UNIVERSITY OF BOTSWANA

Department of Chemistry

Superoxide Dismutase; structure, function and possible role

in cancer prevention

in living cells.

STUDENT NAME: Mogaadile Keorapetse Marrieter P.

SUPERVISOR: Dr N.M Nnyepi

A LITERATURE SURVEY REPORT (CHE 352) SUBMITTED IN

PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE BSc

DEGREE (SINGLE MAJOR) IN CHEMISTRY

April 2008

Gaborone

DEDICATIONS

I will like to dedicate this project to all of the people who

gave me support, the nation of Botswana at large, cancer patients

and Botswana Cancer Association to have a clear vision on cancer

and have a light about it as it has nowadays became a silent

killer which have no cure. Also to dedicate it to my friend who

later this year on March 2008 lost his younger brother because of

blood cancer. My friend Boago “Stix” Sitwane this is to remember

you my man, may your soul rest in peace.

LIST OF UNCOMMON SYMBOLS AND ABBREVIATIONS INCLUDING

NOTATIONS OF SI UNITS

SOD = Superoxide Dismutase

SOD-1 = Superoxide Dismutase-1

SOD-2 = Superoxide Dismutase-2

SOD-3 = Superoxide Dismutase-3

UVB = Ultra violet light B

CuZn-SOD = CopperZinc Superoxide Dismutase

Mn-SOD = Manganese Superoxide Dismutase

EC-SOD = Extra cellular Superoxide Dismutase

GSH = monomeric glutathione

GS-SG = Glutathione Disulphide

NADPH = Nicotinamide adenine dinucleotide phosphate

GSR = Glutathione Reductase

ACKNOWLEDGEMENTS

Thanks to all people who helped me in preparing this literature

project. It was not an easy task to take it alone as it requires

too much research which is based on reading, consulting various

books, internet sources and also journals. All of this requires

much time. In binding the information, a logistic order is

required and thereafter proof reading which helps in correction

of omitted words, sentences which are not clear, grammar and

spellings. All this requires help from various people most

importantly who link with the subject topic. I will start by

thanking my supervisor Dr. N.M Nnyepi who contributed a lot in

this research, for his patience and motivating words. For proof

reading, I will like to thank my medical Doctor Dr Magdi (From

Princess marina hospital), my dad Tlhobogang Mogaadile, Mrs

Kalane, my relative and Lecture at University Of Botswana.

I thank you all, those who supported me that I can make this and

with that from me I say take it up ladies and gentlemen, le ka

moso.

Keorapetse Marrieter P. Mogaadile

ABSTRACT

The enzyme superoxide dismutase (an antioxidant) is an enzyme

which catalyses the dismutation reaction of superoxide to ground

state oxygen and hydrogen peroxide. This enzyme was found by

Irwin Fridovich and Joe McCord. The are three types of superoxide

with superoxide dismutase-1 and superoxide dismutase-3 having

copper and zinc and superoxide dismutase-2 being a manganese

superoxide, however, superoxide dismutase-3 differs from

superoxide dismutase-1.With the help of enzymes involved, such as

Catalase and Glutathione reductase, complete reaction can be

carried out leaving all products which are not harmful to the

body. However no clear studies have been done on whether the role

of superoxide dismutase radicals have any role in aging or life

span but it have been clearly shown that these radicals play an

important role in carcinogenesis as they mutate cancer-related

genes. Superoxide dismutase however follows a very complicated

mechanism due to more enymes being involved. Superoxide Dismutase

plays a role in cancer treatment even though this is not clearly

outstated, however, the enzyme increase the rate of the

dismutation reaction as dismutation reaction can occur out of the

enzyme but the reaction will be relatively slow which can

increase possible activation of cancer cells.

CONTENTS

1. Introduction------------------------------------------------

-------------------------1

1.1 Definition of Superoxide

dismutase-----------------------------------------1

1.2 Discovery of the enzyme superoxide

dismutase---------------------------1

1.3 Role of free radicals of the

enzyme------------------------------------------3

1.3.1 Role of free radicals in

aging----------------------------------------3

1.3.2 Role of free radicals in

carcinogenesis-----------------------------4

2. Main

Finding-----------------------------------------------------

-------------------6

2.1 Structure of the

enzyme(SOD)----------------------------------------------

-6

2.2 Types of

SOD------------------------------------------------------

-------------6

2.2.1 Superoxide Dismutase-1 (SOD-

1)----------------------------------6

2.2.2 Superoxide Dismutase-2 (SOD-

2)----------------------------------8

2.2.3 Superoxide Dismutase-3 (SOD-

3)----------------------------------10

2.3 Dismutation

reaction-------------------------------------------------

----------12

2.3.1 Reaction of Iron and Hydrogen peroxide (Fenton

reagent)------12

2.3.2 Action of enzyme Glutathione

Peroxidase-------------------------13

2.3.3 Action of enzyme

Catalase-------------------------------------------

15

2.4 Reaction catalysed by the enzyme proposed

mechanism-----------------18

2.5 Possible application to cancer treatment or

management-----------------21

3. Conclusions-------------------------------------------------

-------------------------23

4. References--------------------------------------------------

--------------------------24

5. Appendix----------------------------------------------------

-------------------------27

INTRODUCTION

Definition of the Enzyme Superoxide Dismutase

Superoxide Dismutase (abb SOD) is an enzyme which catalyses the

dismutation (disproportionation) of superoxide (O2-) into oxygen

(O2) and hydrogen peroxide (H2O2), which indicates that SOD is an

antioxidant. An antioxidant is a molecule that prevents the

oxidation of other molecules. SOD therefore provides defense

against oxidation in nearly all oxygen-metabolizing cells exposed

to oxygen1. In short SOD can be described as a naturally occuring

enzyme that removes the superoxide (O2-) from living cells2. This

is to say,SOD catalyzes or facilitates the convention of

superoxide (O2-) radical to molecular oxygen as superoxide

radical is harmful or toxic to biological system within the

living cells3. The dismutation reaction can be represented as

shown below;

