Reaction products with peroxynitrite

Fig. 3 Formation of peroxynitrite from nitric oxide and superoxide anion and reaction products with carbon dioxide...

On the other hand, in accord with the free radical mechanism peroxynitrite is dissociated into free radicals, which are supposed to be genuine reactive species. Although free radical mechanism was proposed as early as in 1970 [111], for some time it was not considered to be a reliable one because a great confusion ensued during the next two decades because of misinterpretations of inconclusive experiments, sometimes stimulated by improper thermodynamic estimations [85]. The latest experimental data supported its reliability [107-109]. Among them, the formation of dityrosine in the reaction with tyrosine and 15N chemically induced dynamic nuclear polarization (CIDNP) in the NMR spectra of the products of peroxynitrite reactions are probably the most convincing evidences (see below). 

The inactivation of enzymes containing the zinc-thiolate moieties by peroxynitrite may initiate an important pathophysiological process. In 1995, Crow et al. [129] showed that peroxynitrite disrupts the zinc-thiolate center of yeast alcohol dehydrogenase with the rate constant of 3.9 + 1.3 x 1051 mol-1 s-1, yielding the zinc release and enzyme inactivation. Later on, it has been shown [130] that only one zinc atom from the two present in the alcohol dehydrogenase monomer is released in the reaction with peroxynitrite. Recently, Zou et al. [131] reported the same reaction of peroxynitrite with endothelial NO synthase, which is accompanied by the zinc release from the zinc-thiolate cluster and probably the formation of disulfide bonds between enzyme monomers. The destruction of zinc-thiolate cluster resulted in a decrease in NO synthesis and an increase in superoxide production. It has been proposed that such a process might be the mechanism of vascular disease development, which is enhanced by diabetes mellitus. 

In vivo, peroxynitrite may be intercepted by various cellular agents which will keep its steady-state low (Table 2.4). Not all these interceptors, however, react with peroxynitrite to non-reactive products. For example, carbon dioxide enhances tyrosine nitration and thiyl radical formation. Myeloperoxidase also enhances tyrosine nitration, and in the reactions with GSH and albumin thiyl radicals are formed (for details see Arteel et al. 1999). 

Apoptosis is induced by various intra- and extracellular stimuli, and recently nitric oxide was reported to induce apoptosis in cultured cerebellar granule cells and cultured cortical neurons. The toxicity of nitric oxide is mainly ascribed to peroxynitrite, a reaction product of nitric oxide with superoxide (Figure 13.8). Cells producing an increased amount of SOD (superoxide, superoxide oxidoreductase EC 1.15.1.1) are resistant to nitric oxide-mediated apoptosis. In contrast, superoxide levels that have been increased by downregulation of Cu,Zn-SOD lead to apoptotic cell death in PC 12 cells, which required the reaction with nitric oxide to generate peroxynitrite. Peroxynitrite itself was found to induce apoptosis in PC12 cells and in cultured cortical neurons. 

Another pathway of peroxynitrite-mediated modification of aromatic amino acid residues is hydroxylation. Products of peroxynitrite reaction with phenylalanine include p-, m-, and o-tyrosine. Peroxynitrite also forms dityrosine from tyrosine (V2). Major products of oxidative modifications of tryptophan by peroxynitrite include hydropyrroloindole, oxindole, and IV-formylkynurenine (K4). 

Peroxynitrite, like other oxidants, reacts with proteins, first oxidizing cysteine methionine and tryptophan residues (A7). The reaction products are sulfones, carbonyl moieties, and dityrosines (K23, M29). Formation of protein hydroperoxides and protein fragmentation was also observed (B7, G6). Nitric oxide induces oxidation of methionine residues, thus effecting oxidative damage to proteins (Cl 1). It also reacts with Fe-S clusters of aconitase (D15), though in most cases it is difficult to assess whether these effects are produced by the NO itself, or rather by a more reactive secondary product such as peroxynitrite (C5). At physiological... 

Recently, a novel mechanism for hydroxyl radical production, which is not dependent on the presence of transition metal ions, has been proposed (7). This involves the production of peroxynitrite (ONOO ) arising from the reaction of nitric oxide (NO ) with superoxide (OJ), as shown in the following reactions ... 

FIGURE 3 4-Nitrosophenol formation from the reaction of peroxynitrite (ONOO ), nitric oxide (NO), or both (added simultaneously) with phenol at pH 6.0, 7.4, and 8.0. All reactions contained 2 mM phenol and 0.1 mM DTPA (to minimize redox-active metals) in a final volume of 2 ml of 0.1 M potassium phosphate at the indicated pH at 37"C. ONOO", nitric oxide (210 jU.1 of 1.9 mM solution), or both were added simultaneously to rapidly stuxed solutions. The final pH was measured. Reaction products were measured by high-performance liquid chromatography (8-cm C-18, 100 mM ammonium formate, pH 3.5, linear gradient from 5% to 80% acetonitrile over 10 min). Detection at 280 and 365 nm (nitroso and nitro adducts absorb at 365 nm) was used to identify and quantify reaction products relative to authentic standards. 

Niles JC, Wishnok JS, Tannenbaum SR (2001) Spiroiminohydantoin is the major product of 8-oxo-7,8-dihydroguanosine reaction with peroxynitrite in the presence of thiols, and guanosine photooxidation by methylene blue. Oig Lett 3 763 766... 

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