NEBRASKA REDOX BIOLOGY CENTER EDUCATIONAL PORTAL

Nebraska Redox Biology Center Educational Portal


Peroxynitrite

The peroxynitrite (ONOO-) is a short-lived ROS produced in the reaction of nitric oxide and superoxide radicals [ 1, 2, 3 ]. Peroxynitrite is an important contributor to various forms of cardiovascular, inflammatory and neurodegenerative diseases. Peroxynitrite production has also been implicated in various aspects of ageing, including cellular senescence, as well as the loss of vascular endothelium-dependent responses and the impaired myocardial contractility [ 3, 4, 5, 6, 7 ].

The sites of peroxynitrite formation are assumed to be spatially associated with the sources of superoxide such as the plasma membrane NADPH oxidases or the mitochondrial respiratory complexes. The rates of peroxynitrite production in vivo in specific compartments have been estimated to be as high as 50-100 micromol/per min [ 1, 3, 8 ]. A fundamental reaction of peroxynitrite in biological systems is its fast reaction with carbon, which leads to the formation of carbonate and nitrogen dioxide radicals [ 3, 9 ].

A ) Peroxynitrite anion (ONOO-) is in equilibrium with peroxynitrous acid and either one can undergo direct reactions with biomolecules as indicated (a and b). A fundamental reaction of ONOO- in biological systems is its fast reaction with carbon dioxide (in equilibrium with physiological levels of bicarbonate anion; c) which leads to the formation of carbonate (CO3.-) and nitrogen dioxide (NO2.) radicals. (d) that can readily oxidize amino acids such as cysteine and tyrosine to yield the corresponding cysteinyl and tyrosyl radicals. (d) Alternatively, ONOOH can undergo homolytic fission to generate one-electron oxidants hydroxyl and .NO2 radical. (f) and its decomposition to hydroxyl and .NO2 radical radicals seems to become relevant in hydrophobic phases to initiate lipid peroxidation and lipid and protein nitration processes (g) ONOOH in the membranes may undergo direct reactions with metal centres such as hemin or membrane-associated thiols. B) Pathways in the decomposition of peroxynitrite. Se-GPx, selenium-containing glutathione peroxidase [ 3, 9 ].


Many biomolecules are oxidized and/or nitrated by peroxynitrite-derived radicals, including tyrosine residues, thiols, DNA and unsaturated fatty-acid-containing phospholipids. Tyrosine nitration, dimerization and hydroxylation by peroxynitrite to form 3-nitro-tyrosine, 3,3-dityrosine and 3,4-dihydrophenylalanine, respectively, are entirely dependent on free-radical pathways. Thiols can be oxidized by one-electron reactions by peroxynitrite-derived radicals and initiate radical-dependent chain reactions to produce higher oxidation states of sulphur, including sulphinic and sulphonic acid derivatives [ 3, 10, 11, 12 ]. In DNA, purine nucleotides are vulnerable to oxidation. Also, peroxynitrite can cause deoxyribose oxidation and strand breaks. [ 3, 13, 14 ].

The reaction of peroxynitrite-derived radicals with lipids leads to peroxidation (malondialdehyde, conjugated diene and lipid hydroperoxide formation) and the formation of nitrito-, nitro-, nitrosoperoxo- and/or nitrated lipid oxidation adducts. Peroxynitrite also causes the oxidation of arachidonic acid, and the formation of F2-isoprostanes through the oxidation of low-density lipoprotein. The nitration of fatty acids may lead to the secondary inhibition of protein function via thiol-based modifications but nitrated lipids can also have direct anti-inflammatory functions via peroxisome proliferator-activated receptor-gamma (PPAR-gamma)-dependent and PPAR-gamma-independent pathways. Lipid peroxidation processes may also assist in protein tyrosine oxidation and nitration in biomembranes and lipoproteins [ 3, 15, 16, 17, 18, 19 ]. Reaction of NADH with peroxynitrite can result in the formation of NAD+ and superoxide and, thus, of hydrogen peroxide. This reaction can induce an imbalance in cellular pyrimidine nucleotide levels, and a positive-feedback cycle of cytotoxic oxidant generation. Both the oxidation of BH4 and NADH in vivo must depend on peroxynitrite-derived radicals, because the direct reactions are slow to out-compete with other biological targets [ 3, 20, 21 ]. Peroxynitrite has strong influence on mitochondrial function, modulate inflammation and immune response and trigger of cell death [ 2, 3, 22 ].

Natural scavengers and neutralizers of peroxynitrite.

Various molecules can react directly with peroxynitrite, with peroxynitrite-derived radicals or with both. [ 3, 23, ]. Bacterial and parasitic peroxiredoxins have been shown to readily and catalytically reduce peroxynitrite to nitrite through the reaction of the peroxidatic cysteine, which in turn is reduced back to the resting state via thiol-containing reducing substrates such as thioredoxin or tryparedoxin [ 3, 24, 25 ]. Human peroxiredoxin V is serve as a peroxynitrite/thioredoxin oxido-reductase [ 3, 26 ].

Simple thiol-based antioxidants, such as mercaptoalkylguanidines, N-acetylcysteine and dihydrolipoic acid, have been shown to reduce peroxynitrite-mediated toxicity and reduce 3-nitrotyrosine immunoreactivity in various pathophysiological conditions. [ 3, 27, 28, 29, 30, 31 ].


The major methods for eroxynitrite detection [ 32, 33 ].

Dihydrorhodamine Oxidation [ 34 ]. Dihydrorhodamine 123 (DHR) is a cell-permeant, mitochondrial-avid analog of 2,7 dichlorodihydrofluorescein that can undergo oxidation to the fluorophore rhodamine 123. Peroxynitrite readily oxidizes DHR 123.

Chemiluminescent Reactions [ 35, 36, 37 ]. Peroxynitrite reacts with luminol LumH2 to yield chemiluminescence, a reaction enhanced by CO2 present in bicarbonate-containing systems attributable to formation of nitrosoperoxocarbonate (ONOOCO2-).

Mechanism proposed for luminescence formation during oxidation of luminol-based probe luminol LumH2 [ 37 ].

Bioluminescent detection of peroxynitrite with a boronic acid-caged luciferin. Boronic acid-based bioluminescent system PCL-1 (peroxy-caged luciferin-1), previously reported as a chemoselective sensor for hydrogen peroxide reacts with peroxynitrite stoichiometrically with a bioluminescence detection limit of 16 nM. PCL-1 react with peroxynitrite and produces luciferin rapidly and stoichiometrically [ 38, 39, 40 ].

Mechanism for the detection of peroxynitrite by a peroxy-caged luciferin (PCL-1) and release of luciferin.


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