from their oxygenation are also in singlet states.
• Reaction between the molecular species take place in shorter time than the time
required for conversion from singlet to triplet state
• It suggests that the reaction between the triplet O2 (S =1) and singlet organic
substances(S =0) is forbidden by the spin selection rule, as the spin is poorly
coupled with the moleculer vibration.

• In fact, moleculer vibration is about 104 times faster than spin inversion ( i.e.
triplet to singlet).
• N.B, Atmospheric oxygen is innocent to us, but the singlet oxygen is highly toxic
and the ordinary does not react with the singlet molecules through electro transfer,
but it can react through hydrogen atom abstraction which generates free radicals
to initiate the chain reaction.
• Cl2 is toxic to us because Cl2 is singlet at ground state, so reaction between the Cl2
and organic molecules are both