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Chem. Res. Toxicol. 1998, 11, 716-717
Peroxynitrite Uncloaked? Willem H. Koppenol* Laboratorium fu¨ r Anorganische Chemie, Eidgeno¨ ssische Technische Hochschule Zu¨ rich, Universita¨ tstrasse 6, CH-8092 Zu¨ rich, Switzerland Received March 30, 1998
Introduction. The nitration of tyrosine in vivo by peroxynitrite causes a kinetic problem: nitration, which is first-order in peroxynitrite and zeroth-order in tyrosine, takes place at the rate of decay of peroxynitrite, which is approximately 1.0 s-1 at 37 °C and pH 7.4 (1), while in a cell peroxynitrite disappears much faster through the bimolecular reaction with sulfhydryls. The rate of this process is estimated as follows: the product of an average rate constant of 4 × 103 M-1 s-1 under the same conditions (2) and a cellular concentration of approximately 5-10 mM yields a rate of disappearance of 2040 s-1. Discussion. One concludes that nitration of tyrosine should not be observed, until the sulfhydryls have been depleted. However, we should also investigate the rapid reaction of the peroxynitrite anion with carbon dioxide that yields a nitrating species. With an estimated concentration of 1 mM and a rate constant of 2 × 103 M-1 s-1 at 37 °C and pH 7.4 (3), the rate of disappearance is 2 s-1. Thus, under “normal” circumstances, nearly all peroxynitrite formed in a cell reacts with thiols. When the pH is even slighly lower, then significantly more peroxynitrite reacts with carbon dioxide. In plasma, the concentration of thiols is much lower and carbon dioxide is a major sink for peroxynitrite. The peroxynitritecarbon dioxide adduct, or a product derived from it, is an effective nitrating agent and appears not to be very reactive with sulfhydryls (4). That observation argues against a role for the homolysis products of ONOOCO2(1-carboxylato-2-nitrosodioxidane) (5) in nitration, because CO3•- [trioxocarbonate(1-)] reacts approximately as fast with thiols as with phenolic compounds (6), and ascorbate, which reacts with the other homolysis product, nitrogen dioxide, had little effect on carbon dioxidecatalyzed nitration (4). We are left with a selectively nitrating adduct of carbon dioxide and the peroxynitrite anion, very much the same as the nitrating adduct formed between a metal ion and peroxynitrite. As the yield of nitration, relative to peroxynitrite, could not be increased to 100% in the presence of enough carbon dioxide to ensure complete adduct formation, there must be two reaction paths, one leading to a nitrated product and the other to nitrate and carbon dioxide (7) (Scheme 1). What are the implications of these simple kinetic considerations? 1. The intermediate formed during the isomerization to nitrate, whether that is a hydroxyl and a nitrogen dioxide radical, the nitryl cation and hydroxide, or a twisted trans-peroxynitrite, plays no role in the toxicity of peroxynitrite: peroxynitrite has disappeared before the * Corresponding author. Tel: 41-1-632-2875 or -2852 (Ms. R. Pfister, secretary). Fax: 41-1-632-1090. E-mail:
[email protected].
intermediate can be formed. 2. The adduct that is formed between peroxynitrite and peroxynitrous acid (8) that decays to nitrite and dioxygen is irrelevant for biology: its formation requires unphysiological peroxynitrite concentrations (>50 µM). 3. As stated above, homolysis of the O-O bond in 1-carboxylato-2-nitrosodioxidane is not important for the carbon dioxide-catalyzed nitration of phenolic compounds. 4. For a scavenger to be effective it should react with 90% of all peroxynitrite. Thus, in the presence of 5-10 mM thiols and 1 mM carbon dioxide, the product of the scavenger concentration and its rate constant with either peroxynitrite or peroxynitrous acid should be at least 10 times larger than the rate of disappearance by thiols, 2040 s-1, or carbon dioxide, 2 s-1. A rate of 4 × 102 s-1 at 37 °C and pH 7.4 requires, for instance, that a scavenger has to react with an apparent rate constant of 4 × 107 M-1 s-1 if an intra- and extracellular level of 10 µM scavenger can be achieved. At such a low scavenger concentration it would be advantageous if the scavenger could be recycled. In this respect the reaction of peroxynitrite with metalloporhyrins (9) and glutathione peroxidase1 and also the rapid regeneration of these systems by antioxidants are interesting developments. The considerations given above are simplifications, in that they apply to homogeneous solution. However, the outcome is useful: one sees immediately that because of compartimentalization higher concentrations of scavenger are necessary. Conclusion. As shown above, the chemistry of peroxynitrite is complex. For this reason experiments with peroxynitrite should be carried out carefully to avoid artifacts: (i) the addition of a bolus amount of peroxynitrite may lead to formation of the adduct, and (ii) under continuous irradiation with UV light, homolysis of the N-O bond takes place, which results in the formation of nitrogen monoxide and superoxide (8).
Acknowledgment. This research is supported by the ETH and the Schweizerische Nationalfonds.
References (1) Koppenol, W. H., and Kissner, R. (1998) Can OdNOOH undergo homolysis? Chem. Res. Toxicol. 11, 87-90. (2) Radi, R., Beckman, J. S., Bush, K. M., and Freeman, B. A. (1991) Peroxynitrite oxidation of sulfhydryls. J. Biol. Chem. 266, 42444250. (3) Denicola, A., Freeman, B. A., Trujillo, M., and Radi, R. (1996) Peroxynitrite reaction with carbon dioxide/bicarbonate: Kinetics and influence on peroxynitrite-mediated oxidations. Arch. Biochem. Biophys. 333, 49-58. (4) Gow, A., Duran, D., Thom, S. R., and Ischiropoulos, H. (1996) Carbon dioxide enhancement of peroxynitrite-mediated protein tyrosine nitration. Arch. Biochem. Biophys. 333, 42-48. 1Briviba,
Kissner, Koppenol, and Sies, 1998, submitted.
S0893-228x(98)00060-5 CCC: $15.00 © 1998 American Chemical Society Published on Web 07/20/1998
Forum: Reactive Species of Peroxynitrite
Chem. Res. Toxicol., Vol. 11, No. 7, 1998 717
Scheme 1. Reactions of Peroxynitrite under Physiological Conditions
(5) Mere´nyi, G., and Lind, J. (1997) Thermodynamics of peroxynitrite and its CO2-adduct. Chem. Res. Toxicol. 10, 1216-1220. (6) Ross, A. B., Bielski, B. H. J., Buxton, G. V., Cabelli, D. E., Greenstock, C. L., Helman, W. P., Huie, R. E., Grodkowski, J., and Neta, P. (1994) NDRL-NIST Solution Kinetics Database: Ver. 2, National Institute of Standards and Technology, Gaithersburg, MD. (7) Lymar, S. V., Jiang, Q., and Hurst, J. K. (1996) Mechanism of carbon dioxide-catalyzed oxidation of tyrosine by peroxynitrite. Biochemistry 35, 7855-7861.
(8) Kissner, R., Nauser, T., Bugnon, P., Lye, P. G., and Koppenol, W. H. (1997) Formation and properties of peroxynitrite studied by laser flash photolysis, high-pressure stopped flow and pulse radiolysis. Chem. Res. Toxicol. 10, 1285-1292. (9) Lee, J. B., Hunt, J. A., and Groves, J. T. (1997) Rapid decomposition of peroxynitrite by manganese porphyrin-antioxidant redox couples. Bioorg. Med. Chem. Lett. 7, 2913-2918.
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