**2. Peroxynitrite**

One important reaction of nitric oxide is with superoxide (another free radical) to produce the strong oxidant peroxynitrite (Beckman et al. 1990). The importance of this reaction is highlighted by its diffusion-limited rate (between 6.7x109 and 2x1010 M-1s-1)(Padmaja and Huie 1993; Nauser and Koppenol 2002), meaning that every collision of a molecule of nitric oxide with a molecule of superoxide results in the formation of peroxynitrite (Fig 1). In other words, peroxynitrite will be formed when superoxide and nitric oxide are formed simultaneously. The reason peroxynitrite is not formed in large amounts in normal metabolic conditions is the high intracellular concentration of the enzyme superoxide dismutase (SOD)(Rae et al. 1999), which competes for the superoxide with a rate of 2x109 M-1s-1 (Pacher, Beckman, and Liaudet 2007) (Fig 1). Briefly, when the concentration of nitric oxide is 5 times lower than the concentration of SOD, approximately 50% of the superoxide

Reactive Nitrogen Species in Motor Neuron Apoptosis 315

alters the functionality of structural proteins such as neurofilament L, synuclein, actin, and tubulin (Aslan et al. 2003; Cappelletti et al. 2003; Eiserich et al. 1999; Chang et al. 2002; Crow et al. 1997; Paxinou et al. 2001). Changes in protein function are caused by oxidative modifications of amino acid residues by peroxynitrite (Alvarez et al. 1999). In the case of phosphatases and zinc-thiolate-containing proteins the oxidation of methionine and cysteine residues is critical for the loss of function of the enzymes (Takakura et al. 1999; Crow, Beckman, and McCord 1995). One particular modification of amino acids by peroxynitrite that has driven much attention is the nitration of tyrosine residues (Beckman et al. 1992). The interest is driven by the fact that nitrotyrosine seems to be a universal marker for inflammation and has been detected in a large number of pathological conditions (Ischiropoulos 1998; Greenacre and Ischiropoulos 2001; Ischiropoulos and Beckman 2003; Schopfer, Baker, and Freeman 2003; Radi 2004). It is accepted that nitration by the formation of the decomposition products of peroxynitrite is a major source of biological nitration, in spite that other mechanisms for tyrosine nitration have been described (Ischiropoulos 1998;

Fig. 2. Mechanisms for tyrosine nitration by the nitrative products of decomposition of

peroxynitrite or peroxynitrite donors (Bonfoco et al. 1995; Estévez et al. 1995).

Unsurprisingly, peroxynitrite induces apoptosis or necrosis depending on the concentration of the oxidant (Bonfoco et al. 1995; Estévez et al. 1995), and it has become the accepted mechanism for the toxic effects of nitric oxide in biological systems (Dawson and Dawson 1996; Dawson and Dawson 1996; Dawson and Dawson 1996; Beckman and Koppenol 1996). Although growing evidence suggests that peroxynitrite induces apoptosis by interacting with specific cellular signaling pathways (Estévez et al. 1995; Shin et al. 1996; Spear, Estévez, Barbeito, et al. 1997; Spear, Estévez, Radi, et al. 1997; Shacka et al. 2006; Ye et al. 2007) (Fig 3), the cellular targets responsible for peroxynitrite-induced apoptosis remain unknown. In addition, most studies were performed using exogenously applied stock solutions of pure

Radi 2004; Schopfer, Baker, and Freeman 2003) (Fig 2).

peroxynitrite in the cells.

**3. Peroxynitrite and apoptosis** 

produced would react with nitric oxide to form peroxynitrite. In normal conditions, the intracellular concentration of SOD is in large excess of the concentrations of nitric oxide (Beckman and Koppenol 1996). However, the prediction from the rates is that a small amount of peroxynitrite will always be formed, allowing the speculation that the oxidant may have a physiological function (Go et al. 1999), or otherwise it is efficiently scavenged by small molecular weight antioxidants such as glutathione with rates in the order of 103-104 M-1s-1 (Radi et al. 1991; Alvarez et al. 1999). Other important aspect of the competition between SOD and nitric oxide for superoxide is the presence of membrane cellular compartments, which limit the diffusion of SOD and superoxide but not nitric oxide. SOD can only compete with nitric oxide when the enzyme is in the same compartment than superoxide is being produced, indicating that peroxynitrite can be formed with relatively high efficiency even at low nitric oxide concentrations if superoxide is formed in a compartment SOD cannot access or where the enzyme has been inactivated (Fig 1).

