Role of the Carbonate Radical Anion in Tyrosine Nitration and Hydroxylation by Peroxynitrite Ce ´lio X. C. Santos, Marcelo G. Bonini, and Ohara Augusto 1 Departamento de Bioquı ´mica, Instituto de Quı ´mica, Universidade de Sa ˜ o Paulo, Cx P. 26077, 05513-970, Sa ˜ o Paulo, SP, Brazil Received November 15, 1999, and in revised form February 7, 2000 Peroxynitrite has been receiving increasing atten- tion as the pathogenic mediator of nitric oxide cyto- toxicity. In most cases, the contribution of peroxyni- trite to diseases has been inferred from detection of 3-nitrotyrosine in injured tissues. However, presently it is known that other nitric oxide-derived species can also promote protein nitration. Mechanistic details of protein nitration remain under discussion even in the case of peroxynitrite, although recent literature data strongly suggest a free radical mechanism. Here, we confirm the free radical mechanism of tyrosine modi- fication by peroxynitrite in the presence and in the absence of the bicarbonate– carbon dioxide pair by analyzing the stable tyrosine products and the forma- tion of the tyrosyl radical at pH 5.4 and 7.4. Stable products, 3-nitrotyrosine, 3-hydroxytyrosine, and 3,3- dityrosine, were identified by high performance liquid chromatography and UV spectroscopy. The tyrosyl radical was detected by continuous-flow and spin- trapping electron paramagnetic resonance (EPR). 3-Hydroxytyrosine was detected at pH 5.4 and its yield decreased in the presence of the bicarbonate– carbon dioxide pair. In contrast, the yields of the tyrosyl rad- ical increased in the presence of the bicarbonate– car- bon dioxide pair and correlated with the yields of 3-nitrotyrosine under all tested experimental condi- tions. Taken together, the results demonstrate that the promoting effects of carbon dioxide on peroxynitrite- mediated tyrosine nitration is due to the selective re- activity of the carbonate radical anion as compared with that of the hydroxyl radical. Colocalization of 3-hydroxytyrosine and 3-nitrotyrosine residues in pro- teins may be useful to discriminate between peroxyni- trite and other nitrating species. © 2000 Academic Press Key Words: nitric oxide; peroxynitrite; nitrotyrosine; DOPA; carbonate radical anion; tyrosyl radical. Nitric oxide is produced in vivo by the constitutive and inducible nitric oxide synthase enzymes (1) and probably also by nonenzymatic processes such as the decomposition of nitrite at low pHs (2) as those found in the stomach and the phagosomes of phagocytic cells. Nitric oxide plays important physiological roles but its overproduction has been associated with many patho- logical conditions. Peroxynitrite, 2 which is formed by the fast reaction between nitric oxide and superoxide anion, has been receiving increasing attention as the pathogenic mediator of nitric oxide cytotoxicity (3–7). In many cases, involvement of peroxynitrite has been inferred from the detection of 3-nitrotyrosine residues in injured tissues (8). Initially, nitration of tyrosine residues at neutral pHs was considered to be depen- dent on peroxynitrite formation. More recently, how- ever, other mechanisms have been demonstrated to be possible such as the oxidation of nitrite by hypochlor- ous acid and by peroxidase enzymes (9, 10) and the coupling of nitric oxide with protein tyrosyl radicals followed by further oxidation to 3-nitrotyrosine (11). In addition, the low yields of 3-nitrotyrosine obtained by fluxes of peroxynitrite produced from nitric oxide do- nors and superoxide anion generators has challenged the view that peroxynitrite is physiologically impor- tant as a nitrating agent (12). Enzymatic superoxide anion generator systems, however, are dependent on 1 To whom correspondence and reprint requests should be ad- dressed. Fax: 55-11-8187986 and 55-11-815-5579. E-mail: oaugusto@ iq.usp.br. 2 Unless otherwise specified, the term peroxynitrite is used to refer to the sum of peroxynitrite anion (ONOO - ) and peroxynitrous acid (ONOOH). IUPAC recommended names for peroxynitrite anion, per- oxynitrous acid, and nitric oxide are oxoperoxonitrate (1-), hydrogen oxoperoxonitrate, and nitrogen monoxide, respectively. 146 0003-9861/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 377, No. 1, May 1, pp. 146 –152, 2000 doi:10.1006/abbi.2000.1751, available online at http://www.idealibrary.com on