2O2- + 2H+ → O2 + H2O2 (1)

When SOD comes in contact with O2-, it reacts with it to form

hydrogen peroxide (H2O2). The stoichiometry of this reaction is

that for each 2 superoxide radicals (O2-) encounted by the SOD, 1

H2O2 is formed.4

Discovery of Superoxide Dismutase

SOD was discovered by Irwin Fridovich and Joe McCord,which prior

were known as several metalloproteins with unknown function (for

example, CuZnSOD copper-zinc-SOD was known as erythrocuprein).

Fridovich was born in New York City in 1929. As a child he was

enthralled by what he called the "miracle of insect

metamorphosis" , which led him to an interest in biology (Kresge

N et.al 2006). However, he found that the descriptions provided

by biology were not enough and that he wanted an explanation at

the molecular level. Thus, when he entered the City College of

New York he majored in both chemistry and biology. During his

senior year he enrolled in a biochemistry course taught by

Abraham Mazur. Fridovich found Mazur to be an effective teacher,

and he became interested in biochemistry. Abe Mazur made it clear

that biochemistry was the highest use of chemistry and could lead

to real, testable explanations for the processes of life.

Toward the end of his senior year Fridovich was offered a job

doing research at Cornell Medical School. After graduating in

1951 he spent a year at Cornell isolating vasopressor material

from hog kidneys. When the year was almost over Mazur suggested

Fridovich consider graduate school. When asked where he should

apply, Mazur replied that Fridovich had already been accepted

into the Department of Biochemistry at Duke University School of

Medicine.

After graduating, Fridovich stayed on at Duke as an Instructor in

Biochemistry. He worked his way up the academic ladder at Duke

and became an Assistant Professor (1961), Associate Professor

(1966), and Professor (1971).In 1976 he was named James B.Duke

Professor of Biochemistry, a title he still holds today as an

emeritus professor.

Throughout his time at Duke, Fridovich has continued to focus on

the biology of oxidation. His initial identification of

hypoxanthine led him to further investigate the ability of

xanthine oxidase to catalyze sulfite oxidation. He discovered

that the enzyme could do so, but only while oxidizing its

substrates hypoxanthine or xanthine. Since tens of thousands of

sulfites were oxidized per electron pair removed from

hypoxanthine, he deduced that the reaction was occurring by a

free radical chain. Other studies looking at the oxygen

dependence of cytochrome c reduction suggested that O2- was

conducting electrons from xanthine oxidase to cytochrome c.

 Eventually, Fridovich's graduate student, Joe M. McCord, showed

that xanthine oxidase was releasing O2- into free solution where

it could initiate sulfite oxidation, reduce cytochrome c, or be

intercepted by protein inhibitors of cytochrome c. It became

clear to Fridovich that inhibitors of cytochrome c must be acting

catalytically to eliminate O2- by catalyzing the following

dismutation reaction;

O2- + O2

- + 2H+ → H2O2 + O2 (2)

Using bovine erythrocytes obtained from a gallon of blood from a

slaughterhouse,Fridovich and McCord purified the enzyme that

catalyzed the dismutation of superoxide radicals,superoxide

dismutase (SOD). Fridovich and McCord found that their purified

enzyme contained copper,which was required for its activity,and

that it existed in many tissues including bovine heart,brain and

liver. They also showed that SOD is identical to the copper-

containing erythrocuprein and hemocuprein.

Soon,SODs were isolated from a variety of eukaryotes and

prokaryotes. All of the eukaryotic SODs(1) contained copper and

zinc whereas the prokaryotic SODs(2) contained manganese.

Surprisingly,the mitochondrial SOD contained manganese.Along with

Fred Yost, Fridovich also isolated an iron-containing SOD

(3).Howard M. Steinman and coworkers (Kresge N et.al 2006) later

determined the complete amino acid sequence of the CuZn

dismutase. Steinman and Robert L. Hill (Kresge N et.al 2006)

determined the amino acid sequence of the first 29 residues from

the amino terminus of the mitochondrial manganese dismutase, the

bacterial manganese dismutase, and the bacterial iron dismutase.

Several years later,with Hosni M. Hassan, Fridovich investigated

the effect of paraquat (methyl viologen) on Escherichia coli.They

showed that the herbicide caused a marked increase in the rate of

biosynthesis of Mn-SOD.The cells that had augmented levels of Mn-

SOD also showed an increase in resistance to the toxicities of

oxygen and the quinone streptonigrin.Because paraquat also

increased cyanide-resistant respiration,it seemed likely that it

engaged in a cycle of alternate reduction and autoxidation within

E. coli to increase the production of O2- while providing a

cyanide-insensitive route for electron flow to O2.

