ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 333, No. 1, September 1, pp. 49–58, 1996 Article No. 0363 Peroxynitrite Reaction with Carbon Dioxide/Bicarbonate: Kinetics and Influence on Peroxynitrite-Mediated Oxidations Ana Denicola,* , † Bruce A. Freeman,‡ Madia Trujillo,* and Rafael Radi* ,1 *Department of Biochemistry, Facultad de Medicina and †Department of Physical Biochemistry, Facultad de Ciencias, Universidad de la Repu ´ blica, Montevideo 11800 Uruguay; and ‡Departments of Anesthesiology, Biochemistry and Molecular Genetics and Pediatrics, University of Alabama at Birmingham, Birmingham, Alabama Received April 1, 1996, and in revised form June 3, 1996 adduct (ONOOCO 0 2 ) which can participate in oxidation and nitration processes, thus redirecting the primary Peroxynitrite is a strong oxidant produced in vivo reactivity of peroxynitrite.  1996 Academic Press, Inc. as the reaction product of superoxide anion and nitric Key Words: peroxynitrite; superoxide; nitric oxide; oxide (k Ç 5 1 10 9 M 01 s 01 ) and can be formed and medi- bicarbonate; carbon dioxide; reaction kinetics; ate reactions in the extracellular environment. It has stopped-flow spectrophotometry; free radicals. recently been reported that peroxynitrite and carbon dioxide react in a second-order process (S. V. Lymar and K. Hurst (1995) J. Am. Chem. Soc. 117, 8867–8868). Since one of the most abundant constituents of the ex- tracellular milieu is bicarbonate anion (25 mM in Peroxynitrite 2 production by endothelial cells (1), plasma) which is in equilibrium with carbon dioxide neutrophils (2), and macrophages (3) occurs following (1.3 mM in plasma) we have further studied the kinetics the almost diffusion-controlled reaction (k Ç 5 1 10 9 of the reaction between peroxynitrite and carbon diox- M 01 s 01 ; Refs. 4 and 5) between cell-derived nitric oxide ide/bicarbonate and the effect of bicarbonate on differ- ( • NO) and superoxide (O •0 2 ). Peroxynitrite is capable of ent peroxynitrite-mediated oxidations. The apparent oxidizing a variety of biomolecules including thiols (6), second-order rate constant for the reaction is (2.3 { 0.1) lipids (7), carbohydrates (8), and DNA (9) via complex 1 10 3 M 01 s 01 at 37°C and pH 7.4 and a pH-independent and strongly pH-dependent oxidative reaction mecha- second-order rate constant of (5.8 { 0.2) 1 10 4 M 01 s 01 at nisms. Peroxynitrite anion (ONOO 0 ) directly reacts 37°C was obtained considering peroxynitrite anion and with sulfhydryls (6), a reaction implicated to be a major carbon dioxide as the reacting species. The enthalpy mechanism for peroxynitrite-mediated inactivation of and entropy of activation are DH # Å/10.7 { 0.8 kcal enzymes (10, 11). Once protonated (pK a Å 6.8; Ref. 6) mol 01 and DS # Å06.5 { 0.5 cal mol 01 K 01 , respectively. to peroxynitrous acid (ONOOH), this unstable species The presence of bicarbonate had variable influence on isomerizes to nitric acid with a half-life of less than 1 peroxynitrite-mediated oxidations. While bicarbonate s at pH 7.4 and 37°C (8) and will undergo both one- or significantly enhanced peroxynitrite-mediated nitra- two-electron oxidation to target molecules. The ground tion of aromatics, it partially inhibited the oxidation of state form of peroxynitrous acid reacts with methionine thiols, dimethylsulfoxide, oxyhemoglobin, and cyto- chrome c /2 and totally inhibited the hydroxylation of (12), cytochrome c 2/ (13), ascorbate (14), and trypto- benzoate. Spontaneous chemiluminescence studies phan (15). Peroxynitrous acid also undergoes transition suggest the formation of bicarbonate radicals during to a vibrationally activated state (ONOOH*), 17 kcal the interactions of peroxynitrite with carbon dioxide/ mol 01 above the energy level of the ground state (16), bicarbonate. Our results support that peroxynitrite anion rapidly reacts with carbon dioxide to yield an 2 The term ‘‘peroxynitrite’’ is used to refer to either the anion (ONOO 0 ) or its conjugate acid (ONOOH). When a discussion is spe- 1 To whom correspondence and reprint requests should be ad- cifically limited to either the anion or the free acid, either its name or its structure will be given. IUPAC recommended name for peroxy- dressed at Depto. de Bioquı B mica, Facultad de Medicina, Universidad de la Repu ´ blica, Avda. General Flores 2125, 11800 Montevideo, Uru- nitrite is oxoperoxonitrate (10), for peroxynitrous acid is hydrogen oxoperoxonitrate, and for nitric oxide is nitrogen monoxide. guay. E-mail: RRADI@BQRAD.EDU.UY. 49 0003-9861/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved.