Chapter -1 Introduction of Superoxide dismutase Enzyme (SOD) Superoxide dismutase (EC: 1.15.1.1) is an endogenous metalloenzyme mainly Cu-Zn SOD, Fe SOD and Mn SOD present in all aerobic system. It acts as an antioxidant and protects the cell from toxic, harmful ROS generated in biological systems. SOD plays major role of dismutation of ROS into non harmflil species O2 and H2O2. This is an essential redox enzyme having broad spectrum of applications such cosmetics, medicine, sports nutrition and anti-inflammatory drug etc. However the natural enzyme have certain limitations such as yield, time span, thermal stability and storage condition. This paradigm has been overcome up to certain extent using synthetic chemistry called as artificial enzymes or enzyme mimic. The SOD mimic are the stable, low molecular weight compounds possessing SOD like activity. SOD mimic could either be organic molecules, metal complexes, and nanoparticles, fiinctionalised NPs, or immobilized SOD on various supports. 1.1 Introduction All living beings on planet earth for their survival of life essentially require water, oxygen and light. In an aerobic ecosystem, in the presence of these factors a number of bio transformations takes place in the biological systems. A hundreds of multi-purpose specific enzymes are catalysing these bio transformations to run the smooth and efficient 'Chemistry of life'. Enzymes are a globular proteins made up of linear chains of amino acid residues (ranges from 62 to over 2500 residues) folds to produce a three dimensional primary, secondary, tertiary and quaternary structure of proteins'. The specificity of the enzyme depends on the sequence of these 20 amino acids, coenzymes and cofactor which also determines the structure and catalytic activity. So the enzymes are highly specific in action and play a vital role in life. They are classified on the basis of general class of reactions that they catalyse. Classification of enzyme Table no.l: Classification of enzymes Class Reaction T\T)e Subclass Dehydrogenases, Peroxidases, 1 Oxidoerductase 1 reductases, oxidases, Mono and dioxygenases Glycosyl transferases, 1 Transferases 1 amino tran sfer ase s, AH (. A B t Phosphotransferases Esterases, Glycosidases, •BSCDJB^^^^H ^ n MyO A'^* R I'>»4 Hiosphatases, Peptidases, Amidases C-C , C-O, C-N and C-S bond 1 1 1 Lyases 1 0K-» lyases Epimerases, Cis trans isomerases ..- .. f" y^ 1 J I'.t-t-j^ L (•^ I 1 ! *' X A C-..IJ,C >fM> C-C, C-O, C-N and C-S bond ^ ligases A B The above pie chart^ indicates that, a class of enzymes Hydrolases, Transferases and oxidoreductase have a major contribution of compared to other classes of enzymes. Above all, Oxidoerductase is a major class of enzyme also known as antioxidants, acts as defence system in living beings. Antioxidants are distributed in two types namely Fig. 1.1: Pie chart of enzyme Primary defence antioxidants and Secondary defence antioxidants. Primary defence antioxidants directly react or catalyse the ROS. It includes Superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX), glutathione reductase (GTx) and some minerals like Se, Mn, Cu, and Zn. Secondary defence antioxidants only scavenges the generated ROS. It includes as lipophilic enzymes, phospholipases, proteolytic enzymes, proteases, peptidases, DNA repair enzymes, endonuclease, exonuclease, and ligase enzyme^' '*. In an aerobic system. Superoxide dismutase^'^ (EC: 1.15.1.1) is an endogenous enzyme, acts as primary antioxidant. It protects the cell and other parts from the harmful attack of ROS generated in biological transformation. It is dismutate ROSinto biologically essential dioxygen and hydrogen peroxide. In short, antioxidant SOD scavenges the harmful reactive oxygen species (ROS) and coverts into neutral molecules. Since 1976, there are around 30,701 pubmed-indexed research papers published on SOD enzyme out of which 16609 were published in last decade and 2260 in the last year 2016. There are around 209 US patents granted on SOD^. In present scenario the SOD related inventions and novelties are at its peak due to its property and uses. 1.2 occurrence of SOD i. Occurrence of SOD in plants CallW«» Cu-2nSOO :5 ^ r) C<t-Zii SOD « J-r ) XaiiiiirtV- p \9»m*ei»nt9ooy (\Ukn9iaO J Fig. 1.2: Schematic diagram of SOD in plant SOD is present in almost all parts of plants with varied concentration. Generally it is located in the mitochondria, chloroplast, apoplast, perioxosomes and cell wall of the nucleus^. The SOD enzyme is isolated from different parts of plant like leaves, roots, shoots, seeds, fhiits of numerous sources. The reported SOD sources were tobacco {Nicotiana tabaccumf, mung beans (Vigna mungoY^, watermelon and citrus {Citrullus vulgaris)^^, petals of carnations {Dianthus caryophyllus)^^, peas (Pisum sativumf^, s^mach{Spinacia oleracedf'*'^^, Jackson et al. The rhizomes of the Zingiberaceae (ginger) family rhizomes from C