Almost 40 years after his initial isolation of SOD, Fridovich is

still actively involved in SOD research at Duke.In his current

investigations he has been seeking efficient, stable, and non-

toxic mimics of SOD activity. He has also been tabulating the

biological targets for O2- and the controls on the biosynthesis of

SODs. [5]

ROLE OF FREE RADICALS

Role of free radicals in aging

The free radical theory of aging is that organisms age because

cells accumulate free radical damage with the passage of time.In

general,a "free radical" is any molecule that has a single

unpaired electron in an outer shell.For most biological

structures free radical damage is closely associated with

oxidation damage.Oxidation and reduction are redox chemical

reactions.Oxidation does not necessarily involve oxygen,after

which it was named,but is most easily described as the loss of

electrons from the atoms and molecules forming such biological

structures.The inverse reaction, reduction, occurs when a

molecule gains electrons.As the name suggests,antioxidants like

vitamin C prevent oxidation and are often electron donators.[6]

In biochemistry,the free radicals of interest are often referred

to as reactive oxygen species (ROS) because the most biologically

significant free radicals are oxygen-centered.But not all free

radicals are ROS and not all ROS are free radicals.For

example,the free radicals superoxide and hydroxyl radical are

ROS,but the ROS hydrogen peroxide (H2O2) is not a free radical

species,however the term "Free-radical theory of aging" usually

refers to these compounds as well.[6]

Genetic manipulations that increase CuZn-SOD activity have only a

slight,if any,effect on maximum life span in several species,they

do increase resistance to oxidative stress.However,increasing

both CuZn-SOD and catalase does significantly increase maximum

life span.Decreased SOD in a variety of species increases their

vulnerability to oxidative stress,and in the case of genetically

altered CuZn-SOD,leads to premature death of motor neurons in

humans.However superoxide is among the most abundant reactive

oxygen species (ROS) produced by the mitochondria,and is involved

in cellular signaling pathways.Superoxide and other ROS can

damage cellular macromolecules and levels of oxidative damage

products are positively correlated with aging.Superoxide

dismutase (SOD) enzymes catalyze the breakdown of superoxide into

hydrogen peroxide and water and are therefore central regulators

of ROS levels.[7]

Role of Free radicals in Carcinogenesis

Carcinogenesis meaning literally, the creation of cancer is the

process by which normal cells are transformed or converted into

cancer cells.Oxidant carcinogens interact with multiple cellular

targets including membranes,proteins and nucleic acids.They cause

structural damage to DNA and have the potential to mutate cancer-

related genes.At the same time,oxidants activate signal

transduction pathways and alter the expression of growth and

differentiation-related genes.The carcinogenic action of oxidants

results from the superposition of these genetic and epigenetic

effects. Cancer is,ultimately,a disease of genes.In order for

cells to start dividing uncontrollably,genes which regulate cell

growth must be damaged.Proto-oncogenes are genes which promote

cell growth and mitosis,a process of cell division,and tumor

suppressor genes discourage cell growth,or temporarily halts cell

division from occurring in order to carry out DNA repair.

Typically,a series of several mutations to these genes are

required before a normal cell transforms into a cancer cell.All

cells possess elaborate antioxidant defense systems that consist

of interacting low and high molecular weight components.Among

them,superoxide dismutases (SOD) play a central role.Studies with

mouse epidermal cells have demonstrated that the balance between

several antioxidant enzymes rather than the activity of a single

component determines the degree of

protection.Unexpectedly,increased levels of Cu,Zn-SOD alone in

stable transfectants resulted in sensitization to oxidative

chromosomal aberrations and DNA (Deoxyribonucleic) strand

breaks.Carcinogenesis is caused by this mutation of the genetic

material of normal cells,which upsets the normal balance between

proliferation and cell death.This results in uncontrolled cell

division and tumor formation.The uncontrolled and often rapid

proliferation of cells can lead to benign tumors,some types of

these may turn into malignant tumors (cancer).Benign tumors do

not spread to other parts of the body or invade other tissues,and

they are rarely a threat to life unless they compress vital

structures or are physiologically active for instance,producing a

hormone.Malignant tumors can invade other organs,spread to

distant locations (metastasis) and become life threatening.

More than one mutation is necessary for carcinogenesis.In fact,a

series of several mutations to certain classes of genes is

usually required before a normal cell will transform into a

cancer cell.Only mutations in those certain types of genes which

play vital roles in cell division,apoptosis (cell death),and DNA

repair will cause a cell to lose control of its cell

proliferation.

The cellular antioxidant defense also affects the action of UVB

light (290-320 nm) that represents the most potent carcinogenic

wavelength range of the solar spectrum.UVB light(ultra violet

light B) is known to exert its action in part through oxidative

mechanisms.[8]

MAIN FINDINGS

SUPEROXIDE DISMUTASE

Structure of the Enzyme Superoxide Dismutase

Figure 1: Structure of

Superoxide Dismutase [9]

Types of SOD

Three types of SOD exist; SOD1 which is located in the cytoplasm,

SOD2 which is located in the mitochondria and SOD3 which is

located outside the cells. SOD1 and SOD3 contain copper and zinc,

while SOD2 contains manganese. [10]

SOD1 (Cu-SOD-Zn)