Fig. 1. In the cells, peroxynitrite formation depends on the levels of SOD that competes with nitric oxide for superoxide. Because cellular membranes (in blue) limit the diffusion of SOD and superoxide but not nitric oxide, peroxynitrite formation is also limited by the subcellular localization of the superoxide sources.

In pathological conditions, not only low micromolar concentrations of nitric oxide can be produced, but production of superoxide can also be boosted, increasing the probability for peroxynitrite formation at levels that may overwhelm the intracellular antioxidant defenses (Beckman and Crow 1993; Beckman and Koppenol 1996). Peroxynitrite affects normal cell metabolism by inducing lipid peroxidation (Radi et al. 1991), damage of the DNA (Groves and Marla 1995), and alteration of the mitochondrial function (Radi et al. 2002). In addition, it has been shown that peroxynitrite inhibits the activity in some proteins such as the tyrosine hydroxylase (Ara et al. 1998), mitochondrial manganese SOD, and tyrosine phosphatases (Takakura et al. 1999), activates src kinase (MacMillan-Crow et al. 2000) and

produced would react with nitric oxide to form peroxynitrite. In normal conditions, the intracellular concentration of SOD is in large excess of the concentrations of nitric oxide (Beckman and Koppenol 1996). However, the prediction from the rates is that a small amount of peroxynitrite will always be formed, allowing the speculation that the oxidant may have a physiological function (Go et al. 1999), or otherwise it is efficiently scavenged by small molecular weight antioxidants such as glutathione with rates in the order of 103-104 M-1s-1 (Radi et al. 1991; Alvarez et al. 1999). Other important aspect of the competition between SOD and nitric oxide for superoxide is the presence of membrane cellular compartments, which limit the diffusion of SOD and superoxide but not nitric oxide. SOD can only compete with nitric oxide when the enzyme is in the same compartment than superoxide is being produced, indicating that peroxynitrite can be formed with relatively high efficiency even at low nitric oxide concentrations if superoxide is formed in a compartment SOD

Fig. 1. In the cells, peroxynitrite formation depends on the levels of SOD that competes with nitric oxide for superoxide. Because cellular membranes (in blue) limit the diffusion of SOD

In pathological conditions, not only low micromolar concentrations of nitric oxide can be produced, but production of superoxide can also be boosted, increasing the probability for peroxynitrite formation at levels that may overwhelm the intracellular antioxidant defenses (Beckman and Crow 1993; Beckman and Koppenol 1996). Peroxynitrite affects normal cell metabolism by inducing lipid peroxidation (Radi et al. 1991), damage of the DNA (Groves and Marla 1995), and alteration of the mitochondrial function (Radi et al. 2002). In addition, it has been shown that peroxynitrite inhibits the activity in some proteins such as the tyrosine hydroxylase (Ara et al. 1998), mitochondrial manganese SOD, and tyrosine phosphatases (Takakura et al. 1999), activates src kinase (MacMillan-Crow et al. 2000) and

and superoxide but not nitric oxide, peroxynitrite formation is also limited by the

subcellular localization of the superoxide sources.

cannot access or where the enzyme has been inactivated (Fig 1).

alters the functionality of structural proteins such as neurofilament L, synuclein, actin, and tubulin (Aslan et al. 2003; Cappelletti et al. 2003; Eiserich et al. 1999; Chang et al. 2002; Crow et al. 1997; Paxinou et al. 2001). Changes in protein function are caused by oxidative modifications of amino acid residues by peroxynitrite (Alvarez et al. 1999). In the case of phosphatases and zinc-thiolate-containing proteins the oxidation of methionine and cysteine residues is critical for the loss of function of the enzymes (Takakura et al. 1999; Crow, Beckman, and McCord 1995). One particular modification of amino acids by peroxynitrite that has driven much attention is the nitration of tyrosine residues (Beckman et al. 1992). The interest is driven by the fact that nitrotyrosine seems to be a universal marker for inflammation and has been detected in a large number of pathological conditions (Ischiropoulos 1998; Greenacre and Ischiropoulos 2001; Ischiropoulos and Beckman 2003; Schopfer, Baker, and Freeman 2003; Radi 2004). It is accepted that nitration by the formation of the decomposition products of peroxynitrite is a major source of biological nitration, in spite that other mechanisms for tyrosine nitration have been described (Ischiropoulos 1998; Radi 2004; Schopfer, Baker, and Freeman 2003) (Fig 2).

Fig. 2. Mechanisms for tyrosine nitration by the nitrative products of decomposition of peroxynitrite in the cells.
