Metalloenzymes contain metal ions that help catalyze important biochemical reactions. Antioxidants protect cells from oxidative damage caused by free radicals generated during normal metabolism and environmental exposures. There are many classes of antioxidants including vitamins, minerals, enzymes, carotenoids, flavonoids, and phenolic compounds. Antioxidants act as reducing agents that prevent oxidative chain reactions and thereby protect cellular components from oxidative damage.

1. Metalloenzyme; Antioxidant & Their
Relationship With Aging, Cancer
&Metabolic Disorders
Ms. Latika Yadav (Research Scholar), Dept. of Foods and Nutrition,
College of H.Sc,Maharana Pratap University of Agriculture and
Technology, MPUAT, Udaipur, rajasthan-313001, email.id:
a.lata27@gmail.com

2. Metal plays roles in approximately one-
third of the known enzymes. Metals may be
a co-factor or they may be incorporated
into the molecule, and these are known as
metalloenzymes.
A metalloenzyme is an enzymatic protein
that has a strong link between its protein
part and metal , where the metal is
embedded with in the molecule. In this case
metal ion is bound tightly to the enzyme
and is not dissociated even after several
extensive steps of purification.

3. Metal plays a variety of roles such as :
•They help in either maintaining or producing( or
both), active structural conformation of the
enzyme,
•Formation of enzyme substrate complex
•Making structural changes in substrate molecule,
•Accept or donate electrons,
•Activating or functioning as nucleophiles, and
•Formation of ternary complexes with enzymes or
substrate.

4. Physiological Roles of Metal Ions
• cell replication
• energy production (ATP
synthesis)
• O2 transport and storage
• synthesis of neurotransmitters
• counteracting the effects of aging
• RNA synthesis
• alcohol breakdown in your liver
• hormone synthesis
• dilating blood vessels
• photosynthesis

5. It is estimated that approximately half of all proteins
contain a metal. In another estimate, about one quarter
to one third of all proteins are proposed require metals
to carry out their functions.Thus, metalloproteins have
many different functions in cells, such as
•enzymes, transport and storage proteins, and
• signal transduction proteins.
• 33% of all enzymes contain transition metal ions!
• Metal ions PROMOTE REACTIONS
• bond cleavage
• bond formation
• electron transfer
• atom transfer
• Metal ions PROMOTE PROTEIN FOLDING

6. Coordination chemistry principles
In metalloproteins, metal ions are usually coordinated by
nitrogen, oxygen or sulfur centres belonging to amino acid
residues of the protein.
These donor groups are often provided by side-chains on
the amino acid residues. Especially important are the
imidazole substituent in histidine residues, thiolate
substituents in cysteinyl residues, and carboxylate groups
provided by aspartate.
In addition to donor groups that are provided by amino
acid residues, a large number of organic cofactors
function as ligands. Perhaps most famous are the
tetradentate
N4 macrocyclic ligands incorporated into the heme
protein. Inorganic ligands such as sulfide and oxide are
also common.

7. Storage and transport metalloproteins
Oxygen carriers
Hemoglobin, which is the principal oxygen carrier in
humans has four sub-units in which the iron(II) ion is
coordinated by the planar, macrocyclic ligand
protoporphyrin IX (PIX) and the imidazole nitrogen
atom of a histidine residue. The sixth coordination site
contains a water molecule or a dioxygen molecule.
myoglobin has only one such unit.
The active site is located in an
hydrophobic pocket. This is important as, without it,
the iron(II) would be irreversibly oxidised to iron(III). Haemoglobin
The equilibrium constant for the
formation of HbO2 is such that oxygen is taken up or
released depending on the partial pressure of oxygen
in the lungs or in muscle. In hemoglobin the four sub-
units show a cooperativity effect which allows for
easy oxygen transfer from hemoglobin to myoglobin.