Superoxide dismutase-1(SOD 1)-a soluble cytoplasmic antioxidant

is an enzyme that metabolizes superoxide radicals to molecular

oxygen and hydrogen peroxide thereby being a defence against

oxygen toxicity.The gene is located on chromosomes 21

(21q22.1).This is a copper-and-zinc enzyme found in the

cytoplasm.The protein encoded by this gene binds copper and zinc

ions and is one of two isozymes responsible for destroying free

superoxide radicals in the body.The encoded isozyme is a soluble

cytoplasmic protein,acting as a homodimer to convert naturally-

occuring but harmful superoxide radicals to molecular oxygen and

hydrogen peroxide.The other isozyme is a mitochondrial

protein.Mutations in this gene have been implicated as causes of

familial amyotrophic lateral sclerosis.The enzyme is found in the

eukaryotes as a cytoplasmic form,in plants as a chloroplast

form,also extracellular form in some eukaryotes and periplamic

form in the prokaryotes.Two patterns of the enzyme exist,the

first one contains histidine residues that bind the copper atom

and the second which is located in the C-terminal section of the

enzyme is involved in a disulphide bond.[11]The Figure 4 is of an

SOD1;

Figure 2. Structure of Superoxide Dismutase-1 (SOD1) [12]

Figure 3. Structure of Superoxide Dismutase-1 (SOD1) [13]

The enzyme,Cu/Zn SOD1,copper-zinc superoxide dismutase.The

protein backbone of the molecule is indicated by the white ribbon

in figure 4 .The molecules crucial to keeping the molecule in its

proper shape are shown as colored balls,these include the copper

and zinc ions that help the enzyme do its job of deactivating

reactive oxygen. [14]

SOD2 (Mn-SOD)

Manganese superoxide dismutase (Mn-SOD) protects the mitochondria

against the damaging superoxide (O2-) radicals.Increased levels

of Mn-SOD protects the cells and transgenic animals from various

forms of oxidative stress and also slows down cell growth.[15]This

gene is a member of the iron/manganese superoxide dismutase

family.It encodes a mitochondrial protein that forms a

homotetramer and binds one manganese ion per subunit.The

manganese containing tetrameric enzyme SOD2 is located in the

mitochondrial matrix in close proximity to a primary endogenous

source of superoxide,the mitochondrial respiratory

chain.Mutations in this gene has been associated with premature

aging,sporadic motor neuron disease and cancer.MnSOD,glutathione

peroxidase-1,and catalase are antioxidant enzymes that share a

common detoxification pathway. The gene is located on chromosome

6 (6q25.3). [16]

Figure 4: Structure of the active site of Human Superoxide

Dismutase-2 (SOD2) [17]

Figure 5: Structure of Superoxide Dismutase 2(SOD2) [18]

SOD3 (Cu-SOD-Zn)

SOD3 is an extra cellular SOD (EC-SOD).It is a tetramer (four

subunits) while SOD-1 is a dimer that is consists of two units.It

catalyses the dismutation process in the interstitial spaces of

tissues and extra cellular fluids (e.g plasma).The gene is

located on chromosome 4(4p15.3-p15.1).It eliminates superoxide

radicals from reaching the cell environment and prevents

formation of oxygen species.[19] Extracellular superoxide

dismutase (SOD3) is the primary extracellular enzymatic scavenger

of superoxide (O2-).Like SOD1, SOD3 is a CuZn SOD,however,it is

distinct from SOD1 in its amino acid sequence,antigenic

properties and tissue distribution.The enzyme is synthesized with

a putative 18-amino acid signal peptide preceding the 222 amino

acids in the mature enzyme,indicating that the enzyme is a

secretory protein.In man the highest levels of SOD3 are found in

lung, pancreas, thyroid, and uterus.By RNA gel blot analysis,it

have been determined that the highest levels of EC-SOD expression

are in adult heart,placenta,pancreas and lung followed by

moderate expression in kidney,skeletal muscle and liver.The

characteristic distinguishing SOD3 from SOD1 and SOD2 is the

heparin-binding capacity.SOD3 binds on the surface of endothelial

cells through the heparan sulfate proteoglycan and eliminates the

oxygen radicals from the NADP-dependent oxidative system of

neutrophils.SOD3 is a hearin binding multimer (4-units) of

disulfide linked dimers,primarily expressed in human lungs,vessel

walls and airways.[20]

Figure 6: Superoxide Dismutase 3 [21]

Dismutation Reaction in general

The SOD-catalysed dismutation of superoxide dismutation may be

represented in the following two half equations;

M(n+1)+−SOD + O2- →Mn+ −SOD + O2 (3)

M+−SOD + O2- + 2H+ →M(n+1) −SOD + H2O2 (4)

Where M is Cu (n=1), Mn (n=2), Fe (n=2) and Ni (n=2),oxidation

state of metal oscillates between n and n+1 [22]

The reaction however can be represented as below

2O2- + 2H+ → O2 + H2O2 [23] (5)

Action of Iron as a catalyst (Fenton Chemistry or Reagent)

