Peroxynitrite reaction with superoxide dismutase

The reaction of nitric oxide with superoxide dismutase is a simple reversible equilibrium, whereas the catalytic cycle with superoxide involves a two step sequence. Consequently, superoxide dismutase may be reduced by superoxide and then react with nitric oxide to form nitroxyl anion. Nitroxyl anion may react with molecular oxygen to form peroxynitrite anion (ONOO"). 

Reaction of trans-peroxynitrite with superoxide dismutase. The placement of positively charged amino acids around the active site facilitates the attraction of the negatively charged peroxynitrite anion. Because the copper of superoxide dismutase is buried in a pocket shaped to accommodate superoxide, only peroxynitrite in the trans configuration should be able to fit in the active site. Because the predominant form of peroxynitrite is the cis form, the isomerization from the cis to trans geometry limits the reaction of peroxynitrite with superoxide dismutase. 

Superoxide anion and nitrogen monoxide engender peroxynitrite at almost diffusion-controlled rates (6.7x10 M" . s" Huie and Padmaja 1993 for review see Ducrocq et al. 1999). Its high rate constant means that NO competes effectively with superoxide dismutase for reaction with O2 " (Hogg et al. 1992). 

Guanine is the most easily oxidizable natural nucleic acid base [8] and many oxidants can selectively oxidize guanine in DNA [95]. Here, we focus on the site-selective oxidation of guanine by the carbonate radical anion, COs , one of the important emerging free radicals in biological systems [96]. The mechanism of COs generation in vivo can involve one-electron oxidation of HCOs at the active site of copper-zinc superoxide dismutase [97, 98], and homolysis of the nitrosoperoxycarbonate anion (0N00C02 ) formed by the reaction of peroxynitrite with carbon dioxide [99-102]. 

Although hydroxyl radical is commonly assumed to be the most toxic of the oxygen radicals (with little direct evidence), other direct reactions are more likely to be important for understanding the cytotoxicity of peroxynitrite. A second oxidative pathway involves the heterolytic cleavage of peroxynitrite to form a nitronium-like species (N02 ), which is catalyzed hy transition metals (Beckman et al., 1992). Low molecular weight metal complexes as well as metals bound in superoxide dismutase and other proteins catalyze the nitration of a wide range of phenolics, including tyrosine residues in most proteins (Beckman et al., 1992). 

Peroxynitrite reacts with the active site of superoxide dismutase (SOD) to form a nitronium-like species (Fig. 37), analogous to the Fe EDTA reactions described earlier. However, copper in the active site of superoxide dismutase was necessary for the formation of the adduct. Removing copper from the active site by reduction with borohydride and dialysis against 50 mM KCN resulted in no adduct being formed, while restoration of copper to the active site gave back full enzyme activity. To account for the essential role of copper in the active site and the subsequent formation of 3-nitrotyrosine located 18-21 A distal from the active site, we proposed that peroxynitrite is attracted by the same electrostatic force field that draws superoxide into the active site (Beckman et al., 1992 Ischiropoulos et al., 1992b). Peroxynitrite appears to bind to copper in the active site to form a transient cuprous adduct as shown. 

Trapping of peroxynitrite from rat alveolar macrophages by superoxide dismutase. Although the fotmation of peroxynitrite is drawn as superoxide reacting with nitric oxide in the extracellular space, the actual reactions may be a combination of the pathways shown in Fig. 40. 

The direct reaction of superoxide with nitric oxide is only one of at least four possible pathways that can form peroxynitrite (Fig. 40). For example, superoxide should also efficiently reduce nitrosyldioxyl radical to peroxynitrite. Alternatively, nitric oxide may be reduced to nitroxyl anion, which reacts with oxygen to form peroxynitrite. Superoxide dismutase could even catalyze the formation of peroxynitrite, since reduced (Cu or cuprous) superoxide dismutase can reduce nitric oxide to nitroxyl anion (Murphy and Sies, 1991). Thus, superoxide might first reduce superoxide dismutase to the cuprous form, with nitric oxide reacting with reduced superoxide dismutase to produce nitroxyl anion. A fourth pathway to form peroxynitrite is by the rapid reaction of nitrosonium ion (NO" ) with hydrogen peroxide. This is a convenient synthetic route for experimental studies (Reed et al., 1974), but not likely to be physiologically relevant due to the low concentrations of hydrogen peroxide and the difficulty of oxidizing nitric oxide to nitrosonium ion. 

It was recently reported that the tryptophan residues of proteins could be nitrated by the action of peroxynitrite (67). This reactive nitrogen species (RNS) is generated from the reaction of nitric oxide with superoxide at a rate that is ten times greater than the destruction of superoxide by dismutases. The authors propose that the nitration of tryptophan, although less common than tyrosine nitration, could serve to modulate the function of some proteins. However, at this time the in vivo evidence for tryptophan nitration by RNS has yet to be reported. 

Fig. 3. Production of reactive species. (A) ROS can be produced from the weak radical oxygen in the mitochondria and endoplasmic reticulum, by various enzymatic reactions, and from oxyhemoglobin. Normally, nontoxic hydrogen peroxide can give rise to the powerful hydroxyl radical in the presence of transition metals (R5). Oxygen can also be induced to react with biomolecules by transition metals and enzymes. RNS can be produced by reaction of superoxide anion radical with the weak radical nitric oxide. These can react to form the powerful oxidant peroxynitrite/peroxynitrous acid, which can cause formation of other radicals, some with longer lives. See the text for details. SOD, superoxide dismutase. (B) Myeloperoxidase in leukocytes can produce the reactive species hypochlorous acid and tyrosyl radical. Unpaired electrons are indicated by the dense dots and paired electrons by the light ones.

Mn(III)TMPyP is a manganese porphyrin that acts as a superoxide dismutase (SOD) mimetic and peroxynitrite decomposition catalyst (Han et al., 2001). SOD mimetics described to date are unstable and are capable of catalyzing undesired side reactions in addition to the dismutation of the superoxide radical. Mn(III)TMPyP is an SOD mimetic with increased stability to pH and hydrogen peroxide. The rate constants for superoxide dismutation and peroxynitrite decomposition are 3.9 X 10 M s and... 

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