ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 345, No. 1, September 1, pp. 160–170, 1997 Article No. BB970240 Carbon Dioxide Modulation of Hydroxylation and Nitration of Phenol by Peroxynitrite Jean-Noe ¨l Lemercier, Sarojini Padmaja, Rafael Cueto, Giuseppe L. Squadrito, Rao M. Uppu, and William A. Pryor 1 The Biodynamics Institute, Louisiana State University, Baton Rouge, Louisiana 70803-1800 Received April 4, 1997; and in revised form June 9, 1997 nitrocarbonate; carbon dioxide; tyrosine; nitrotyro- sine; radical. We have examined the formation of hydroxyphenols, nitrophenols, and the minor products 4-nitrosophenol, benzoquinone, 2,2 -biphenol, and 4,4-biphenol from the reaction of peroxynitrite with phenol in the pres- ence and absence of added carbonate. In the absence Peroxynitrite 2 can be formed in vivo from the diffu- of added carbonate, the product yields of nitrophenols sion-controlled reaction of nitric oxide and superoxide and hydroxyphenols have different pH profiles. The radicals (1, 2). In vitro, peroxynitrite has been shown to rates of nitration and hydroxylation also have differ- react with almost all classes of biomolecules, including ent pH profiles and match the trends observed for the proteins, lipids, carbohydrates, antioxidants, and nu- product yields. At a given pH, the sum of the rate con- cleic acids (3 – 12). The nitration of tyrosine residues in stants for nitration and hydroxylation is nearly identi- proteins, which may be involved in signal transduction cal to the rate constant for the spontaneous decompo- (13), is often taken as evidence for the formation of sition of peroxynitrite. The reaction of peroxynitrite peroxynitrite in vivo (14), since tyrosine and other phe- with phenol is zero-order in phenol, both in the pres- nolics are nitrated by peroxynitrite (14–16) but not by ence and absence of added carbonate. In the presence nitric oxide (17, 18). of added carbonate, hydroxylation is inhibited, Peroxynitrite is known to nitrate and hydroxylate whereas the rate of formation and yield of nitrophe- aromatic compounds (4, 15, 16, 19, 20). Halfpenny and nols increase. The combined maximum yield of o- and Robinson (19), who first studied these reactions, sug- p-nitrophenols is 20 mol% (based on the initial concen- gested a radical mechanism. Hydroxyl and • NO 2 tration of peroxynitrite) and is about fourfold higher than the maximal yield obtained in the absence of radicals have been suggested to be formed during the added carbonate. The o/p ratio of nitrophenols is the decomposition of peroxynitrite (21) and have been sug- same in the presence and absence of added carbonate. gested as the species responsible for the peroxynitrite- These results demonstrate that hydroxylation and ni- mediated hydroxylation and nitration of phenylalanine tration occur via two different intermediates. We sug- and tyrosine (15). However, several lines of evidence, gest that the activated intermediate formed in the including the lack of effect of typical hydroxyl radical isomerization of peroxynitrous acid to nitrate, ONOOH*, scavengers (3), experiments performed using spin is the hydroxylating species. We propose that interme- traps (e.g., 5,5-dimethylpyrroline-N-oxide) (22–25), diate 1, O|N{OO{CO 0 2 , or secondary products de- and a study on the effect of viscosity on the rate of rived from it, is (are) responsible for the nitration of decomposition of peroxynitrite (26), do not indicate the phenol. The possible mechanisms responsible for ni- formation of free hydroxyl radical during the decompo- tration are discussed.  1997 Academic Press sition of peroxynitrous acid. The nitronium ion, / NO 2 , Key Words: peroxynitrite; nitrophenol; hydroxy- formed from peroxynitrous acid at low pH (16) or from phenol; nitration; kinetics; nitrosoperoxycarbonate; 2 The term peroxynitrite is used to refer to the sum of both peroxy- 1 To whom correspondence should be addressed at 711 Choppin nitrite anion (ONOO 0 ) and its conjugated acid, peroxynitrous acid (ONOOH). The term carbonate represents the sum of all carbonated Hall, The Biodynamics Institute, Louisiana State University, Baton Rouge, LA 70803-1800. Fax: (504)-388-4936. E-mail: wpryor@ species (CO 2 ,H 2 CO 3 , HCO 0 3 , and CO 0 3 ). If a particular carbonated species is referred to, it is represented by its chemical formula. lsu.edu. 160 0003-9861/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.