Metallobiomolecules

Metallobiomolecules

METALLOBIOMOLECULES

CONTENTS

1. Introduction to
Metallobiomolecules
2. Classification of
Metallobiomolecules
2.1. Transport and
Storage Proteins
2.2. Oxygen binding
Metallobiomolecules Today scientists try to explore the chemistry basis behind the
2.3. Electron Transfer biological processes. As a result of this, new areas have evolved such as
bioinorganic chemistry and bioorganic chemistry. In this section we will
Proteins talk about an important concept in bioinorganic chemistry called
2.3.1. Cytochromes “Metallobiomolecules”.

2.3.2. Iron-Sulphur 1.0 Introduction to Metallobiomolecules
Proteins
As we already know, biomolecules are molecules appear in
2.4. Zinc Metalloproteins biological systems to perform a specific function, like carbohydrates,
proteins, lipids and nucleic acids. Metallobiomolecules are molecules
associated with metal ions which play a major role in regulating
biological processes, like biomolecules do. Characteristic feature of
metallobiomolecule is as the name implies association of metal ion with
molecular part. Classification of metallobiomolecules can be done
based on several criteria. Let us consider the classification component.

2.0 Classification of Metallobiomolecules

As previously mentioned, association of metal ion is the
characteristic feature of metallobiomolecules. That mean metal ion part
is common for all metallobiomolecules. Depending on the nature of the
other molecular part, metallobiomolecules can be divided into three
categories.

W.K.B.P.M.Weerawarna University of Colombo 1

Metallobiomolecules

1. Transport and Storage Proteins:
Molecular part is belongs to the group of Proteins, but they are not enzyme and perform the
transport and storage function. Myoglobin (Fe), Hemoglobin (Fe), Hemocyanin (Cu),
Cytochromes (Fe) and Blue copper (Cu) Proteins are some examples.

2. Enzymes:
Molecular part is belongs to the group of Proteins, and also they are enzymes. They perform
the catalytic function. According to the type of reaction they catalyzed, they can further
classified into three groups.
Hydrolases : Carboxypeptidases (Zn)
Oxido-reductases : Oxidases (Fe, Cu, Mo)
Isomarases and synthestases : Coenzymes (Co)

3. Nonproteins
Molecular part is a nonprotein group. Best example is Chlorophyll (Mg).

Let us discuss about transport and storage proteins first.

2.1.0 Transport and Storage Proteins

According to the function they perform, transport and storage proteins can be further divided into
three groups like below.

1. Oxygen binding:
Their function is to bind with oxygen and transport and storage the oxygen in body.

2. Electron carriers:
Their function is to act as electron carriers and facilitate the electron transfer in biological
processes.

3. Metal storage, carrier and structural
Their function is to store metals and act as metal carriers.

2.1.1 Oxygen binding Metallobiomolecules

Oxygen binding metallobiomolecules are molecules with metal ions associated with protein but non
enzymatic molecular part and perform the oxygen transport and storage functions. During the evolutionary
history of life, organisms have evolved these types of metallobiomolecules to make sure the efficient
transportation of oxygen in their body which fulfill the oxygen demand of the body. In early evolution history
of life transportation of oxygen is mainly through simple diffusion. Best example is amoeba. Simple diffusion
as a transportation way is mainly governed by two factors:

1. Solubility of oxygen in water
2. Surface area to volume ratio

Solubility of oxygen in water is very few. Therefore concentration of oxygen in water is less. Also when
animal get bigger, surface area to volume ration get decrease. Diffusion of oxygen is mainly through body
surface. Therefore simple diffusion is not enough when animal get bigger. Therefore animals evolved
transport system to overcome this problem, where all the body cells meet their oxygen requirement.
However still they used water as the transport medium of oxygen although they developed transport
system. Due to the low solubility of oxygen in water, they have evolved special medium called blood, where
the solubility of oxygen is higher than that of water. This more solubility of oxygen in blood is due to the

Metallobiomolecules
presence of blood pigments. Oxygen can preferentially bind with these pigments; hence solubility of oxygen
in blood is very higher than that of water. But in Antarctic fish blood does not contain blood pigments
(haemoglobin). This is because of the low Antarctic temperature. Due to the low temperature, oxygen can
highly dissolve in water than in tropical countries. That is why Antarctic fish blood does not contain blood
pigments. This is an exceptional case. However in the evolutionary history, after they evolve blood with
blood pigments as the oxygen transportation medium, they have faced another problem. Blood pigments
are large molecules. Presence of large molecules in blood causes the production of high osmotic pressure. To
overcome this problem, higher animals have shifted blood pigments into cells. In humans and other
vertebrates, these cells are known as red blood cells (RBC).
Above is a little description of how animals evolved blood pigments or dioxygen transport and storage
metallobiomolecules in the evolutionary history of life. In this chapter we will discuss about three dioxygen
transport and storage molecules.

