Hybridoma Technology ( Production , Purification , and Application )
Structure and Catalytic Function of Superoxide Dismutase.pptx
1. Structure and Catalytic
Function of Superoxide
Dismutase (SOD)
Guided By: Dr. Rakesh Kumar Pathak
Presentatinon By: Soumyajit Banik (20144)
2. Introduction:
Superoxide is produced in aerobes as a product of metabolism.
Superoxide, O2
·-, is toxic when in the protonated form, HO2
. , cause damage to the cells
Superoxide Dismutase (SOD) is a metalloenzyme that can catalyze the breakdown of
superoxide (O2
−
) radical to molecular O2 and hydrogen peroxide (H2O2):
2O2
- + 2H+ = H2O2 + O2
Cells use superoxide dismutase in order to prevent oxidative damage by removing
superoxide free radicals.
Oxidative stress is harmful and it is caused by radicals, such as superoxide, that can attack
lipids, proteins, and DNA within the body.
Provides cellular defense against the oxidative stress.
Oxidative stress is linked to many human diseases including hypertension, diabetes
mellitus, ischemic diseases, and malignancies.
It is an 8-stranded "Greek key" beta-barrel, with the active site held between the barrel
and two surface loops.
A dismutation reaction is a reaction in which one substance is oxidized and the other
is reduced simultaneously (disproportionation).
Greek Key Beta Barrel motif
3. Types of SOD:
There are three major families of superoxide dismutase, depending on the protein fold and
the metal cofactor:
1. The Cu/Zn type (which binds both copper and zinc)
2. Fe and Mn types (which bind either iron or manganese)
3. Ni type (which binds nickel).
4. Copper-Zinc SOD (Cu-Zn-SOD):
Most commonly used by eukaryotes, including humans.
Cu-Zn-SOD available commercially is normally purified from bovine red blood cells.
The ligands of the copper and zinc are six histidine and one aspartate side-chains; one histidine is bound
between the two metals.
There are two redox reactions that occur in the copper zinc superoxide dismutase system, and copper
catalyzes these reactions.
5. In one reaction Cu2+ is reduced to Cu1+, while the superoxide molecule, O2
-, is oxidized to O2. In the second step
Cu1+ is oxidized to Cu2+, while a second superoxide molecule is reduced to hydrogen peroxide, H2O2. A
simplified catalytic cycle can be seen in the figure given below:
Simplified Catalytic Cycle
6. Ribbon diagram of the reduced form of Cu-Zn SOD with two
subunits differentiated in blue and red. Active site side chains
and metal cofactors are shown with superoxide bound.
(Modified from PDB 1sxz).
A "snapshot" of the Cu,Zn SOD active site, adapted from PDB
structure 1SXZ, is shown below. In the PDB structure, 1SXZ,
azide is bound to the reduced copper center. We have
converted azide to superoxide.
7. 1. Structure 1 represents, copper(II) metal center with a
four histidine ligands and a water ligand. It is a five
coordinate system
2. Then the water molecule is displaced as the
superoxide molecule binds to copper(II). It is a ligand
substitution reaction.
3. When copper(II) is reduced to copper(I), there are only
three histidine ligands that are attached. This
copper(I) structure in a trigonal planar geometry is
illustrated in Structure 3.
4. Then superoxide binds near copper in the active site,
and the copper(I) is oxidized to copper(II), while the
superoxide molecule is reduced to hydrogen
peroxide.
5. A water binds to the oxidized Cu+2, to return to
structure 1.
6. This continuous cycle converts the superoxide
molecule to hydrogen peroxide and dioxygen.
This catalytic cycle alternates between electron counts of 19
and 16. The four steps of the detailed catalysts cycle are
shown below:
8. Iron (Fe-SOD):
Used by prokaryotes (bacteria) and protists, and in mitochondria and chloroplasts
(plants)
E. coli contain both Fe-SOD and Mn-SOD.
They have the same arrangement of alpha-helices, and their active sites contain
the same type and arrangement of amino acid side-chains.
They are usually dimers, but occasionally tetramers.
9. FeSOD is thought to be the first SOD to evolve due
to the abundance of iron and low levels of oxygen
in Earth’s primitive atmosphere.
It was first discovered in 1973 in yeast (E. coli) by Yost
et. al, and has since been found in archaea, bacteria,
protists, and some primitive plants within the
cytosol, glycosomes, and mitochondria.
During the dismutation reaction, the iron ion at the
active site cycles between Fe(III) with five d-
electrons, and Fe(II), with 6 d-electrons.
It is clear from this figure that the ligand
configuration around Fe(III) involves three His
groups, one Asp group, and one water molecule in
a slightly distorted from trigonal bipyramidal
configuration.
The active site for FeSOD is characterized by two
equatorial Histidines, an axial Histidine, an
equatorial Aspartic Acid, and an axial water
coordinated to the iron in a trigonal bipyramidal
geometry.
The fourth nitrogen of each His group, the oxygen
of the water, and the negative oxygen of the Asp
are the specific donor atoms.
The Iron Superoxide Dismutase Molecule in 3-dimensions
10. The half reactions of the catalytic mechanism of FeSOD are as follows:
O2
- + H++HO--Fe+3SOD → O2 + H2O- Fe+2SOD (2)
O2
- + H++H2O-Fe+2SOD → H2O2 +HO- - Fe+3SOD (3)
In this mechanism, the reduction of Fe(III) creates a proton gradient which supplies the necessary
protons, via hydrogen bonding, for neutralizing the superoxide radical.
11. Manganese (Mn-SOD):
Overall quaternary structure of MnSOD. Adapted using
Chimera PBD 1VEW. Each monomer is depicted in a
different color.