8. Hemerythrin is another iron-containing oxygen carrier. The oxygen
binding site is a binuclear iron center. The iron atoms are
coordinated to the protein through the carboxylate side chains of a
glutamate and aspartate and five histidine residues. The uptake of
O2 by hemerythrin is accompanied by two-electron oxidation of
the reduced binuclear center to produce bound peroxide (OOH-).
Hemocyanins carry oxygen in the blood of most molluscs, and
some arthropods such as the horseshoe crab. They are second only
to hemoglobin in biological popularity of use in oxygen transport.
On oxygenation the two copper(I) atoms at the active site are
oxidised to copper(II) and the dioxygen molecules is reduced to
peroxide, O22-.

9. Cytochromes
Iron(II), can easily be oxidized to iron(III). This functionality is
used in cytochromes which function as electron-transfer vectors.
The presence of the metal ion allows metalloenzymes to
perform functions such as redox reactions that cannot easily be
performed by the limited set of functional groups found in
amino acids. The iron atom in most cytochromes is contained in
a heme group. The differences between those cytochromes lies
in the different side-chains.
For instance Cytochrome a has a heme a
prosthetic group and cytochrome b has a heme b prosthetic
group. These differences result in different Fe2+/Fe3+ redox
potentialssuch that various cytochromes are involved in the
mitochondrial electron transport chain.
Cytochrome P450 enzymes perform the function of inserting an
oxygen atom into a C—H bond, an oxidation reaction

10. Rubredoxin
Rubredoxin is an electron-carrier found in
sulfur-metabolizing bacteria and archaea.
The active site contains an iron ion which
is coordinated by the sulphur atoms of
fourcysteine residues forming an almost
regular tetrahedron. Rubredoxins perform
one-electron transfer processes. The
oxidation state of the iron atom changes
between the +2 and +3 states. In both
oxidation states the metal is high spin,
which helps to minimize structural
changes.
rubredoxin active site

11. Plastocyanin
Plastocyan is one of the family of blue
copper proteins which are involved in electron
transfer reactions. The copper binding site is
described as a ‘distorted trigonal pyramidal’.The
trigonal plane of the pyramidal base is composed
of two nitrogen atoms (N1 and N2) from separate
histidines and a sulfur (S1) from a cysteine. Sulfur
(S2) from an axial methionine forms the apex.
The ‘distortion’ occurs in the bond lengths
between the copper and sulfur ligands. Plastocyanin copper
binding

12. Metal-ion storage and transfer
Iron
Iron is stored as iron(III) in ferritin. The exact nature of the
binding site has not yet been determined. The iron appears to
be present as an hydrolysis product such as FeO(OH). Iron is
transported by transferrin whose binding site consists of two
tyrosines, one aspartic acid and one histidine The human body
has no mechanism for iron excretion. This can lead to iron-
overload problems in patients treated with blood transfusions,
as, for instance, with β-thallasemia.
Copper
Ceruloplasmin is the major copper-carrying protein in the
blood. Ceruloplasmin exhibits oxidase activity, which is
associated with possible oxidation of Fe2+ (ferrous iron) into
Fe3+ (ferric iron), therefore assisting in its transport in the
plasma in association with transferrin, which can only carry
iron in the ferric state.

13. Metalloenzymes all have one feature in common, namely, that the metal ion is bound
to the protein with one labile coordination site. As with all enzymes, the shape of the
active site is crucial. The metal ion is usually located in a pocket whose shape fits the
substrate.
Carbonic anhydrase
Active site of carbonic anhydrase. The three coordinating histidine residues are
shown in green, hydroxide in red and white, and the zinc in gray.
CO2 + H2O H2CO3
This reaction is very slow in the absence of a catalyst, but quite fast in the presence of
the hydroxide ion
CO2 + OH- HCO3-

14. Vitamin B12-dependent enzymes
Vitamin B12 catalyzes the transfer of methyl (-CH3) groups
between two molecules, which involves the breaking of C-C
bonds, a process that is energetically expensive in organic
reactions. The metal ion lowers the activation energy for the
process by forming a transient Co-CH3 bond.This is a naturally
occurring organometallic compound, which explains its
function in trans-methylation reactions, such as the reaction
carried out by methionine synthase.