1. Haemoglobin:
These are carried in RBC (erythrocytes). Interior of the RBC is field with haemoglobin in the
case of vertebrates. Each RBC contain 250 million molecules of Hb. Each Hb can carry four
oxygen molecules. Therefore each RBC carries one billion oxygen molecules. There is a best
surface area to volume ration in RBC to absorb more oxygen in to RBC. Hb is a tetramer.
There is another molecule in muscles called myoglobin which is a monomer and function is
to store oxygen rather than carrying oxygen like in Hb.

2. Haemoerythrin
Found in certain marine invertebrates
3. Haemocyanin
Haemocyanin is in certain arthropods and mollusks

Let us talk about haemoglobin and myoglobin

Structure of Haemoglobin

It is made out of two components, a protein component called globin and a non-protein haeme group

Protein component

The protein portion consist with four polypeptide chains
Two of them are alpha chains and other two are beta chains
The four polypeptide chains fit together approximately a tetrahedral geometry
One haeme group is bound to each polypeptide chain
Hence one haemoglobin molecule contain four haeme groups
The four haeme groups are far apart, the distance between the closest atoms being 25 A
Therefore haemoglobin is a tetramer consists of four polypeptide chains and four haeme groups

Non-Protein component-(haeme group)

Haeme group consist of an organic portion and an iron atom
The organic portion is a porphyrin derivative
The porphyrin in haeme with its particular arrangement of four methyl, two propionate and two vinyl
substituents is known as protoporphyrin IX
CH3
CH2CH3

CH2CH3
H3C

N HN

N
NH

H3C CH3

CH2CH2COO- CH2CH2COO-


Metallobiomolecules

Haeme is then protoporphyrin IX with a centrally bound Fe atom
In Mb and Hb the iron atom s in the Fe(II) oxidation state and is positioned near the center of the
protoporphyrin ring
In oxygenated Mb and oxygenated Hb the iron atom is six coordinate
It bids to the four nitrogens f the protoporphyrin, nitrogen atom of an imidazole ligand of the proximal
histidine residue and dioxygen
In the deoxygenated form iron atom is five coordinate as the dioxygen binding site is vacant
In deoxy Hb and deoxy Mb the Fe atom is places 0.36-0.42 A and 0.42 A respectively from the plane of
porphyrin ring towards the proximal histidine residue
Because in the deoxygenated form ionic radius of Fe is high therefore it can not go and fit into the ring
In the oxygenated forms of Hb and Mb the Fe atom moves to within 0.12A and 0.18A respectively form
the ring plane and fit to the ring whole
Because in oxygenated form electrons in Fe(II) attract towards more electronegative oxygen atom
Therefore it is now like Fe(III) (but actually it is not Fe(III))
Ionic radius of the iron atom reduced and now it can move into the ring

Dioxygen binding and dissociation curves for Hb and Mb

A plot of fractional saturation (Y) for oxygen vs the partial pressure of oxygen is called an oxygen
dissociation curve
The fractional saturation Y is defined as the fraction of oxygen binding site occupied
Therefore value of Y can be ranging from 0 (all sites empty) to 1 (all sites occupied)
Oxygen dissociation curve for Mb is a hyperbolic curve
Oxygen dissociation curve for Hb is a sigmoidal curve
The special property of Hb molecule that makes it an effective oxygen carrier can be illustrated by
comparing the oxygen dissociation curves of Hb and Mb

Differences of oxygen dissociation curves of Hb and Mb

This differs in two ways

1. The curve for Mb is hyperbolic while that of Hb is sigmoidal. (Hb shows a cooperative binding of
oxygen)
2. For any given pO2, saturation is higher for Mb than for Hb

Cooperative binding of oxygen by Hb enables Hb to deliver 1.83 times more oxygen under typical
physiological conditions than if the sites were independents
This cooperative binding is the reason for the sigmoidal oxygen dissociation curve for Hb


Metallobiomolecules

This can be explain by drawing hyperbolic curve for Hb having same P50 (26 torr) YO2 value
At P50, percentage fractional saturation of oxygen is 55%
If Hb oxygen dissociation curve is hyperbolic the highest percentage fractional saturation it can achieve is
79%
But due to the sigmoidal shape the highest percentage fractional saturation it shows is 95%
That means if the curve is hyperbolic, only 24% can be saturated from P50 value towards highest
saturation
But due to the sigmoidal curve it shows 40% saturation from P50 value towards highest saturation
Therefore curve of the Hb is being a sigmoidal curve rather than being a hyperbolic enables to deliver
(40/24) 1.83 times more oxygen under typical physiological conditions

Comparison of Hb and Mb oxygen dissociation curve

For this, we have to consider three situations

1. At Lungs

Now the partial pressure of the oxygen is very high
According to the curves both Hb and Mb are completely saturated

