Nebraska Redox Biology Center Educational Portal

Superoxide Dismutases and Superoxide Reductases

Superoxide (O2.-) is highly reactive compounds produced when oxygen is reduced by a single electron [ 1, 2, 3, 4, 5 ].

Superoxide is formed in all organisms as result of contact with oxygen. Depending upon its localization and concentration it may act as a signaling agent or a toxic substance. Its levels are controled in vivo by two different types of enzymes, superoxide reductase (SOR) and superoxide dismutase (SOD) [ 1, 2, 3, 4, 6, 7, 8 ]. Nearly all type of cells and intracellular organelles may generate superoxide anion using the enzymatic complexes or as result of by external stress such as radiation, xenobiotics, etc [ 1, 2, 3, 4, 5 ]. Several sources for superoxide radical are known: 1) the mitochondrial electron transport chain, 2) cytosolic xanthine and xanthine oxidase, 3) nitric oxide synthetizes, 4) membrane-associated NADPH oxidase complex, 5) hemoglobin in erythrocytes, 6) homocysteine. Superoxide can be transformed to the radical or non-radical type of ROS

Reactive species oxidation from starting superoxide radical [ 3 ].

Superoxide Production in Biological Systems

1) Mitochondrial ROS production. There are two respiratory chain complexes (I and III) involved in superoxide production. From quantitative data obtained on isolated mitochondria, it has been suggested that about 2-5% of oxygen consumption is due to superoxide anion generation and that about 70-80% of superoxide formation is is connected with the operation of the Q cycle in complex III. Another predictions estimate that, under physio- logic conditions, the superoxide production is around 0.1% of the respiratory rate [ 9, 10, 11 ]. Electrons (e-) donated from NADH and FADH2 pass through the electron transport chain and ultimately reduce O2 to form H2O at complex IV. ROS are produced from the leakage of e- to form superoxide (O2.-) at complex I and complex III. O2.- is produced within matrix at complex I, whereas at complex III O2.- is released towards both the matrix and the intermembrane space. Once generated, O2.- is dismutated to H2O + O2 by superoxide dismutase 1 (SOD1) in the intermembrane space and by SOD2 in the matrix. Afterwards, H2O2 is fully reduced to water by glutathione peroxidases (GPX) [ 9, 10, 11, 12 ].

Mitochondrial sourses of ROS. OM: outer membrane; IM: inner membrane [ 12 ].

2) Xanthine oxidase is a ubiquitous enzyme involved in a variety of physiological and pathophysiological processes. It plays a critical role in purine catabolism producing uric acid and hydrogen peroxide thereby contributing to other reactive species generation. XO can use as substrate either oxygen, in normal conditions or hyperoxia, and nitrate in hypoxia. In hypoxia XO shifts from oxygen consumption to nitrite consumption [ 13, 14, 15 ].

Xanthine oxidase (XO) is able to generate both, NO or superoxide depending on the cellular changing conditions [ 3 ].

3) Nitric oxide synthases are a family of enzymes catalyzing the production of nitric oxide (NO) from L-arginine. There are three nitric oxide synthases isoforms in human: nNOS (neuronal NOS), iNOS (inducible NOS) and eNOS (endothelial NOS). Endothelial NOS may generate superoxide depending the availability of its substrates within cell. The endothelial nitric oxide synthase activity is regulated by a combination of mechanisms that allow eNOS to modulate its activity under physio-pathological condition [ 16, 17, 18, 19 ].

Endothelial Nitric oxide synthase (eNOS) is generating either NO or superoxide depending on the substrate availability. [ 3 ].

4) NADPH Oxidases. The nicotinamide dinucleotide phosphate oxidases (NOX or NADPH Oxidases) proteins are integral membrane proteins sharing several conserved structural features. It is made up of six subunits: one of them has GTP-ase activity while the others five have oxidase activity. NADPH oxidase utilizes NADPH as the electron donor to reduce molecular oxygen and to produce superoxide. Activation of this enzyme requires the assembly of both cytosolic and membrane bound subunits to form a functional enzyme complex [ 20, 21, 22 ]. The C-terminal tails of all these proteins contain a catalytic subunit consisting of FAD and NADPH-binding sites, whereas the N-terminus is made up of six transmembrane domains and two haem groups which form a channel to allow successive transfer of electrons [ 23, 24, 25 ]. The general reaction catalyzed by phagocytic/non-phagocytic NADP oxidase is:

NADPH + 2O2 ↔ NADP + 2O2.- ;

ROS production is achieved through the removal and transfer of an electron from an NADPH substrate to FAD, then haem and finally to molecular oxygen, generating superoxide (O2.- ), which is rapidly converted into H2O2 by superoxide dismutase. H2O2 is then capable of freely diffusing across membranes due to the activity of aquaporin channel proteins [ 23, 24, 26 ].