aeruginosa in dietary- based medical applications'^, Fe-SOD was isolated from tomato leaves {Lycopersicon esculentumY^, A peroxisomal Mn SOD was isolated in pea {Pisum sativumy^ for the removal 3 of O2 formed as a result of xanthine oxidase action. The seeds and seedHngs of com (Zea mays)'^, oats (Avena sativay^, and peas {Pisum sativumY^ were used for SOD isolation. Also SOD was isolated Soyabean roots nodule'^, Tissues of Spinach, kiwi Fruit^^ and apples and were used for assay of SOD enzyme. Occurrence of SOD in animals and human beings In an abundant molecular oxygen environment during body metabolism spin restriction and reduction of oxygen may occur generating a hazardous ROS as by-product. Superoxide dismutase catalyse, these ROS into O2 and H2O2. SOD is present in eukaryotes and prokaryotes of aerobic ecological system. Fe and Mn SOD are characteristic of prokaryotes. Cu-Zn SOD are characteristics of cytosol eukaryotes. There three types of SOD's are present in humans. The SOD activity varies with tissues. The highest SOD activity was observed in liver, adrenal gland, kidney and spleen. Mn SOD in mitochondrial matrix was isolated from yeast, chicken liver, rat liver, human liver, baboon liver and pig heart ^'. In cytosol both Cu-Zn SOD and Mn SOD were observed. 1.3 Classification of SOD's SOD (EC: 1.15.1.1) was first isolated in 1938 by Mann and Kleilin. Earlier it was considered as copper storage protein but in 1963, I. Fridovich^^ isolated a protein from eukaryotes and reported as SOD enzyme. Broadly SOD enzyme is classified into three types encompasses Fe- SOD, Cu-Zn SOD and Mn SOD depending on metal ion as cofactors. Copper-Zinc Superoxide dismutase (Cu-Zn SOD) Fig.l .3: Ribbon diagram of Cu-Zn SOD In plant cells, Cu-Zn SOD is a soluble enzyme and is located in the stroma, nucleus and apoplast ^^••^*. In green plants, several isozymes of Cu Zn-SOD have been detected and isolated from both extracellular and subcellular locations, such as the chloroplasts, cytosol, mitochondria and peroxisomes^^'^^. The Cu-Zn SODs in chloroplasts or the cytosol have been isolated from various green plants observed the difference in their amino acids residues, absorption spectra in visible light, circular dichroism spectra, sensitivity to inactivation by hydrogen peroxide and immunological properties'^. In animals'^ Cu-zn SOD is denoted as (SODl). A Cu-Zn SOD were found in both eukaryotes and prokaryotes. They are p barrel proteins with either homodimeric or monomeric binuclear metal ions at the centre. Iron Superoxide dismutase (Fe SOD): Fig. 1.4: Ribbon diagram of Fe SOD Fe SOD is almost present in chloroplast in many plants, may associate with the plastid nucleoid and participate in signalling or gene regulation. It is also located in cytoplasm of cowpea. The presence of Fe SOD activity in Gingkoaceae, Nymphaceae, and Cruciferae was concluded to be a random occurrence of the enzyme in the plant kingdom'^. Comparative crystallography studies with Fe SODs showed that three primary chemical functions are necessary for Fe SOD to catalyse a dismutation reaction^": 1. Availability of at least one coordination site of Fe centre for binding O2" between two adjacent states. 2. The ion redox couple must be lie between redox potentials of O2" oxidation (0.16 v) and O2" reduction (0.89 v) in primary coordination sphere surrounded by protein matrix. 3. The rapid conversion between the Fe (II) and Fe (III) oxidation states is must at Fe centre than the spontaneous dismutation reaction^'. Manganese Superoxide dismutase (Mn SOD): Fig. 1.5: Ribbon diagram of Mn SOD Mn SOD's are located in mitochondria, chloroplasts and in the peroxisomes with homo di or tetrameric subunit at its active centre^^. Mn and Fe SODs have a similarity in the structure, but differ in their activity^^. The mechanism of Mn SOD was well explained by Bowler et al. 1994. Catalysis by Mn SODs is through the attraction of negatively charged O2" molecules to a site formed from positively charged amino acids present at the active site of the enzyme, similar to Cu-Zn SODs^"*. The metal present in the active site reduces one O2" molecule on an electron donation, which in turn forms H2O2 with a proton. Nickel Superoxide dismutase (Ni SOD): Fig. 1.6: Ribbon diagram of Ni SOD An entirely new class of SOD is established as Ni SOD found in Streptomyces and cyanobacteria with a different metal centre and distinct protein fold. Ni SOD is small protein having 117 amino acids with no any homology sequence compared to other SOD's^^. A tetramer of Ni is functioned as Ni SOD where its monomeric form is helpless. It was observed from X-ray and EPR studies that Ni is in oxidised Ni^^ form in Streptomyces seoulensis^^. Its biological function helps in regulation of reactive oxygen species. 1.3 Applications of SOD In aerobic life, nature has SOD enzyme as most potent antioxidant enzyme to fight against ROS.