15. Nitrogenase (nitrogen fixation)
The fixation of atmospheric nitrogen is a very energy-intensive
process, as it involves breaking the very stable triple bond
between the nitrogen atoms. The enzyme nitrogenase is one
of the few enzymes that can catalyze the process. The enzyme
occurs in certain bacteria.
There are three components to its action:
1. a molybdenum atom at the active site,
2. Iron-sulfur clusters which are involved in transporting the
electrons needed to reduce the nitrogen and
3. an abundant energy source.
The energy is provided by a symbiotic relationship between the
bacteria and a host plant, often a legume. The relationship is
symbiotic because the plant supplies the energy by
photosynthesis and benefits by obtaining the fixed nitrogen.

16. Superoxide dismutase
The superoxide ion, O2- is generated in biological systems by reduction of
molecular oxygen. It has an unpaired electron, so it behaves as a free radical. It is
a powerful oxidising agent. These properties render the superoxide ion very toxic
and are deployed to advantage by phagocytes to kill invading micro organisms.
Otherwise, the superoxide ion must be destroyed before it does unwanted damage
in a cell. The superoxide dismutase enzymes perform this function very
efficiently.
Oxidation: M(n+1)+ + O2− → Mn+ + O2
Reduction: Mn+ + O2− + 2H+ → M(n+1)+ + H2O2.
This type of reaction is call a dismutation reaction. It involves both oxidation and
reduction of superoxide ions. The superoxide dismutase group of enzymes,
abbreviated as SOD, increase the rate of reaction to near the diffusion limited
rate.

17. Calmodulin
Calmodulin is an example of a signal-transduction protein. It is a small protein
which contains four EF-hand motifs, each of which can bind a Ca2+ ion.
In an EF-hand loop the calcium ion is coordinated in a pentagonal bipyramidal
configuration.
The protein has two approximately symmetrical domains, separated by a flexible
"hinge" region. Binding of calcium causes a conformational change to occur in
the protein. Calmodulin participates in an intracellular signalling system by
acting as a diffusible second messenger to the initial stimuli.

18. The EF hand is a helix-loop-helix structural domain found in a large family of
calcium-binding proteins. The EF-hand motif contains a helix-loop-helix topology,
much like the spread thumb and forefinger of the human hand, in which the
Ca2+ ions are coordinated by ligands within the loop. It consists of twoalpha helices
positioned roughly perpendicular to one another and linked by a short loop region
(usually about 12 amino acids) that usually binds calciumions. The motif takes its
name from traditional nomenclature used in describing the protein parvalbumin,
which contains three such motifs and is probably involved in muscle relaxation via
its calcium-binding activity. EF hands also appear in each structural domain of the
signaling protein calmodulin and in the muscle protein troponin-C.

19. Regulation and Control
Metalloenzyme Inhibition
•Approximately one-third of the known enzymes have metals as part
of their structure, require that metals be added for activity, or are
further activated by metals.
•In enzymes where a metal has been built into the structure of the
enzyme molecule, the metal cannot be removed without destroying
that structure. Such enzymes include the metalloflavoproteins, the
cytochromes, and the ferredoxins.
•Metals resemble protons (H+) in that they are electrophiles that are
capable of accepting an electron pair to form a chemical bond. In
doing so, metals may act as general acids to react with anionic and
neutral ligands. This characteristic of metals is helpful in enzymatic
structure and function but makes the enzyme it is part of pH
dependent. Changes in pH can disrupt this electron flow that the metal
would normally help facilitate and thus inhibit the overall
effectiveness of the metalloenzyme.

20. ANTIOXIDANT
An antioxidant is a molecule capable of slowing or
preventing the oxidation of other molecules.
Oxidation is a chemical reaction that transfers electrons
from a substance to an oxidizing agent. Oxidation
reactions can produce free radicals, which start chain
reactions that damage cells.