NOX enzymes reduce molecular oxygen to superoxide as a primary product, and this is further converted to various reactive oxygen species. Different isoforms require additional subunits to form functional reactive oxygen species-generating NADPH Oxidase [ 27 ].

5) Hemoglobin oxidation. Oxygen binds to hemoglobin at the ferrous iron. The ferrous state (Fe2+) of iron is a condition for hemoglobin normal function. However a small percent of Fe2+ is slowly converted by O2 to ferric form (Fe3+) in resulting methemoglobin. An enzymatic system, methemoglobin reductase quickly restores Fe3+ to Fe2+ and reduces methemoglobin back to hemoglobin. Binding of oxygen to the iron in the hem is considered not to change the oxidation state of the metal. However oxygenated hem has some of the electronic characteristics of Fe3+ - O2- peroxide anion. Fe3+ and O2- complex is able to generate superoxide. Hemoglobin auto-oxidation causes superoxide formation within erythrocyte [ 28, 29, 30 ].

6) Homocysteine methabolism may result in generation of superoxide radicals thus promoting vasoconstriction [ 31 ].

Major Representatives of Superoxide Dismutases and Reductases

Superoxide dismutase (SOD) represents a group of enzymes that use metal cofactor (nickel, iron, manganese, or copper/zinc ions). Superoxide dismutase detoxify superoxyde by catalyzing its dismutation. One superoxide molecule is being oxidized to O2 and another is reduced to H2O2. In the first step of reaction the ion metal is reduced by superoxide which loses its electron and is converted to molecular oxygen. As result, metal cofactor changes oxidation state (Ni3+ → Ni2+, Fe3+ → Fe2+, Mn3+ → Mn2+ and Cu2+ → Cu1+). In second step of reaction the ion metal is oxidized by superoxide which accept electron from tetal and is converted to hydrogen peroxyde (Ni2+ → Ni3+, Fe2+ → Fe3+, Mn2+ → Mn3+ and Cu1+ → Cu2+). As result, the enzyme is returned to its initial reactive state [ 3, 6, 7, 8, 32 ].

NiSOD was discovered in the cytosol of Streptomyces and cyanobacteria as well as in a few green algae. NiSOD contains one Ni per monomer and may for tetramers or hexamers [ 6, 33 ].

Ni3+ + O2.- + H+ ↔ Ni2+ + O2 ;
Ni2+ + O2.- + H+ ↔ Ni3+ + H2O2 ;

Crystal structure of nickel superoxide dismutase (NiSOD). Ni atoms are shown as blue spheres.

FeSOD present in archaea and in the chloroplasts of plants, as well as in the cytosol, glycosomes, and mitochondria of protists. FeSODs have monomers of ~200 amino acids and occur as dimers or tetramers [ 6, 34, 35 ].

Fe3+ + O2.- + H+ ↔ Fe2+ + O2 ;
Fe2+ + O2.- + H+ ↔ Fe3+ + H2O2 ;

Crystal structure of iron superoxide dismutase from Pseudoalteromonas haloplanktis (FeSOD). Fe atoms are shown as blue spheres. .

MnSOD was identified in the cytosol of archaea and bacteria, and eukaryotic cells typically contain MnSOD in the mitochondrial matrix. In many eukaryotic organisms, such as humans and Saccharomyces cerevisiae, MnSOD is located exclusively in the mitochondrial matrix, while in Candida albicans and many crustaceans, an additional isoform of MnSOD is present in the cytosol. Similarly, plant cells express additional MnSODs in their peroxisomes and chloroplasts. MnSODs have monomers of ~200 amino acids and occur as dimers or tetramers [ 6, 36, 37, 38 ].

Mn3+ + O2.- + H+ ↔ Mn2+ + O2 ;
Mn2+ + O2.- + H+ ↔ Mn3+ + H2O2 ;

Crystal structure of Mn(III) superoxide dismutase from Thermus thermophilus (MnSOD) . Mn atoms are shown as blue spheres. .