Peroxide formed can also be dangerous to the cells,more

especially as it transforms to a hydroxyl radical (OH.) when

reacting with Fe2+ (Fenton reaction),a combination of H2O2 and Fe2+

is known as Fenton's reagent[24].Fenton chemistry was developed in

1890s by Henry John Horstman Fenton.In this type of

reaction,ferrous iron(II), Fe2+ is oxidized by hydrogen peroxide

(H2O2) to ferric iron (III), Fe3+,a hydroxyl radical (OH.) and a

hydroxyl anion (OH-).A hydroxyl radical (OH.) is the neutral form

of the hydroxide ion (OH-).A hydroxyl is a molecule of oxygen and

hydrogen in a covalent bond.This hydroxyl radical is highly

reactive and short lived.In addition to the Fenton reaction,

iron (III) can then be reduced back to Fe2+,a peroxide radical

(OOH.) and a proton by the same hydrogen peroxide via a

disproportionation process[25].The reaction can be represented as

below;

Fe2+ + H2O2 → Fe3+ + OH. + OH- (6)

Fe3+ + H2O2 → Fe2+ + OOH. + H+ (7)

Note: OH- + H+ → H2O (8)

The net reaction results in two molecules of H2O2 hydrogen

peroxide being converted to water and two hydroxyl radicals. This

clearly shows that iron is truly catalytic.The generated hydroxyl

radicals then are engaged in secondary reactions.[26] Below is the

net reaction;

2H2O2 → OH. + OOH. + H2O (9) (net reaction)

However in the net equation it is stated that;

2H2O2 → 2OH. + H2O [27] (10)

Two hydrogen peroxide give two hydroxyl radicals and water.

Action of Glutathione Peroxidase

However,this reaction (10) is dangerous as the hydroxyl radical

generated can engage in secondary reactions like convention of

benzene to phenol and can also destroy organic compounds e.g

trichloroethylene.The conversion of hydrogen peroxide to generate

hydroxyl radicals is disturbed by glutathione peroxidase which

reduces hydrogen peroxide by energy transfer from reactive

peroxide to a very small sulfur-containing protein called

glutathione. [28]

Figure7. Structure of Enzyme Glutathione Peroxidase [29]

Figure 8: Structure of Glutathione

Glutathione peroxidase is an enzyme found in the cytoplasm of

nearly all mammalian tissues,whose preferred substrate is

hydrogen peroxide. Glutathione peroxidase is the general name of

an enzyme family with peroxidase activity whose main biological

role is to protect the organism from oxidative damage. The

biochemical function of glutathione peroxidase is to reduce lipid

hydroperoxides to their corresponding alcohols and to reduce free

hydrogen peroxide to water. [30]

In this type of reaction, glutathione Peroxidase catalyze H2O2 as

given below;

2GSH + H2O2 → GS−SG + 2H2O (11)

GSH is monomeric glutathione and GS−SG is glutathione disulphide,

formed by oxidation of glutathione through loss of the thiolic

hydrogens and formation of an S-S bond.

Then Glutathione Reductase (in figure 9 below) regenerates the

enzyme by reducing the oxidized glutathione to complete the cycle

as below;

GS−SG + NADPH + H+ → 2GSH + NADP+ (12)

NADPH is the reduced form of nicotinamide adenine dinucleotide

phosphate.

Glutathione reductase,also known as GSR,is a human gene.It is an

enzyme which reduces glutathione disulfide (GSSG) to the

sulfidryl form GSH,which is an important cellular antioxidant. [31]

For every one mole of GSSG one mole of NADPH is required.

Figure 9. Structure of Glutathione Reductase [32]

Action of Catalase

Catalase (an enzyme found in all living organisms, Figure 10)

which is highly concentrated in peroxisomes which are located

everywhere in the cells also reacts with hydrogen peroxide to

form oxygen and water.[33] The reaction of catalase in the

decomposition of hydrogen peroxide is as follows;

2H2O2 → 2H2O + O2

This is to say 2 hydrogen peroxide decompose to give 2 moles of

water and 1 mole of oxygen.

Figure 10. The enzyme Catalase [34]

SOD is found in almost all living organisms in the presence of

oxygen including some anaerobic bacteria.In aerobic cells,free

radicals are constantly produced,mostly as reactive oxygen

species, and once produced,free radicals are removed by the

antioxidants defense system,which includes the enzyme SOD.Since

cancer cells produce more oxidants than normal cells,they can

literally oxidize themselves to death.If SOD is inhibited to

cancer cells,they are damaged,thereby controlling their growth.[19] The reaction of superoxide with non-radicals is spin

forbidden.In biological systems,this means its main reactions are

with itself (dismutation) or with another biological radical such

as nitric oxide (NO).The superoxide anion radical (O2-)

spontaneously dismutes to O2 and hydrogen peroxide (H2O2) quite

rapidly (~105 M-1 s-1 at pH 7).SOD is biologically necessary

because superoxide reacts even faster with certain targets such

as NO radical,which makes peroxynitrite.Similarly,the dismutation

rate is second order with respect to initial superoxide

concentration.Thus,the half-life of superoxide,although very

short at high concentrations (e.g. 0.05 seconds at 0.1mM) is

actually quite long at low concentrations (e.g. 14 hours at 0.1

nM).In contrast, the reaction of superoxide with SOD is first

order with respect to superoxide

concentration.Moreover,superoxide dismutase has the fastest

turnover number (reaction rate with its substrate) of any known

enzyme (~109 M-1 s-1), this reaction being only limited by the

frequency of collision between itself and superoxide.That is the

reaction rate is "diffusion limited".[35]

The general picture of the role of SOD can then be summarized as

the below picture;

Figure 11: A picture showing overview of all dismutation

reactions and steps taken [36]