2. At peripheral Tissues

Now due to the consumption of oxygen, partial pressure of the oxygen is near 30 torr
At this partial pressure of oxygen, percentage oxygen saturation of Mb is still 100%
That means it is still storing oxygen rather than giving oxygen to the tissues
But the percentage oxygen saturation of Hb is 60%
That means it has given oxygen to tissue

3. Extra Tired Tissues

Now due to the over consumption of oxygen, partial pressure of oxygen is very small
Cording to the curve when the partial pressure of oxygen is about 5 torr percentage oxygen saturation of
Hb is less than 5%
That means now under high metabolism, all the oxygen in Hb have given to tissue
Now according to the curve percentage oxygen saturation of Mb is about 75%
That means now Mb begins to release oxygen to tissue
This is the importance of having two different curves for Hb and Mb
If both are sigmoidal, then there will be no molecule to give oxygen under extra tired conditions
So Hb cat as a oxygen carrier while Mb act as a oxygen storage molecule

The role of the hindered environment in Hb and Mb

The hindered environment at haeme is essential for reversible oxygen binding
Distal histidine group of the globular protein play major role
In Hb and Mb a histidine residue of the globin polypeptide chain is positioned close to the sixth
coordination sited of the iron
The steric hindrance caused by the distal histidine make sure the sixth coordination site of iron is bound
predominantly by oxygen rather than carbon monoxide
When haeme group is isolated form globin protein, carbon monoxide bind 25.000 times more strongly
than oxygen
However in Hb and Mb due to the distal histidine group the binding affinity is of oxygen is 200 times
more then carbon monoxide


Metallobiomolecules

This can be explain by hybridization of oxygen atoms in CO and oxygen sp2 hybridized
In carbon monoxide, oxygen atom is sp hybridized
Therefore the preferred angle of binding with Fe is 180
That means it preferred to bind Fe with linearly O O
In oxygen, oxygen atom is sp2 hybridized sp hybridized
Therefore the preferred angle of binding with Fe is about 120
In isolated form linear binding of carbon monoxide with Fe is more
stable than the angular binding of oxygen with Fe atom C O
That is why when haeme is isolated carbon monoxide binding is 25000
times higher than that of oxygen binding
But in Hb and Mb, due to the distal histidine residue, there is a steric hindrance
Therefore now carbon monoxide is forced to bind with Fe in a angular (bent) geometry which is less
stable than the angular (bent) binding of oxygen
That is why when haeme is with its globular protein (in Hb and Mb) oxygen binding is 200 times more
than carbon monoxide

C
O
0
180 O O
1200
Isolated haeme

C O
O O
1200
Haeme in Hb and Mb

What more globin does?

Globin prevent the auto oxidation of oxygenated haeme
That means globin does not allow haeme-oxygen-haeme complex to be formed
Once this complex is formed oxygen can not bind reversibly at the iron centre
Globin prevents the formation of this complex by creating a steric hindrance around the sixth
coordination site of the iron atom where the oxygen binds

Quaternary structure of Haemoglobin (functionality of Hb)

Hb has two conformation
Relax conformation which is denoted as R and Tens conformation which is denoted as T
The R and T forms are in the equilibrium
The tense form contain more salt bridges than relaxed form
Salt bridges are one type of interaction which facilitate the protein folding and form more folded
conformation
Due to this more folded conformation in tense form the proximal histidine residue pulls Fe atom form
porphyrin ring towards proximal histidine
Therefore iron atom is not situated on the middle of porphyrin ring and situated slightly away form it
towards proximal histidine
Therefore the affinity towards oxygen of tense form is lower than R form
Tense form favour the deoxygenated state

Metallobiomolecules

Relax form has less salt bridges than tense form
Therefore it is less folded than relax form and proximal histidine residue push Fe atom towards
porphyrin ring
Therefore affinity to oxygen of Relax form is higher than that of tense form
Relax form favour the oxygenated state
Therefore T form switch to R form as oxygen binds to the haemoglobin

How this T form switch to R form as oxygen binds to the haemoglobin

In haemoglobin Fe atom in its Fe(II) state
It has d6 configuration
When oxygen is not bound to Fe, it is in the high spin state and repulsion of d electron causes the
increasing ionic radius
Therefore it is difficult to move and fit to the porphyrin ring
Now Hb is in the tense form having regular number of salt bridges
But when oxygen binds to the Fe, d electrons in Fe atom attract toward oxygen
Now it is like Fe(III) state (but actually it is Fe(II) state [no change in oxidation state])
Therefore the ionic radius is reduced and it move in to porphyrin ring
This also cause the movement of proximal histidine residue towards porphyrin ring
As a result of this, entire polypeptide chain will move breaking more salt bridges
Now Hb is in R form
This is how T form switch to R form

Factors that influence the oxygen binding to haemoglobin

1. pH
2. CO2
3. 2,3-bisphosphoglycerate (BPG)

