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Superoxide dismutases (SODs) can be classified into three classes according metal ion content; Cu/Zn-SODs, Mn-SOD or Fe-SODs and Ni-SODs. Cu/Zn-SODs contain both Cu and Zn ions in their active sites. Mn- and Fe-SODs contain Fe or Mn in their active sites. Mn-SOD or Fe-SODs can be further subdivided into three classes: enzymes that are active with Mn and Fe or either of the two metal ions in their active site [137]. The mechanisms and redox regulation of active sites of Mn- SOD and Fe-SODs appear to incorporate crucial differences [138]. However, their active sites, structure, and amino acid sequence are quite similar to each other. H2O2

inhibits the Fe and the Cu/Zn, but not the Mn-containing superoxide dismutase. Ni– SODs are entirely different from well-known Cu/ Zn-, Fe-SOD and Mn-SODs [137].

4.3.1. Cu/Zn-SODs

Cu/Zn-SOD (erythrocuprein) was the first SOD to be identified. The oxidized form of Cu/Zn-SOD has a Cu2+ _{ion coordinated in a distorted square pyramid by}

four histidines, one of which is also a ligand for a distorted tetrahedral Zn2+ _{ion and a}

solvent molecule, while Zn2+ _{is coordinated by three additional ligands two histidines}

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Page | 100 shown in Figures 4.1 and 4.2, respectively. Nearly all cells in eukaryotes contain Cu/Zn-SOD [139]. In yeast such as Saccharomyces cerevisiae and mammals, Cu/Zn- SOD is found mainly in the cytosol with a lower fraction in the mitochondrial intermembrane space. Also, it has been detected in nuclei, lysosomes, peroxisomes

using immunocytochemical methods. Several bacteria also have been reported to

contain Cu/Zn-SODs and have been demonstrated to be embedded in the periplasm. In prokaryotes, Cu/Zn-SOD is important for their survival especially in late

stationary phase and their growth ability in aerobic environments [140]. Most of the

reported Cu/Zn-SODs are from Gram-negative bacteria including E. coli, and they are localised in the periplasm, found only in Gram-negative bacteria [141].

Figure 4.1. Crystal structure of Cu/Zn superoxide dismutase from bovine erythrocyte with copper

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Page | 101

Figure 4.2. The catalytic cycle of Cu/Zn superoxide dismutase. Superoxide radical anion displaces the

axial water and binds to Cu2+ _{centre for electron transfer. This binding is stabilised by a positively}

charged arginine residue. Reduction of Cu(II) leads to the release of dioxygen and protonation of imidazolate group (His61) loses its binding to Cu(I). The second superoxide radical coordinates with Cu(I). Hydrogen peroxide is formed by electron transfer from inner-sphere and is protonated. Additional protonation results in the release of hydrogen peroxide, and a water molecule coordinates with Cu(II) again, reforming the imidazolate bridge [143],[144].

4.3.2. Fe-SODs and Mn-SODs

FeSODs are found mainly in prokaryotes while Mn-SODs are occurring in both prokaryotic and eukaryotic cells, in a high frequency [139]. The Mn-SOD and Fe-SOD family was first discovered by Fridovich in 1970 and 1973, respectively [140]. The catalytic mechanism of Fe-SOD and Mn-SODS are similar to that of Cu/ZnSODs (see Figure 4.3 for more detail). Inside the active side of each subunit is a single manganese or iron center bound to three histidines, one aspartate (Figure 4.4) and water molecule or hydroxide anion in a trigonal bipyramidal geometry [139],[140]. Therefore, their active site is completely unrelated to Cu/ZnSODs [138].

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Page | 102 The Fe-SOD and Mn-SOD enzymes share a well-conserved protein fold and significant sequence similarity across different phyla of archaea, eubacteria and eukaryotes. They encompass a dimer or tetramer of about 21 kDa subunits. Usually, both metal forms are expressed in the same cells. The dimeric forms are expressed by bacteria, but tetrameric forms are found in a limited number of prokaryotes, such as hyperthermophiles. Generally, in eukaryotes only tetrameric Mn-SODs are found [140].

The location of Fe-SOD and Mn-SOD in cyanobacteria and photosynthetic bacteria is different. Mn-SOD is embedded in the plasma and thylakoid membranes while Fe-SOD is localised in the cytosol. In photosynthetic organisms, the expression profile of Fe-SOD and Mn-SOD show differences as well. Expression of Mn-SOD is strongly regulated and inducible by stress conditions. In contrast, expression of Fe- SOD has been revealed to be regulated developmentally and by light and is changed by environmental stimuli. It has been reported that FeSOD is important for protection of photosynthetic organisms from damage as a consequence of chilling, sulphur dioxide stress, drought, Cu toxicity, and superoxide radicals produced by the electron transport chain [137].

In the cyanobacterium Anabaena sp. strain PCC7120, two SOD enzymes (FeSOD and MnSOD) have been isolated and purified: the FeSOD enzyme is cytosolic while MnSOD is localised in the thylakoid membrane. MnSOD has a leader peptide and a membrane attachment motif in the N-terminal sequence. In addition, it has been reported that the leader peptide is essential for its activity and membrane localization. The Anabaena sp. strain PCC 7120 genome contains no Cu/ZnSODs [145].

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Page | 103

Figure 4.3. The catalytic cycle of Mn-SOD and Fe-SOD, M represent Fe and Mn ions [146].

Figure 4.4. The active site of superoxide dismutase (a) inMnSOD from E. coli (PDB code: 1EN5)

and (b) in FeSOD from Thermosynechococcus elongatus (PDB code: 1MY6). PyMol was used for display.

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Page | 104 Studies indicate that FeSOD enzymes from E coli and Pseudomonas ovalis can be inactivated by H2O2. Cu/ZnSODs can also be inactivated by H2O2 but MnSODs

are not. The inactivation of FeSOD from E. coli by H2O2 has been correlated with

loss of tryptophan and some loss of iron, but not histidine or another amino acid as detected in FeSOD from Pseudomonas ovalis. The proposed mechanism of Cu/Zn SOD inactivation is through a reduction of Cu(II) to Cu(I) by H2O2, followed by a

Fenton-like reaction between Cu(I) and H2O2. The strong oxidant (Cu(II)-O) is

probably generated at the Cu site by the latter reaction, and then this oxidant attacks an adjacent histidine, leading to the loss of one histidine residue per subunit during inactivation [147].

4.3.3. NiSODs

Ni-dependent SODs are completely unrelated to the well-known Cu/ZnSOD, FeSOD, and MnSODs. It has been shown that Cyanobacteria [139] and several

Streptomyces species produce nickel containing SODs. The active site of NiSODs contain one Ni per monomer [138].