REACTION CATALYZED BY THE ENZYME-PROPOSED MECHANISM

Both copper ions and copper-containing enzymes have been shown to

catalyze NO release from GSNO.It have been observed that copper-

zinc superoxide dismutase (Cu,ZnSOD) in the presence of H2O2

caused a rapid decomposition of GSNO,forming oxidized glutathione

(GSSG) and (*)NO.The cupric ions (Cu(2+) released from Cu,ZnSOD

were bound to the glutamate moiety of GSNO,yielding a 2:1 (GSNO)

(2)Cu(2+) complex.Strong chelators of cupric ions,such as

histidine and diethylenetriaminepentaacetic acid,inhibited the

formation of (GSNO)(2)Cu(2+) complex,GSSG and (*)NO.GSSG alone

inhibited Cu(2+)-induced decomposition of GSNO.This effect is

attributed to complexation of copper by GSSG.Binding of copper to

GSNO is obligatory for NO release from GSNO,however,the rate of

this reaction was considerably slow due to binding of Cu(2+) by

GSSG.The glutamate moiety in GSNO and GSSG controls copper-

catalyzed NO release from GSNO.Cu,ZnSOD and H2O enhanced

peroxidation of unsaturated lipid that was inhibited by GSNO.The

antioxidant function of GSNO is related to the sequestering of

copper by GSNO and its ability to slowly release NO. [37]

Further mechanism can be represented as below Figures;

The catalytic mechanism is shown below illustrating the oxidation

of the Mn3+enzyme to Mn2+ with one superoxide anion and the

reduction of the enzyme back to the ground state Mn3+.Also shown

is the entry of the human enzyme into an “inactive form” which we

have designated the product inhibited form of the enzyme.

NADH

Succinate2e- Succinate Dehydrogenase

(3Fe-S)

CO M PLEX II

NADHDehydrogenase

(5Fe-S)

CO M PLEX I

UQ cyt. b cyt. c1CO M PLEX III

cyt. c cyt. a-a3e- e-CO M PLEX IV

O2O2

-.

e-

e-

4e- O2

H2O

O2O2

-.

Amobarbital

AntimycinAM yxothiazole

HQNO

2e-

Figure 12: Kinetic mechanism of Mn-SOD [38]

Kinetic M echanism of M n-SOD

P-M n3+O 2·- P-M n3+: O2·-

P-M n2+

O 2k1k-1

k2

P-M n2+: O 2·-H 2O 2O 2·-

k-3k3

k-4k4

Inactive formk-5 k5

Figure 13: Mechanism involved in NADH [38]

This anti-oxidant enzyme functions primarily to protect

mitochondrial components from superoxide liberated as a normal

byproduct of respiration.Complex I and Complex III release

superoxide radical as a consequence of normal respiration,with

estimates of 1-5% of the oxygen consumed being liberated as

superoxide anion.MnSOD is therefore the cells primary defense

against free radical mediated damage.In addition,stimulated

levels of the enzyme have evolved to address increased free

radical production during an inflammatory episode. [38]

 

POSSIBLE APPLICATION OF SUPEROXIDE DISMUTASE IN CANCER TREATMENT

Cancer may affect people at all ages but risk for the more common

varieties tends to increase with age.Critics of the role of

superoxide in cancer have pointed out that it is not known

whether the loss of Mn-SOD is a cause or effect of cancer.After

all,there are numerous enzyme changes that occur in cancer (both

additions and deletions).In contrast to most other enzymes,the

principal role of SOD seems to be to act as a protective

enzyme.Thus,its absence can lead to widespread metabolic

consequences.In order to understand these consequences,it is

necessary to consider what is known about the chemistry of the

superoxide radical.Proposed pathways are shown in Chart 1

below.It should be emphasized that there is considerable evidence

for each of these pathways,but none have been proven beyond

doubt.Three pathways are of particular interest in the field of

cancer.Superoxide can oxidize SH groups to S−S via RS (Pathway 1)

dismute to form H2O2 + ground-state oxygen (O2) (Pathway 2),react

with ferric ion to form ferrous ion (Pathway 3).Each of these

pathways can probably lead to large changes in cell

metabolism.Considering Pathway 1 initially,sulfidryl groups are

constituents of many proteins.Oxidation of these groups to

disulfides can result in protein conformational changes with

possible activation or inactivation of key enzymes.Thesecond

important pathway,dismutation to form H2O2 and ground-state

oxygen O2, occurs either with or without SOD.Dismutation occurs

much faster in the presence of SOD than in its absence.

