Nebraska Redox Biology Center Educational Portal


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

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 ROS that can be either radical or non-radical

Reactive species oxidation from starting superoxide radical [ 3 ].

Superoxide Production

1) Mitochondrial superoxide anions is mainly generated by complex I and III as a by-product. Superoxide is produced in mitochondria by slippage of an electron from the electron transport chain to molecular oxygen during oxidative phosphorylation. In the process of electrons transport chain is considered that only 3% of total oxygen is consumed to generate superoxide radical [ 3, 6, 7 ].

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. [ 8, 9, 10 ].

Xanthine oxidase 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 [ 11, 12, 13, 14 ].

Endothelial Nitric oxide synthase differently behaves generating either NO or superoxide depending on the substrate availability. BH4 is tetrahidrobiopterine [ 3 ].

4) The NADPH oxidase is a membrane-bound enzyme complex. 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 [ 15, 16, 17 ]. The general reaction catalyzed by phagocytic/non-phagocytic NADP oxidase is:

NADPH + 2O2 ↔ NADP + 2O2.- ;

5) 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 [ 18, 19, 20 ].

6) Homocysteine methabolism may result in generation of superoxide radicals thus promoting vasoconstriction [ 21 ].

Superoxide Detection

Most of commonly used superoxide detection methods employ indicating compounds, a compounds which reacts with superoxide, producing a detectable product. The most commonly used indicating scavengers are cytochrome c, lucigenin and luminol [ 22, 23, 24 ].

1) Cytochrome C is reduced by reaction with superoxide, producing ferrocytochrome c, which has a detectable absorbance at 550 nm. This assay is relatively insensitive, and is subject to a number of interferences from other chemicals and from enzymes which reduce the cytochrome directly [ 22, ].

Fe3+ Cyt C + 2O2.- ↔ Fe2+ Cyt C + 2O2 ;

2) Lucigenin is compound, which emits light on reaction with superoxide. The reaction involves an initial reduction of the lucigenin to a radical. The lucigenin radical can then react with either oxygen, producing superoxide, or with superoxide in an addition reaction, leading to the decomposition of the lucigenin into two acridones, one of which is in an excited state, and decays to produce light. Such hemiluminescent reaction is more sensitive than cytochrome c reduction assay. A disadvantage of lucigenin assay is related to measure of superoxide because lucigenin itself can react to produce superoxide [ 22, 23, 24 ].

Detection of superoxide with lucigenin chemiluminescent reaction.

3) Luminol, unlike cytochrome c and lucigenin, is oxidized by superoxide. This leads into a complex series of reactions between luminol, luminol radicals, oxygen and superoxide. Ultimately these reactions will produce a luminol endoperoxide, which decomposes with the release of a photon. Again, because superoxide is involved as both initiator and intermediate in the reaction, objections have been raised to the use of luminol as a quantitative measure of superoxide production [ 22, 23, 24 ].

Detection of superoxide with luminol chemiluminescent reaction.

4) Cellular superoxide can be visualized by dihydroethidium. This compound exhibits a blue fluorescence in the cytosol until oxidized primarily by superoxide. With oxidation the compound intercalated with cellular DNA, staining the nucleus a bright fluorescent red with reported excitation and emission wavelengths of 535 nm and 635 nm respectively. The mitochondrial superoxide can be visualized using fluorescence microscopy with similar to dihydroethidium MitoSOX Red reagent. MitoSOX Red reagent is a cationic derivative of dihydroethidium that permeates live cells where it selectively targets to mitochondria [ 22, 23, 24 ].

Detection of superoxide with dihydroethidium oxidation assay.

Superoxide Scavengers - SOD and SOR

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, 25, 26, 27 ].

NiSOD was discovered in the cytosol of Streptomyces and cyanobacteria as well as in a few green algae [ 25, 28 ]. FeSOD present in archaea and in the chloroplasts of plants, as well as in the cytosol, glycosomes, and mitochondria of protists [ 25, 29, 30 ]. 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 [ 25, 31, 32, 33 ]. 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 [ 25, 33, 34, 35 ]. Superoxide Reductases (SORs) are present in all three domains of life, especially in anaerobic archaea and bacteria. It was also identified in unicellular eukaryotes [ 25, 27, 36 ].

The major similarity between these enzymes 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 [ 25, 26, 27 ].

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