Bacterial CuZnSOD is located in the periplasm. In eukaryotic cells, CuZnSOD is primarily cytosolic but is also present in the mitochondrial intermembrane space and nucleus. Plants contain additional CuZnSODs in their chloroplasts and peroxisomes, and mammals and many plants secrete an extracellular isoform of CuZnSOD. CuZn SODs have monomers of ~150 amino acids and mostly occur as dimers [ 6, 38, 39, 40 ].

Cu2+Zn2+ + O2.- + H+ ↔ Cu1+Zn2+ + O2 ;
Cu1+Zn2+ + O2.- + H+ ↔ Cu2+Zn2+ + H2O2 ;

Crystal structure of bovine CuZn superoxide dismutase (CuZnSOD). Cu atoms are shown as orange spheres and Zn atoms are shown as blue spheres. .

Superoxide Reductases (SORs) are present in all three domains of life, especially in anaerobic archaea and bacteria. It was also identified in unicellular eukaryotes. The active site of SOR consists of an Fe2+ center. SORs reacts specifically at a nearly diffusion-controlled rate with superoxide, generating hydrogen peroxide and the oxidized active site Fe3+. The electrons involved in the recycling reaction can be provided to the oxidized center iron by rubredoxin in the case of sulfate reducing bacteria or by NADpH-dependent cellular reductases in the absence of rubredoxin [ 6, 8, 41, 42 ].

SOR (Fe2+) + O2.- + 2H+ → SOR (Fe3+) + H2O2 ;
SOR (Fe3+) + 1e- → SOR (Fe2+) ;

There are three classes of SOR depending on whether their sequence contains or not a small N-terminal domain that binds a desulforedoxin-like iron center. Homodimeric desulfoferrodoxin (Dfx) protein from Desulfovibrio desulfuricansare known as first class of SOR. Dfx contains the two iron centers [ 6, 8, 41, 42 ].

Crystal structure of superoxide reductase from Desulfovibrio desulfuricans (SOR). Fe atoms are shown as blue spheres. .

The homotetrameric neelaredoxin protein (Nlr) such as from Pyrococcus furiosus, Archaeoglobus fulgidus, and Desulfovibrio gigas are representatives of the second class of SOR. Each polypeptide contains only one iron center, structurally almost identical to the iron center of Dfx. Nlrs present large sequence homologies with the C-terminal domain of Dfx, but differs by the absence of the second domain to bind the iron [ 6, 8, 41, 42 ].

Crystal structure of superoxide reductase from Pyrococcus furiosus (SOR). Fe atoms are shown as blue spheres. .

A third type of SOR has high sequence homology to Dfx, but containing a single iron center. This class of SORs has been identified in the spirochete Treponema pallidum. This class of SORs contains the N-terminal domains, but lacks three of the four cysteine residues involved in the chelation of the iron. No structural data are available for this class of SOR [ 6, 8, 41, 42 ].

The major similarity between these SODs and SORs is based on presence of redox-active metal ions at their active sites: Ni2+/3+ in NiSOD, Fe2+/3+ in FeSOD and SOR, Mn2+/3+ in MnSOD, and Cu1+/2+ in CuZnSOD. The SOD enzymes all catalyze superoxide disproportionation where superoxide acting alternately to reduce the oxidized metal ion and then to oxidize the reduced metal ion. The SOR enzymes is based on Fe2+ oxidation step but not the Fe3+ reduction step [ 6, 7, 8 ].

Method for measurement of Superoxide Dismutase activity

Xanthine oxidase assay is the best method for SOD activity estimation. Xanthine oxidase is used to generate superoxide and nitroblue tetrazolium (NBT) reduction is used as an indicator of supeoxide production. SOD will compete with NBT for superoxide and the percent inhibition of NBT reduction will be a measure of SOD activity [ 43, 44 ]. NBT can be replased with another substances used in comercially avaliable kits.

SOD WST Assay is a convenient and highly sensitive SOD assay utilizing the highly water-soluble tetrazolium salt, WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfo-phenyl)-2H-tetrazolium, monosodium salt), which produces a water-soluble formazan dye upon reduction with a superoxide anion. The rate of WST-1 reduction by superoxide anion is linearly related to the xanthine oxidase activity and is inhibited by SOD for accurate colorimetric determinations at 450 nm [ 45, 46, 47, 48 ].

SOD Inhibition Assay Mechanism [ 45 ].

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