The last pathway may lead to perhaps the most widespread

changes.Superoxide can denote electrons to metals to change their

oxidation state.For instance,it has been shown that O2- can react

with Fe3+ to produce Fe2+.Reactions of this sort can lead to vast

metabolic consequences due to large changes in the oxidation-

reduction potential of the cell.Many enzymatic reactions require

metals as cofactors,and a change in their oxidation-reduction

state will surely affect these reactions.Fridovich has recently

shown that O2- is apparently a very diffusible substance able to

migrate large distances and even through membranes if they do not

contain SOD.Thus,in the cancer cell,which is low in SOD,changes

may occur far from where the O2- was originally produced.The

evidence that SOD plays a much important role in cancer

treatment.SODs catalyses the dismutation of O2-,an essential step

in eliminating the toxic free radical.This critical function

makes SOD an attractive target for pharmacological

intervention.Although several small molecules,including cyanide

ion (CN-),hydroxyl ion (OH -) and azide ion (N3-),were found to

inhibit SOD by competing with O2- at the catalytic site,these

chemicals are highly toxic and their potential for cancer therapy

is limited.Inhibition of SOD leads to free-radical-mediated

damage to mitochondrial membranes and the release of cytochrome

c.This is probably a major pathway,although other mechanisms such

as inhibition of tubulin polymerization and angiogenesis may also

contribute to its activity.O2- may also give rise to hydroxyl

radicals and cause DNA damage.The correlation between the ability

of oestrogen derivatives to inhibit SOD and their ability to

cause apoptosis indicates that inhibition of SOD may be an

important mechanism by which it kills cancer cells.However,the

role of O2- in causing apoptosis may vary among different cell

types,and thus inhibition of SOD should not be considered as a

general approach to cancer therapy.Because inhibition of SOD

impairs the ability of cancer cells to cope with O2 - radical

stress,it may also provide a mechanism for the design of new

strategies for cancer treatment. [39]

Chart 1: Reaction Pathways of

Superoxide [40]

CONCLUSIONS

Superoxide dismutase catalyses the superoxide (O2-) to

hydrogen peroxide (H2O2) and oxygen (O2)

There are three types of superoxide dismutase

Superoxide dismutase-1 differs from superoxide dismutase-3

even though they are both copper-zinc-SOD and apart from

that the other one (SOD-1) is in the cell cytoplasm and SOD-

3 in outside the cell

Fenton reagent is stopped by glutathione reductase and

catalase as hydrogen peroxide is dangerous to the cells by

generating the hydroxyl radicals.

There is no clarifications on whether superoxide dismutase

have possible role in cancer treatment.

Loss of superoxide dismutase have not been shown on whether

is a cause or effect of cancer and such studies should be

taken for future reference to clarify this

References

1. Superoxide dismutase, February 23,2008,MediaWiki,Wikimedia

Foundation, Inc

http://en.wikipedia.org/wiki/superoxide_dismutase,retrieved

28 February 2008

2. Superoxide Dismutase,April 2001,Karl Harrison,

http:www.3dchem.com/molecules.asp,retrieved 08-03-08.

3. Superoxide Dismutase,11 March 2008,The Center for Cancer

Education,University of Newcastle,

http://cancerweb.ncl.ac.uk/cgi-bin/omd?

superoxide+dismutase,retrieved 15 March 2008

4. Griendling K.K. and Weber D.S.,2003, The Yin/Yang of

superoxide dismutase mimetics: potential cardiovascular

therapies? British Journal of Pharmacology, 139, 1059–1060

5. Kresge N.,Hill R.L. and Simoni R.D., June 2, 2006,Forty

Years of Superoxide Dismutase Research: the Work of Irwin

Fridovich, Journal of Biochemistry,Vol. 281(22),01-03

6. What are free radicals,Free radicals and aging,Copyright

1999,The National Health Museum,

http://65.79.226.34/LC/ST/bgfreerad.html,retrieved 17 March

2008

7. Cause of Aging,Reactive oxygen Species,11 March

2008,MediaWiki,Wikimedia Foundation Inc,

http://en.wikipedia.org/wiki/Reactive_oxygen_species,retriev

ed 18 March 2008

8. Amstad P.,Cerutti P.,Ghosh R.,Oya Y.,December 1994,The role

of the cellular antioxidant defense in oxidant

carcinogenesis, Environ Health Perspect,Vol 102(Supply

10),123–129

9. Image:SOD.gif,28 November 2006,Mediawiki,Wikimedia

Foundation Inc,

http://en.wikipedia.org/wiki/Image:SOD.gif,retrieved 27

February 2008

10. General,Types,Superoxide dismutase, 23 February

2008,Mediawiki,Wikimedia Foundation Inc,

http://en.wikipedia.org/wiki/Superoxide_dismutase,retrived

01 March 2008

11. Copper/Zinc superoxide dismutase signatures,April 2006,

Swiss Institute of

Bioinformatics,http://www.expasy.org/prosite/PDOC00082,retri

eved 01 March 2008

12. Image:PBB Protein SOD1 image.jpg,9 October 2007,

Mediawiki,Wikimedia Foundation Inc,

http://en.wikipedia.org/wiki/Image:PBB_Protein_SOD1_image.jp

g,retrieved 01 March 2008

13. SOD1 (copper zinc superoxide dismutase 1) and

ALS,2004,The ALS Association,

http://www.alsa.org/research/article.cfm?,retrieved 03 March

2008

14. SOD1 (copper zinc superoxide dismutase 1) and

ALS,2004,The ALS Association,

http://www.alsa.org/research/article.cfm?,retrieved 03 March

2008

15. Carlson E.J.,Carra S and Epotein C.J.,October

2001,Strain-dependent high-level expression of a transgene

for manganese superoxide dismutase is associated with growth

retardation and decreased fertility, Free Radic. Biol.