Let us consider about the effect of pH and CO2

1. Effect of pH and CO2 (Bohr effect)

The influence of H+ and CO2 on oxygen binding by Hb is known as the Bohr effect
This mechanism is important as it allows oxygen to be supplied to tissue in need of oxygen as in for
example rapidly metabolizing tissue
In rapidly metabolizing tissues, carbon dioxides are accumulated
Therefore H+ concentration of tissue is increase
Hence pH of the tissue decrease
According to the oxygen dissociation curves for Hb under different pH values, at any given pO2 value
decreasing pH cause the less oxygen saturation of Hb
That means at any given pO2 value, Hb can supply more oxygen to tissues with decreasing pH
Deceasing pH cause the reduction of oxygen affinity of Hb
Reason for this is when H+ concentration increase, Hb takes more H+ in and more salt bridges are
formed and Hb is in tense form
Therefore high amount of oxygen can be given by Hb to tissues due to pH shift

Carbon dioxide transport in RBC

Hb also plays an important role in the transportation of carbon dioxide by blood
Most of CO2 in the blood is carried in the form of HCO3- dissolve in plasma
Other are transported by RBC


Metallobiomolecules

In tissue Cells

In tissue cells CO2 concentration is high and CO2 take up into RBC
Therefore H+ concentration inside the RBC increase
So Hb take more H+ (more Hb protonated) in and more salt bridges are formed switching relaxed
formed to tense formed
More salt bridges are formed because of the positively charged histidine due to H+
Therefore affinity of Hb for oxygen is reduced and more oxygen will released to tissues
In lungs reverse thing is happening
But here instead of CO2, oxygen concentration is high
Therefore more oxygen molecules are take into RBC
Due to the high concentration of oxygen, oxygen binds to Hb breaking more salt bridges and Hb is
converted to R form
Therefore more Hb are deprotonated and H+ concentration inside the RBC increase
This cause the shifting of carbonate-bicarbonate equilibrium towards carbonate and produced more
carbon dioxide
Then CO2 concentration increases inside the RBC and it is then diffuse into lungs
Try to show this like an equation

2. 2,3-bisphosphoglycerate (BPG)

How does BPG affect the oxygen affinity for Hb?

One molecule of BPG fits nearly into the central
cavity of deoxy Hb
There are number of positively charge residues
around the central cavity of deoxy Hb
So BPG stabilize the quaternary structure of
deoxy Hb
Because of this oxygen affinity of Hb is lowered
In the fetal Hb two alpha chains and two
gamma chains are there instead of two beta
chains
In the fetal Hb, one positively charged residue
histidine is replaced by uncharged serine
residue
This cause the reduction of salt bridges, forming
relaxed form and increasing the affinity to
oxygen
Therefore fetus can get oxygen more efficiently
form mother
Therefore oxygen saturation of HbF is higher
than that of HbA and this can clearly see by comparing the oxygen dissociation curve of HbF and HbA
In order to adapt to this condition, the number of RBC and the amount of Hb per RBC is increased
Since BPG is synthesized in the RBC this results in a rapid increase in erythrocyte BPG concentration
As a result of this, the oxygen affinity for Hb decreases and hence more oxygen is unloaded in the
capillaries


Metallobiomolecules

Abnormal Hemoglobin

Normal RBC are rounded shape and baso-laterally flattened
Therefore they can travel through capillaries without blocking it
But in the sickle-cell disease, they tend to aggregate (clump together) and block the capillaries in
different organs
Then the blood flow is stopped causing multiple organ damage
The life span of RBC is 120 days
But in sickle cell diseases life span is 60 days
So bone marrow can not produce enough RBC to keep the level up
Therefore anemic condition occur
Sickle cell disease is a genetically transmitted disease
See more among black population
In normal oxy Hb (Oxy A) does not contain any receptor or sticky patches
But deoxy Hb (deoxy A) contain receptors for sticky patches
In sickle cell oxy Hb (Oxy S) it contain sticky patches
In deoxy sickle cell Hb (deoxy S) it contain both sticky patches and receptors
Therefore deoxy S serve as chain propagation unites
Then under low oxygen concentration, this deoxy S polymerize into a fibrous structure
But presence of deoxy A will terminate the polymerization by binding with deoxy S
This fibrous structure blocks the capillaries

Model Chemistry of Biomolecules

Models are needed as the exact biomolecules are too small to be observed
The model should be well characterized
One type is structurally similar model and other type is functionally similar model

1. Functionally similar model for Hb (Co complex)

Occupation of fifth coordination site by a base makes the orbital along th Z axis of Co metal ion to retain
an electron
This makes its structure form square planer to square pyramidal
This electron interacts with incoming oxygen along the z axis and facilitates the binding
Similar thing happens by imidazole group at Fe atom in Hb
ESR also said that in FE-O2 bond electrons are more towards oxygen hence has superoxide like
properties


Metallobiomolecules

2.1.2 Electron Transfer Proteins (Metallobiomolecules)

So far we have discussed about oxygen binding metallobiomolecules and their functions. In this section we
will talk about metallobiomolecules, the function of them is to act as electron carriers. Mainly there are
three types of electron transfer proteins. In this section, we only talk about first two proteins.