21. HISTORY
• The term antioxidant originally was used to refer
specifically to a chemical that prevented the
consumption of oxygen. In the late 19th and early 20th
century, extensive study was devoted to the uses of
antioxidants in important industrial processes, such as
the prevention of metal corrosion, the vulcanization of
rubber, and the polymerization of fuels in the fouling of
internal combustion engines.
• Early research on the role of antioxidants in biology
focused on their use in preventing the oxidation of
unsaturated fats, which is the cause of rancidity.
Antioxidant activity could be measured simply by
placing the fat in a closed container with oxygen and
measuring the rate of oxygen consumption. However, it
was the identification of vitamins A, C, and E as
antioxidants that revolutionized the field and led to the
realization of the importance of antioxidants in the
biochemistry of living organisms.

22. • The possible mechanisms of action of antioxidants were first
explored when it was recognized that a substance with anti-
oxidative activity is likely to be one that is itself readily
oxidized.Research into how vitamin E prevents the process of
lipid peroxidation led to the identification of antioxidants as
reducing agents that prevent oxidative reactions, often by
scavenging reactive oxygen species before they can damage
cells.

23. GENERATION OF EXCESS FREE RADICALS
• Faulty dietary habits
• Diet high in animal fats
• Sunlight
• Toxic Drugs
• Cigarette smoking
• Over-exercise
• Environmental Pollution

24. CLASSIFICATION OF ANTIOXIDANTS
1.) VITAMINS
a) Vitamin E - It is fat soluble, alpha tocopherol is in a unique
position to safeguard cell membranes largely composed of
fatty acids from damage by free radicals. Alpha tocopherol
also protects the fats in low density lipoproteins from
oxidation.
b)Vitamin C - It scavenge free radicals that are in an aqueous
(watery) environment, such as inside cells. Vitamin C works
synergistically with vitamin E to quench free radicals.
c) Vitamin A -Vitamin A (retinol), also synthesized by the body
from beta-carotene, protects dark green, yellow and orange
vegetables and fruits from solar radiation damage, and is
thought to play a similar role in the human body.

25. 2.MINERALS :
a) Selenium
b) Manganese
c) Copper
d) Zinc
These are components of antioxidant enzyme like glutathione
peroxidase, superoxide dismutase and catalase.
3.VITAMIN COFACTORS :
a) Coenzyme Q10
4.CAROTENOIDS:
a) Beta- carotene : It is the best quencher of single oxygen(an
energized but uncharged form of oxygen that is toxic to cells).
Beta- carotene is also especially excellent at scavenging free
radicals in low oxygen peroxidase.
b) Lycopene
c) Lutein

26. 5. a) Flavonoid polyphenolic
• Flavones 7.Other nonflavonoid phenolics
• Apigenin •Flavonolignans
• Luteolin •Xanthones
• Tangeritin
b) Flavonols 8.Other Organic Antioxidants
• Myricetin •Bilirubin
• Proanthocyanidins •Citric acid
•Lignan
c) Flavanones: •R alphalipoic acid
• Hesperetin •Uric acid
d) Flavanols and their polymers The antioxidant enzymes are
• Isoflavone phytoestrogens •Superoxide dismutase,
• Anthocyanins •Catalase
•Glutathione peroxidase
6. Phenolic acids and their esters They serve as primary line of
• Chlorogenic acid defense in destroying free radicals.
• Ellagic acid
• Gallic acid
• Rosmarinic acid

27. Other Classifications
1.Preventive Antioxidants :- They inhibit the initial production of
free radicals. They include catalase,glutathione
peroxidase,diethyltriamine pentaacetate and ethylene diamine
tetra-acetate(EDTA).
2.Chain breaking Antioxidants :- They inhibit the damaging phase
of free radicals. They include superoxide dismutase, uric acid and
Vitamin E. Alpha tocopherol act as the most effective naturally
occurring chain breaking antioxidants in body tissues.
3.Water soluble antioxidants:- Water soluble antioxidants are
referred to as hydrophilic antioxidants. Basically, they are able to
assist the body in the process of cell cytosol and help out in the
blood plasma. In other words they take a hands on approach to
ridding the body of harmful free radicals and pollutants. Most
common water soluble antioxidants are:
• Ascorbic acid
• Glutathione
• Lipoic acid
• Uric acid