Med.,Vol 31(8),1018-1030

16. SOD2,6 April 2008, Mediawiki,Wikimedia Foundation Inc,

http://en.wikipedia.org/wiki/SOD2,retrived 7 April 2008

17. Human,Superoxide dismutase,23 February

2008,Mediawiki,Wikimedia Foundation Inc,

http://en.wikipedia.org/wiki/Superoxide_dismutase,retrieved

28 February 2008

18. Image:PBB Protein SOD2 image.jpg,1 November

2007,Mediawiki,Wikimedia Foundation

Inc,http://en.wikipedia.org/wiki/Image:PBB_Protein_SOD2_imag

e.jpg,retrieved 28 February 2008

19. Human,Superoxide Dismutase,23 February

2008,Mediawiki,Wikimedia Foundation Inc,

http://en.wikipedia.org/wiki/Superoxide_dismutase,retrieved

29 February 2008

20. Superoxide Dismutase, Extracellular EC-SOD,Superoxide

Dismutase,Elevated Extracellular,Included,Superoxide

Dismutase 3; SOD3,30 May 2007,

http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?

id=185490,retrieved 29 February 2008

21. Image:PBB Protein SOD1 image.jpg,9 October 2007,

Mediawiki,Wikimedia Foundation Inc,

http://en.wikipedia.org/wiki/Image:PBB_Protein_SOD1_image.jp

g,retrieved 01 March 2008

22. Reaction,Superoxide Dismutase,23 February

2008,Mediawiki,Wikimedia Foundation Inc,

http://en.wikipedia.org/wiki/Superoxide_dismutase,retrieved

28 February 2008

23. Superoxide: superoxide oxidoreductase, 2008,

Worthington Biochemical Corporation, http://www.worthington-

biochem.com/SODBE/default.html,retrieved 28 February 2008

24. Decomposition,Hydrogen Peroxide, 14 April

2008,MediaWiki,Wikimedia Foundation Inc,

http://en.wikipedia.org/wiki/Hydrogen_peroxide,retrieved 14

April 2008

25. Fenton's reagent, 21 February 2008,MediaWiki,Wikimedia

Foundation Inc, http://en.wikipedia.org/wiki/Fenton

%27s_reagent ,retrieved 28 February 2008

26. Ashraf S.S., Alhadrami S and Rauf M.A.,2006,

Degradation of Methyl Red using Fenton's reagent and the

effect of various salts,Science Direct,Vol 69(1-2),74-78

27. Fenton's reagent, 21 February 2008,MediaWiki,Wikimedia

Foundation Inc, http://en.wikipedia.org/wiki/Fenton

%27s_reagent ,retrieved 28 February 2008

28. Glutathione Peroxidase,20 March

2008,MediaWiki,Wikimedia Foundation Inc,

http://en.wikipedia.org/wiki/glutathione_peroxidase,retrieve

d on 22 March 2008

29. Image:GlutPeroxidase-1GP1.png,20 March

2008,MediaWiki,Wikimedia Foundation

Inc,http://en.wikipedia.org/wiki/image:GlutPeroxidase-

1GP1.png, retrieved;28 March 2008

30. Glutathione peroxidase, 20 March 2008,

MediaWiki,Wikimedia Foundation Inc,

http://en.wikipedia.org/wiki/Glutathione_peroxidase,retrieve

d 28 March 2008

31. Glutathione reductase,20 March 2008,

MediaWiki,Wikimedia Foundation Inc,

http://en.wikipedia.org/wiki/Glutathione_reductase,retrieved

28 March 2008

32. Image:Glutathione reductase.png, 7 May 2007,

MediaWiki,Wikimedia Foundation Inc,

http://en.wikipedia.org/wiki/Image:Glutathione_reductase.png

,retrieved 28 March 2008

33. Catalase,12 April 2008,MediaWiki,Wikimedia Foundation

Inc, http://en.wikipedia.org/wiki/Catalase,retrieved 13

April 2008

34. Image:PBB Protein CAT image.jpg, 7 November 2007,

MediaWiki,Wikimedia Foundation Inc,

http://en.wikipedia.org/wiki/Image:PBB_Protein_CAT_image.jpg

, retrieved 28 March 2008

35. Biochemistry,Superoxide dismutase,23 February

2008,Mediawiki,Wikimedia Foundation Inc,

http://en.wikipedia.org/wiki/Superoxide_dismutase,retrieved

29 March 2008

36. Cell Damage,2008, Sigma-Aldrich Co.,

http://www.sigmaaldrich.com/Area_of_Interest/Biochemicals/En

zyme_Explorer/Cell_Signaling_Enzymes/

Superoxide_Dismutase.html,retrieved 29 March 2008

37. Antholine W.E.,Goss S.P.A.,Hogg N.,1 December

1999,Mechanism of Superoxide Dismutase/H2O2-Mediated Nitric

Oxide Release from S-Nitrosoglutathione— Role of

Glutamate,Archieves of Biochemistry and Biophysics,Vol

372(1),8-15

38. The enzyme,its reaction and mechanism,Manganese

Superoxide Dismutase (MnSOD),

http://www.ufbi.ufl.edu/~hnick/Manganese%20Superoxide

%20Dismutase.htm,retrieved 8 April 2008

39. Feng L.,Huang P.,Keating M.J.,21 September

2000,Superoxide dismutase as a target for the selective

killing of cancer cells,Nature journal,407,390-395

40. Buettner G.R. and Oberley L.W.,April 1979,Role of

Superoxide Dismutase in cancer,A review,Cancer

Research,39,1141-1149

APPENDIX

SECTION A

Possible diagrams that helps in undetrstanding the dismutation

reaction better