1. Cytochromes
2. Iron-Sulphur Proteins
3. Blue-Copper Proteins

Theses electron transfer proteins are important in energy production in organisms and they carry electron
form one place to another place. Main energy storing way of plant is photosynthesis. They use some of this
energy for their survival through respiration. Also other animals use respiration to fulfill their energy
requirement. Definition of electron transfer protein is “electron transfer proteins are molecules involved in
respiration and photosynthesis which carry electrons form one place to another place”. Therefore above
mentioned electron transfer proteins are involved in respiration and photosynthesis. Let us talk about each
protein one by one.

2.1.2.1 Cytochromes

Introduction

Cytochromes are found in mitochondria and chloroplasts. There are three types of cytochromes.

1. Cytochrome a
2. Cytochrome b
3. Cytochrome c

Classification is based on the substituents of the porphyrin ring and the way in which the porphyrin ring is
attached to the polypeptide chain. In cytochromes all six coordination sites are occupied by porphyrin ring
and the polypeptide chain. They are so stable. Therefore the only way they can react is oxidation and
reduction of metal center. In cytochrome, metal centre is an iron atom. So iron atom in centre oxidized form
Fe(II) to Fe(III) by giving one electron to near by Fe(III). This process is happening like a chain. This is how
electron transfer is happening in the cytochromes.

Basic Structure of Cytochromes

Cytochromes are haeme proteins. They contain haeme group where the porphyrin is attached to iron centre.
But the structure of the iron-porphyrin complex in the various types of cytochromes is different. The iron-
porphyrin complex in b type is identical to that found in myoglobin and haemoglobin. In cytochrome-a,
methyl substituent on protoporphyrin IX is replaced by a formyl group and one of the vinyl group is replaced
by a long hydrophobic tail of isoprene units. Haeme group in C type is different because the way it is
attached of the polypeptide chain. The haeme group is covalently attached to the protein through the
thioether links of cysteine residues.

Physical Characters of Cytochromes

Redox potential is the main physical character of Cytochromes. In general, redox potential is given as a
reduction potential. If it is high (positive value) that means its potential to reduction is very high. Therefore
that species is easily reduced. Also if the reduction potential is low (negative value), it is difficult to reduced.
In cytochromes, iron centre has varying redox potentials in small range (G=nFE). Protein component in
cytochrome play major role in fine tuning the redox potential. Because if potential range is large and if it is
going to be used large energy for the electron transfer, total system will burn out. Therefore small amount of
different should be there. Key to the functionality of Cytochrome is the variation in redox potential.

Metallobiomolecules
Cytochrome function as one electron carrier by shifting one electron between Fe(II) and Fe(III) oxidation
states at the active site. As mentioned earlier, redox potential indicates that how it is easy to remove
electron. If reduction potential is high, easy to remove an electron. Basically this redox potential is depend
on the electron density around the metal ion. If electron density around the metal ion is high, it is easy to
remove an electron. Therefore major factor which determine the redox potential of a metal atom is electron
density around it. If electron density changes, redox potential will also change. There are some factors that
determine the electron density around central metal atom.

1. Protein
a. Axial Ligands
Axial ligands play major role in determination of electron density around the metal
atom (Fe). This can be explained by using hard and soft acid, base theory. Most of
the time metal ions act as acids and if the charge is high, they are considered as hard
acids. However Fe(II) is a borderline case where as Fe(III) is a hard acid. Hard acids
tend to bind with hard bases (Ligands) and stabilities of these complexes are high.
Soft acids tend to bind with soft bases and stabilities of these complexes are also
high. Sulphur in methionine is a soft base than that of nitrogen in histidine. Histidine
imadazole group has borderline hard, soft characters. So sulphur in methionine
more tend to bind with Fe(II) than Fe(III), because sulphur in methionine is a soft
base which preferred to bind with soft acid Fe(II). Therefore cytochrome with
methionine as an axial ligand more preferred to have its metal iron centre as Fe(II)
state than Fe(III). Therefore it is easy to reduced Fe(III) ion centre to Fe(II) state in
cytochromes having methionine as an axial ligand. Therefore these cytochromes
have high redox potentials of reduction.

b. Composition of amino acids and state of protonation of ligands (Amino acids) in the protein
Composition and the protonation state of amino acids cause the electrostatic
interaction with metal ion centre. These interactions can neutralize some charges on
the metal ion centre. This also influences the easiness of removal of electron form
the central metal atom.

c. Protein environment
Protein environment also influence the electron density around the central metal
atom hence the redox potential. Due to the hydrophobic interactions of proteins,
most of the water is removed from the interior of the protein and therefore interior
is almost like water free environment, due to the protein. Water has high dielectric
constant. Electrostatic interactions are inversely proportional to the dielectric
constant. Therefore if protein interior is filled with water, then the electro static
interactions will get weaken. But due to the water free environment (no shielding
form water), electrostatic interactions inside the protein is high compared to
surrounding. Therefore central metal ion feel protein charges more because they
appear inside of the protein. So protein environment also influence the removal of
electron form iron atom.