Each monomer contains an active site between its N-
and C-termini. Two His residues from the N-terminal
domain (purple) and an Asp and His residue from the
C-terminal domain (green and orange) compose the
metal-center active site of MnSOD
12. Nearly all mitochondria, and many bacteria, contain a form with manganese (Mn-SOD): For example, the Mn-
SOD found in human mitochondrial matrix.
The ligands of the manganese ions are 3 histidine side-chains, an aspartate side-chain and a water molecule
or hydroxy ligand, depending on the Mn oxidation state (respectively II and III).
These monomers each contain N- and C-terminal domains, which pack tightly together, resembling a dimeric
structure shown. Each monomer contains an active site bound to a Mn3+
ion.
Mitochondria are the location of many biological processes reducing O2, producing an influx of O2˙ radicals.
The cofactor of the MnSOD protein is manganese (Mn). The Mn changes between its 3+ and 2+ oxidation states
within the dismutase mechanism, consisting of two half-reactions.
Electron exchange in metal complexes can happen by either an inner-sphere or outer-sphere mechanism.
In inner-sphere electron transfer, the metal cofactor is oxidized or reduced by a ligand bound directly to the
metal.
The catalysis of superoxide anions by MnSOD occurs by an inner-sphere mechanism.
Mn3+
SOD+O2
∙ −2
↔Mn2+
SOD+O2
Mn2+
SOD+O2
∙ −2
+2H+
↔Mn3+
SOD+H2O2
13. Blue nitrogens are part of the three
histidine ligands.
The purple oxygen indicates aspartate
ligand. The superoxide anion (red) binds
directly to Mn metal centers (green) by
inner-sphere redox.
Intermediate 1 to 2: Superoxide binds to
the Mn3+SOD complex.
The electron from the oxidized
superoxide is transferred to the reduced
Mn3+, producing Mn2+SOD and O2.
Intermediate 3 to 4: Electron from
oxidized Mn2+SOD is transferred to the
reduced superoxide, and when reacted
with two hydrogen ions, produces
hydrogen peroxide and Mn3+SOD.
14. Nickel SOD:
3. Nickel –
Found in prokaryotes.
This has a hexameric (6-copy) structure built from right-handed 4-helix bundles, each containing N-terminal
hooks that chelate a Ni ion.
The Ni-hook contains the motif His-Cys-X-X-Pro-Cys-Gly-X-Tyr
It provides most of the interactions critical for metal binding and catalysis and is, therefore, a likely diagnostic
of NiSODs.
15. The structure of Ni-SOD is important in catalyzing
superoxide dismutation.
Ni-SOD is a product of the sodN gene and is comprised
of a 117 amino acid sequence that has no homology
to other SODs.
Ni-SOD is a globular protein.
In other words, the overall shape of the protein is
relatively spherical.
As is common with globular proteins, Ni-SOD is soluble
in aqueous solutions.
In addition to its spherical shape, it has a hollow center
and is considered a homohexamer, that is, it is
comprised of six identical subunits. Structure of Ni-SOD (PDB: 1T6I). The active site (Ni-SOD complexed with the nickel cofactor) is emphasized in the
figure. Nickel cofactors are shown in yellow. The structure of Ni-SOD contains 6 subunits, or monomers (shown in
different shades of blue), each containing a nickel cofactor. There are 6 nickel cofactors bound, one Ni active site
in each of the six monomers. The monomer of Ni-SOD with the bound nickel cofactor emphasized.
16. Ni3+SOD + O2
- → Ni2+SOD + O2
Ni2+SOD + O2
- + 2H+ → Ni3+SOD + H2O2
Net Reaction: 2O2
- + 2H+→ O2 + H2O2
However, nickel does not catalyze superoxide dismutation in neutral, aqueous solutions.
For instance, other common SOD cofactors are more redox active and can cycle between their oxidation states in
biological fluids, thus dismutating superoxide, while Ni does not dismutate superoxide in the absence of the Ni-
SOD protein.
This is due to the fact that the reduction potential of Ni3+ to Ni2+ is proposed to be ~2V near pH~7.
This value is much larger than the proposed acceptable range for catalysis of superoxide dismuatation, which
includes reduction potentials ranging from -0.160-0.870V near pH~7.
In order to account for this large reduction potential, Ni-SOD utilizes uncommon active site ligands, Cys-2 and
Cys-6, to tune the reduction potential of Ni3+ to Ni2+ to ~0.30V (pH~7), which falls in the acceptable range for
catalysis (-0.160-0.870V near pH~7).
It is proposed that these Cys residues tune Ni reduction potential through bonding. The covalent nature of the
Ni-S(Cys) bonds is due to the bonding interactions.
Moreover, studies have proposed that the S-donor of Cys-2 is the protonation site, or the proton (H+) source, for
the oxidative half of the reaction, due to the greater electron density of its side chain, compared to that of Cys-6.
Therefore, due to redox tuning via its active site ligands, nickel is a redox active metal ion when it is bound to Ni-
SOD, making superoxide dismutation possible.
17. Inner sphere mechanisms refer to mechanisms in
which the metal is oxidized or reduced by a ligand
bound directly to the metal
Outer sphere mechanisms employ redox reactions
more indirectly, that is, via ligands that are not directly
directly bound to the metal center.
Electron transfer in Ni-SOD’s catalytic cycle occurs
between the superoxide substrate and the active site
site Ni, to which it binds.
Hence, Ni-SOD’s mechanism is considered to be an
inner sphere mechanism.
Cyclic mechanism of Ni-SOD facilitated dismutation of
of superoxide. The cycle contains 4 steps.
Species that are 18 electrons or above, proceed to
dissociation of a ligand.
Species that are less than 18 electrons gain electrons
electrons in a ligand association step, driving the
cyclic superoxide mechanism forward.