28. 4 .Lipid Soluble Antioxidants:
Unlike water soluble antioxidants, the lipid soluble version do not
actively go out seeking to destroy rogue cells in the human body.
These are the antioxidants which in fact have a much more passive
role in keeping the human body healthy. Basically, these antioxidants
work by clinging on to damaged cells, and injecting valuable nutrients
which support the replenishment and health of that particular
individual cell. In this way, antioxidants are able to promote the health
of cells on a celllular level,by working in sync with the cells
themselves.For e.g. :- Carotenes , Ubiquinol
Deficiency of antioxidants
A shortage of antioxidants could cause, or assist in causing Alzheimer's
disease, cancer, cardiovascular disease, cataracts, diabetes,
hypertension, infertility, macular degeneration (eye lens
degeneration) , mental illness, respiratory tract infection.
By adding enough antioxidants to the diet, there is less oxidation stress,
and aging is also slowed down.

29. FOOD SOURCES OF ANTIOXIDANTS
• Beta-carotene- Green vegetables, ripe yellow fruits &
vegetables like papaya ,mango ,pumpkin and carrots.
• Vitamin A – Milk fat, Egg yolk,liver,kidney & fortified
vanaspati.
• Vitamin C – Fresh & Citrus fruits-
amla,orange,lemon,sweet lime,guava and gooseberry,
green leafy vegetables & sprouted pulses.
• Vitamin E – Oil seeds,cereals,nuts,cereal products,
vegetable oils & egg yolk.
• Selenium & Zinc – Meat,Sea-food,Cereals & pulses.
• Copper – Oysters, Mushroom, Liver & nuts.
• Iron- Green leafy vegetables, cereals,
millets,pulses,nuts,meat and liver

30. Non-Nutrient antioxidants sources:
Flavonoid, flavonols, phenolic acids, non flavonoid phenolics.
Such rich sources of these compounds are bean, cloves, oats,
tea, coffee, grapes, turmeric, mustard,walnut,
tomato, brown rice, oak bark, red wine.

31. Antioxidants and aging
Aging is an irreversible phenomenon for all living organisms. With aging, cell
division and replacement of dead or damaged cells slows down. Cell death,
mutation or damage is partly
caused by the free radicals.
Free radicals affect the skin in three main ways:
• They can alter the fatty layers in cellular membranes. These fatty layers provide
structure to the cell, and control which nutrients and other agents can pass in
and out.
• They can alter the DNA within cells, which aside from the potential to develop
into serious illnesses, can make skin inclined to wrinkles and sagging before its
natural biological time.
• Altered DNA creates a blueprint for collagen and elastin fibers that don't
function as healthy, normal ones would.

32. Free radicals also lead to a process called the cross-linking of
collagen fibers. This occurs in the skin's dermis, as a result of
collagen and elastin fibers becoming hard, thick, and then
binding together. Cross-linked fibers create wrinkles, skin sag,
and cause regular expression lines to become etched in face as a
permanent fixture.
With healthy collagen and elastin fibres these expression lines would
simply disappear once moved facial muscles in a different way.
And enzymes that metabolize collagen are encouraged by free
radicals, which, given the importance of collagen in youthful
looking skin, is best minimized.
Antioxidant and aging relationship
• They strengthen the capillaries that supply important nutrients to
the skin cells, as well as supporting cellular membranes.
• Healthy cell membranes regenerate quickly and slow the aging
process.
• Antioxidants' anti-aging benefit is due to their anti-inflammatory
effect.