2. Substituents in the porphyrin ring
Depending on the type of substituents, electron density around the central metal
atom will vary. This different would alter the redox potential.

When we talk about the electron transfer, there are two mechanisms under theoretical treatment of
electron transfer.

1. Inner-Sphere Mechanism
2. Outer-Sphere Mechanism


Metallobiomolecules

Inner-Sphere Mechanism Outer-Sphere Mechanism

Electron transfer occurs via bridge Electron transfer occurs via adduct formed by
intermediate formed between the two diffusion of the two reactants together. In
reactants. biological systems, only way that electron
transfer can occur is outer-sphere mechanism.
Outer-sphere mechanism also consists of two
parts.
1. Inter molecular pathway
2. Intra molecular pathway
In biological systems, electron transfer has to be
in inter molecular pathway.

In cytochromes, electron transfer is a inter molecular outer-Sphere process. Absolute requirements for
electron transfer is that it has to be,
Extremely RAPID
Very SPECIFIC
Therefore in outer-sphere electron transfer, above requirements should be fulfilled. To occur a rapid
electron transfer, the energies of the electron donor and acceptor (reactants) should be matched prior to
the electron transfer. This is the pre requirement that reactants should fulfilled prior to the electron transfer.
Frank-Condon principle said that “electron transfer occurs so rapidly that nuclei can be considered as
stationary, until the rearrangement is completed”. Further more, the energies of the two levels should be
the same at the moment that electron transfer begins. However if energy levels don’t match, electron
transfer can occur, but it is more successful when energies are matched.
Together with Frank-Condon principle, it follows that the rate of electron transfer and activation energy will
depend on the ability of nuclei to adopt arrangements in which their energies will be matched. Therefore if
greater the reorganization required for match the energies, slower the reaction rate, hence greater the
activation energy. Major barrier (obstacle) for a rapid electron transfer is the geometric differences between
the oxidized and reduced forms of molecules. As an example consider the below self exchange reaction.

∗ ∗
[Fe(H2O)6 ]3+ + [Fe(H2O)6 ]2 + → [Fe(H 2O)6 ]2 + + [Fe(H2O)6 ]3 +
This reaction is very fast and bond length of Fe(III)-O is 2.05 A and Fe(II)-O is 2.21 A. For this reaction to
become a fast reaction, Fe-O bond lengths of both complexes are required to assume an intermediate value
prior to the transfer and this reorganization should be a small one. Then only this will be a fast reaction.

Therefore factors contribute to the activation energy (reaction rate) are:

1. The adjustment of bond lengths in both complexes to a common value (reduced and oxidized forms)
2. Re-organization of solvent molecules to reflect the changes in bond length and the charges on the
complexes when reaction is completed.
3. The electrostatic energy between two reactants.

In practical situation, the crystal structures of the oxidized and the reduced forms of cytochrome-c of tuna
are very similar. Therefore reorganization required prior to the electron transfer is very small, so activation
energy will reduced and rapid transfer will occur. Cytochromes therefore are considered as being in an
entatic state. According to the exited state theory, the ground state of molecule is situated between the
typical structures for each of the individual redox states. (it is closer to the transition state). Hence
cytochromes do not show a significant conformational change during electron transfer and are therefore
able to achieve rapid electron transfers.


Metallobiomolecules

Iron-Sulphur Proteins

Introduction

Iron-sulphur proteins are found in all organisms, in mitochondria and chloroplasts. Especially in
mitochondria, they are found in terminal electron transport pathways of respiration. They involve in
respiration, photosynthesis and nitrogen fixation. Approximately 1% of iron content in mammals is present
as iron-sulphur proteins. They act as one electron donors and one electron acceptors in electron transfer
process.
Eg: Iron-Sulphur clusters cycles between the Fe(II) (reduced) and Fe(III) (oxidized) state.

Structure of Iron-Sulphur Proteins

Fe-S proteins are non haeme proteins. That means it does not contain haeme group. Central iron atom is not
connected to porphyrin ring as in the case of cytochromes. Instead of that these proteins are bound by S
atoms. These S atoms are in the form of inorganic sulphide (S2-) or cysteine residues of the protein chain.

