Peroxynitrite

2 Peroxynitrite. - In terms of its ability to induce biomolecular injury, by far the most important reaction of NO is its combination with 02 to form the oxoperoxonitrate(— 1) ion (ONOO , peroxynitrite ) [k = (6.7-19) x 10 M s ]. Whereas ONOO is relatively stable, peroxynitrous acid (ONOOH, pKa 6.5-6.S) undergoes rapid decay to the harmful OH and NO2 radicals at a yield of ca. 30% (see Tsai et al. and references therein ). Although there are several cellular sources of 02 for combination with NO, spin trapping studies have shown that NOS itself may generate the radical, but not without attracting controversy. 

Xu has reported the detection of DMPO/-OOH in reaction mixtures containing various cofactors required for NOS activity, including NADPH, FAD, FMN, tetrahydrobiopterin (BH4) and calmodulin (CaM). The inclusion of native, boiled or even trypsinised nNOS had no effect on the intensity of the 

It is apparent, therefore, that NOS can generate both NO and 02. Generation of the latter species is enhanced when the enzyme is uncoupled due to BH4 deficiency. L-Arg also suppresses 02 formation, as well as electron transfer to artificial acceptors, such as paraquat, which is reduced to its radical cation by NOS. Peroxynitrite can also be formed via the combination of NO with 02  

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). 

Oxidations by peroxynitrite can take place either directly by ground-state peroxynitrous acid, ONOOH, or indirectly by ONOOH where ONOOH is an activated form of peroxynitrous acid (Goldstein et al. 1996). In the direct oxidation pathway the reaction is first order in peroxynitrite and first order in substrate, and the oxidation yield approaches 100%. In the indirect oxidation pathway the reaction is first order in peroxynitrite and zero order in substrate. In the presence of sufficient concentrations of a substrate that reacts by the indirect oxidation pathway, about 50-60% of the ONOOH directly isomerize to nitric acid, and about 40-50% of the ONOOH is converted to ONOOH. The involvement of hydroxyl radicals in indirect oxidations by peroxynitrite is ruled out on the basis of kinetics and oxidation yields. 

Peroxynitrite itself is not a free radical because the two unpaired electrons of superoxide and nitric oxide have combined to form a new bond. Peroxynitrite is an isomer of nitrate, but is 36 kcal x mol-1 higher in energy (Koppenol et al. 1992). The half-life of peroxynitrous acid (HOONO), which is 7 s at 0 °C and 1 s at 37 °C, should be long enough to allow the molecule to diffuse inside the cell and react with DNA. The decomposition pathway of HOONO, leading to the formation of nitric acid, remains 

Aldehydes, like CO2, react rapidly with peroxynitrite and catalyse its decompensation (Uppu et al. 1997). The pH dependence of the reaction is consistent with the addition of ONOO (not ONOOH) to the carbonyl carbon atom of the free aldehyde forming a 1-hydroxyalkylperoxynitrite anion adduct, which structurally resembles the nitrosoper- 

Aconitase is readily inactivated by peroxynitrite, but not by its precursor, nitric oxide (Castro et al. 1994). 

In the vascular system, a particularly important fate of peroxynitrite may be its rapid reaction with Hb-Fe(II)-02. Studies by Romero et al. have led to the proposal of a mechanism for the formation of Hb-Fe(III), involving the displacement of 02 - from Hb-Fe(TI)-02 by ONOO, with the formation of Hb-Fe(III)-ONOO. This transient decays mainly to NO and Hb-Fe(III), but ca. 10 % gives Hb-Fe(IV) = 0 plus N02. Tyrosyl and cysteinyl radicals, trapped using MNP and DMPO, respectively, were proposed to arise via electron transfer from the protein moiety to the ferryl haem.97 In an earlier study, a mechanism involving N02 generation had been suggested for the formation of Hb-Fe(IV) = 0 from Hb-Fe(II)-02 by ONOOH.98 

---

Okamoto T, Akaike T, Sawa T et al (2001) Activation of matrix metaUoproteinases by peroxynitrite-induced protein S-glutathiolation via disulfide S-oxide formation. J Biol Chem 276 29596-29602... 

Not all oxidants formed biolc cally have the potential to promote lipid peroxidation. The free radicals superoxide and nitric oxide [or endothelium-derived relaxing aor (EDRF)] are known to be formed in ww but are not able to initiate the peroxidation of lipids (Moncada et tU., 1991). The protonated form of the superoxide radical, the hydroperoxy radical, is capable of initiating lipid peroxidation but its low pili of 4.5 effectively precludes a major contribution under most physiological conditions, although this has been suggested (Aikens and Dix, 1991). Interestingly, the reaction product between nitric oxide and superoxide forms the powerful oxidant peroxynitrite (Equation 2.6) at a rate that is essentially difiiision controlled (Beckman eta/., 1990 Huie and Padmaja, 1993). 

Graham, A., Hogg, N., Kalyanaraman, B., O Leary, V.J., Darley-Usmar, V. and Moncada, S. (1993). Peroxynitrite modification of low density lipoprotein leads to recognition by the macrophage scavenger receptor. FEBS Lett. 330, 181-185. 

Van Der Vliet, A., Smith, D., O Neill, C.A., Kaur, H., Darley-Usmar, V.M., Cross, C.E. and Halliwell, B. (1994). Interactions of peroxynitrite with human plasma and its constituents oxidative damage and antioxidant depletion. Biochem. J. 303, 295-301. 

Beckman, J.S., Beckman, T.W., Chen, J., Marshall, P.A. and Freeman, B.A. (1990). Apparent hydroxyl radical production by peroxynitrite implication for endothelial injury from nitric oxide and superoxide. Proc. Natl Acad. Sci. USA 87, 1620-1624. 

During ischaemia, NOS is activated by calcium influx or by cytokines like tumour necrosis factor (TNF) or by lipopolysaccharide (LPS) and NO is produced in excess. It has been proposed that the excitotoxic effect of glutamate, which contributes to ischaemia-induced neuronal damage, is mediated by increased production of NO via a chain of events that includes increases in intracellular calcium (via glutamate activation of NMDA receptors), calcium activation of NOS, production of NO and peroxynitrite, and induction of lipid peroxidation. In fact, N-nitro-L-atginine, a selective inhibitor of NOS, has been shown to prevent glutamate-induced neurotoxicity in cortical cell cultures (Dawson rf /., 1991). 

---