33. ANTIOXIDANT AND CANCER
Cancer – a disease that affects so many around the world and
continues to be studied earnestly in order to finally identify a
cure. But, in the meantime, researchers, in an effort to take
control of the spread of this disease, promote programs of
prevention. Diet, exercise, and the avoidance of controllable
environmental pollutants are all part of the effort to prevent
cancer.
It has been shown that cancer derives from good cells gone bad.
Affected by poor diet, environmental factors, and chemical
substances, molecules inside the body lose electrons in
response. The molecules become free radicals and, as such,
they begin their attack on healthy cells to take back electrons.

34. Antioxidants and cancer:
• Antioxidants bolster the immune system and work alongside
healthy cells to combat free radicals.
• Cancer works against the cells in the body while antioxidants
work on behalf of cells.
Studies related to antioxidants and cancer:
• The first large randomized trial on antioxidants and cancer
risk was the Chinese Cancer Prevention Study, published in
1993. This trial investigated the effect of a combination of
beta-carotene, vitamin E and selenium significantly reduced
incidence of both gastric cancer and cancer overall.
• A 1994 cancer prevention study entitled the Alpha-
Tocopherol/Beta Carotene Cancer Prevention Study
demonstrated that lung cancer rates of Finish male smokers
increased significantly with beta-carotene and were not
affected by vitamin E.

35. ANTIOXIDANT AND DIABETES
Diabetes is a metabolic disorder. The disorder is due to a
deficiency or diminished effectiveness of the hormone
insulin.
Antioxidants are beneficial for diabetes suffers, not only to
maintain antioxidant levels in the body but also to treat the
long term complications that can arise.
• Neuropathy
• Retinopathy
• Nephropathy
Multiple sources of oxidative stress in diabetes including -
• Non enzymatic,
• Enzymatic
• Mitochondrial pathways.

36. Antioxidants and Cardiovascular Diseases
Atherosclerosis is a condition where the walls of the arteries
are damaged and narrowed by deposits of cholesterol and
other fatty substances, calcium, fibrin, and cellular wastes ,
eventually blocking off the flow of blood. High blood levels
of cholesterol - particularly the cholesterol carried by low-
density lipoprotein ("LDL", a protein found in blood) - are
associated with an increased risk of atherosclerosis.
Oxidation of LDL is believed to contribute to the development
of atherosclerosis (Frei 1995). Macrophage cells
preferentially take up oxidized LDL, become loaded with
lipids, and convert into "foam cells" (Aviram 1996). Foam
cells accumulate in fatty streaks, early signs of
atherosclerosis. Humans produce auto-antibodies against
oxidized LDL.

37. The identification of LDL oxidation as a key event in
atherosclerosis suggests that it may be possible to reduce the
risk of atherosclerosis by antioxidant supplementation (Ylä-
Herttuala 1991). Vitamin E is the major naturally-occurring
antioxidant in human lipoproteins (Bowry et al. 1992). Most
circulating carotenoids are associated with lipoproteins in
plasma (Clevidence and Bieri 1993). Bieri 1993).
The largest fraction of total carotenoids is found in LDL, as
evidenced by the typically yellow color of this lipoprotein
fraction (Clevidence and Bieri 1993). The largest fraction of
hydrocarbon carotenoids (e.g., beta-carotene and lycopene),
as well as most vitamin E and other tocopherols, is
transported by LDL ( Oshima et al. 1997), suggesting that
these compounds in particular may play an important role in
preventing oxidative modification of this lipoprotein
fraction.

38. REFERENCE
•Chatterjea M.N., Shinde,Rana, Textbook of Medical
Biochemistry. Jaypee Brothers Medical Publishers, New
Delhi,1999.
•Deb,A.C., Fundamentals of Biochemistry. New Central Book
agency(P)Ltd., Kolkata, 2008.
•http://en.wikipedia.org/wiki/Metalloprotein
•http://depts.washington.edu/chemcrs/bulkdisk/.../notes_Lecture_3
.pdf
•www.sciencemag.org/content/261/5122/701.full.pdf
•http://en.wikipedia.org/wiki/Antioxidant

39. THANK YOU