Classification

Categorization is based on the number of iron atoms and number of inorganic sulphur atoms present. They
are generally represent as [nFe-mS*]. Protein contain single iron atom are called Rubredoxins and proteins
contain 2Fe and 4Fe clusters are known as Ferredoxins.

Rubredoxins [1Fe-0S*]

These are found only in bacterias and the simplest type of Fe-S protein. Rubredoxin contains a single high
spin iron (II) or (III). Fe centre is coordinated to the sulfhydryl groups of four cysteine residues. Geometry is
tetrahedral. Therefore crystal field splitting energy is very low. So Fe irons are always in high spin state. The
main Fe-S distances in all Rubredoxins are nearly identical. Distance is slightly increase in reduced form of
iron (Fe(II) state). The difference between the reduced and oxidized forms is only 2-3%. No change in
coordination number or spin state. Therefore rubredoxins can be considered as being an entatic state.
Reduction potential is ranging form +50 to -50 mv.

Ferredoxins
a. [2Fe-2S*] Proteins

They are dinuclear clusters that contain two iron atoms (Cys)S S S(Cys)
bridging by two inorganic sulphides. The two iron atoms are
tetrahedrally coordinated. The remaining four coordination sites are Fe Fe
n+
occupied by cysteine residues. The charge of [Fe2S2] core can be in (Cys)S S S(Cys)
three forms, 0[Fe(II)Fe(II)], +1[Fe(II)Fe(III)] and +2[Fe(III)Fe(III)]. One
electron can be transfer form 0[Fe(II)Fe(II)] to +1[Fe(II)Fe(III)] and mixed valence form to +2[Fe(III)Fe(III)].
In biological systems, only latter two forms are involved in electron transfer process (+1 to +2). In the
oxidized form of the metal center which has Fe(III), Fe(III) has the Fe-Fe separation of 2.70 A. The reduced
form which has Fe(II) and Fe(III) has the Fe-Fe separation of 2.76 A. Increment is only 0.06 A. Hence as with
the rubredoxins, the iron centre does not undergo a significant structural change. Therefore the structure of
the oxidized and the reduced forms are nearly identical. This can also be considered as being an entatic
state. Reduction potentials for proteins with dinuclear sites are more negative (difficult to reduced) than
mono nuclear sites. Reduction potential is ranging form -280 to -490mv.


Metallobiomolecules

b. Rieske Proteins [2Fe-2S*]
(Cys)S S N(His)
This is a subclass of ferredoxin proteins. The centre is consist with
unsymmetrical structure where one Fe atom linked to two cysteine Fe Fe
residues and other Fe atom is linked to two histidine residues. Reduction
(Cys)S S N(His)
potentials of these proteins are very high (easy to reduced). Reason for
this observation can be explain by using hard, soft-acid, base theory. As mentioned previously, Fe(III) is a
hard acid and Fe(II) is a borderline case. S of cysteine residue is a weak base. Therefore it is not
preferentially binds with either Fe(II) or Fe(III). However N in histidine residue is a borderline case base and it
preferentially binds with Fe(II). Therefore N in histidine residue stabilize Fe(II) more than Fe(III). Therefore
reduction of Fe(III) in Rieske proteins to Fe(II) is easier than in other ferredoxin proteins. Hence the reduction
potential is higher than other ferredoxins. It is ranging from -150 to +350 mv.

c. [4Fe-4S*]

This is the most common and most stable iron-sulphur cluster. These S
clusters are cubic and contain four iron atoms and four inorganic Fe
Fe
sulphides. Iron atoms occupied altered corners of the cube. Remaining S
corners are occupied by inorganic sulphides which are triply bridged. Fe
Resulting geometry of the iron centers are distorted tetrahedrally. Mean S S
Fe-Fe distance is approximately 2.75 A which is some what similar to [2Fe- Fe
2S*]. This can exist three rather than two oxidation levels compared to
other units. (Hi potential Iron Protein [HiPIP])

HiPIP (oxi) HiPIP (red)/Fdox Fdred
+3[3Fe(III)1Fe(II)] +2[2Fe(III)2Fe(II)] +1[1Fe(III)3Fe(II)]

Therefore one electron transfer can happen between

HiPIP (oxi) → HiPIP (red)/Fd ox
HiPIP (red)/Fd ox → Fdred

In biological systems, only one redox pair is employed. Therefore never undergo all three oxidation level
transfers. Different oxidation pairs exist in different organisms.
As three oxidation states are available, redox potentials of these clusters vary widely.
HiPIP (oxi) → HiPIP (red)/Fdox (close to + 350mv )
HiPIP (red)/Fdox → Fdred (ranging from − 650mv to − 289mv )

These multi nuclear clusters have achieved much more similarity between the oxidized and the reduced
structure, when compared to mononuclear systems to achieve high electron transfer rate. (The change of
structure at electron transfer is just 1.3% per electron where as it is 2-3% for mononuclear). Electron transfer
of [4Fe-4S*] clusters are the fastest known.

*Plastocyanins and Azurins are also important electron transfer proteins*

Let us move on to the other section, which is metallobiomolecules related to enzymes. In this section we will
mainly talk about the Zinc metalloproteins, because Zn is the second most abundant trace element in human
and it a integral compound of over 100 metalloproteins.


Metallobiomolecules

2.2.0 Zinc Metalloproteins (Enzymes)

Introduction

Metal elements in human can be classified into two groups.
1. Bulk metals
2. Trace metals
Bulk metals form 1-2% of human weight. The trace elements represent 0.01% of human body weight. Trace
metals also can be classified into two groups
1. Fe, Cu, Zn group
2. Remaining six group (V, Cr, Mn, Co, Ni, Mo) these are ultra trace metals.

Of the trace metals, Zn is the second most abundant transition element in human. First one is iron. Zinc is a
integral component of over 100 metalloproteins in a number of different species. Play an important role in
enzymatic reactions.

Why use a metal in enzymatic reactions?

A metal can be represent as positively charge species even in an adequate concentration even at pH 7, when
H+ concentration is not enough to catalyze an enzymatic reaction. Metal ions have several coordination
numbers, so that they can act as “collecting points” of reactants. It facilitates the reaction by setting
reactants close proximity and enhance the rate of reaction.

Why use Zn in enzymatic reactions?

Zinc has one stable oxidation state. So reactions between metal ions does not interfere the activity of
enzyme. Also crystal field stabilization energy for zinc is zero due to d10 configuration in Zn(II) and it can
adopt any conformation. (Any geometric shape depending on the number of ligands bound). Also the
complexes are kinetically labile and rapidly go from 4 to 5 to 6 coordination numbers.

Role of Zinc

Zinc has two roles
1. Structural role
2. Catalytic role

Structural Role of Zinc Catalytic Role of Zinc

Zinc plays structural, conformation determining In chemical point of view, most effective
role in some biomolecules function of zinc in biological system is its ability
to act as a lewis acid. It can polarize substrate
Eg: Superoxide dismutase including water at physiological pH.
Alcohol dehydrogenase
Zinc fingers

Ligands bind to Zn(II)

Zn(II) has a borderline hardness. So it binds well with O, N or even with S. so it binds to residues of histidine,
glutamate, aspartate and cysteine. When Zn(II) involves in catalytic function, it expose to solvent and
generally one water molecule coordinate directly to Zn(II). In such cases the dominant ligands are histidine
residues.


Metallobiomolecules

After the electron transfer reaction, electrons are transferred to oxygen and it is converted to water.

4H + + 4e + O 2 → 2H 2O
During this process harmful intermediate products can be formed, superoxides and peroxides. Superoxides
can be converted to peroxide by exchanging one electron. So the enzyme that catalyzes this reaction should
have oxidation states of one unit higher. This enzyme is known as superoxide dismutase. In lower
organisms, Fe(II)/Fe(III), Mn(II)/Mn(III) involves and in higher organisms Cu(I)/Cu(II) involves. Funnel like
arrangement of amino acids in the superoxide dismutase enzyme sucks superoxides ions to the system or
bottom part of the funnel where the Zn(II) is present. Also there is a positive charge gradient to wards the
pocket. Then these peroxides can be converted to non harmful products like below

2H 2O 2 → 2H 2O + O 2 (Catalases )
H 2O 2 + 2H + → 2H 2O (Peroxidase s )

These catalase and peroxidase are haeme proteins.

Human Carbonic Anhydrase (II) (HCA)

This is found predominantly in RBC. It catalyses the reversible hydration of carbon dioxide to form
bicarbonate ion and a proton. Therefore it is essential for respiration

CO 2 + H 2O ⇔ H 2CO 3 ⇔ H + + HCO 3−

In HCA enzyme, Zn(II) center at the active site is bound with neutral ligands. Because of that it is highly
positive. This is the one of most positive Zn active site. This high positive metal center produce (or results)
OH- at neutral pH (pH 7).

Zn − H 2O ⇔ Zn − OH − + H +
(Zn − OH − )
pH = pK a + log
(Zn − H 2O )

pKa value of water is reduced form 14 to 7 due to Zn iron. If Zn iron is not present at pH 7, no OH- irons at
neutral pH. But due to Zn iron, now OH- is present at pH 7. This Zn-OH- is a powerful nucleophile. It helps to
orient the CO2 well for the reaction.


Metallobiomolecules

O

H C
H O
O CO2
O
Zn2+
Zn2+

O
O

H
H C
O C O O

O Zn2+
Zn2+

O H
O

C
H C O O
O O
2+
Zn
Zn2+

O H

H2O HCO3- H H
C O
O O

Zn2+ Zn2+

H H H
O
O
+ H+
Zn2+
Zn2+


Metallobiomolecules

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