Distance-Dependent Diffusion-Controlled Reaction of • NO and O 2 •- at Chemical Equilibrium with ONOO - Horacio Botti,* ,†,‡ Matı ´as N. Mo ¨ller, ‡,§,| Daniel Steinmann, #,⊥ Thomas Nauser, # Willem H. Koppenol, # Ana Denicola, ‡,§ and Rafael Radi* ,‡,∇ Unidad de Cristalografía de Proteı ´nas, Instituto Pasteur de MonteVideo, MonteVideo, 11400, Uruguay, Center for Free Radical and Biomedical Research, MonteVideo, 11800, Uruguay, Laboratorio de Fisicoquı ´mica Biolo ´gica, Instituto de Quı ´mica Biolo ´gica, Facultad de Ciencias, UniVersidad de la Repu ´blica, MonteVideo, 11400, Uruguay, Department of Chemistry and Vanderbilt Institute of Chemical Biology, Vanderbilt UniVersity, NashVille, Tennessee 37240, United States, Institute of Inorganic Chemistry, Department of Chemistry and Applied Biosciences, ETH Zu ¨rich, CH-8093, Switzerland, and Departamento de Bioquı ´mica, Facultad de Medicina, UniVersidad de la Repu ´blica, MonteVideo, 11800, Uruguay ReceiVed: June 17, 2010; ReVised Manuscript ReceiVed: August 29, 2010 The fast reaction of • NO and O 2 •- to give ONOO - has been extensively studied at irreversible conditions, but the reasons for the wide variations in observed forward rate constants (3.8 e k f e 20 × 10 9 M -1 s -1 ) remain unexplained. We characterized the diffusion-dependent aqueous (pH > 12) chemical equilibrium of the form • NO + O 2 •- ) ONOO - with respect to its dependence on temperature, viscosity, and [ONOO - ] eq by determining [ONOO - ] eq and [ • NO] eq . The equilibrium forward reaction rate constant (k f eq ) has negative activation energy, in contrast to that found under irreversible conditions. In contradiction to the law of mass action, we demonstrate that the equilibrium constant depends on ONOO - concentration. Therefore, a wide range of k f eq values could be derived (7.5-21 × 10 9 M -1 s -1 ). Of general interest, the variations in k f can thus be explained by its dependence on the distance between ONOO - particles (sites of generation of • NO and O 2 •- ). I. Introduction It is currently accepted that the main route to peroxynitrite anion [oxoperoxonitrate(1-), ONOO - ] formation in biological systems is the spin-allowed, termination reaction between superoxide anion (O 2 •- ) and nitric oxide (nitrogen monoxide, • NO), reaction 1A 1-3 The first kinetic report supported a fast reaction between • NO and O 2 •- with a rate constant (k f ) on the order of 10 7 M -1 s -1 . 4 As a consequence of that report, the in vivo occurrence of peroxynitrite was proposed at that time and ONOOH was portrayed as a biologically relevant metal-independent source of the highly damaging HO • . 5-7 Currently, most authors agree that the reaction is diffusion limited. 3 Therefore, it is widely accepted that it will occur to some extent anytime these two radicals are available in the same compartment, 3,8 despite the facts that superoxide dismutases (SODs) compete with • NO for O 2 •- and that peroxynitrite has never been directly detected. Rate constants reported later that support this view were determined under irreversible conditions and are within a broad range, 3.8 × 10 9 M -1 s -1 e k f irr e 20.0 × 10 9 M -1 s -1 . 9-13 Nauser and co-workers proposed that the statistically significant discrepancy arises because of the inadequateness of pulse radiolysis protocols to produce • NO and O 2 •- rapidly enough to allow simple and accurate determinations of the rate constant, recommending a value of 1.6 ( 0.3 × 10 10 M -1 s -1 that resulted from a weighted average of flash photolysis determinations that varied between 1.3 ( 0.2 × 10 10 and 2.0 ( 0.4 × 10 10 M -1 s -1 with different experimental designs. 13 In contrast, a flash photolysis experiment-derived value of 6.7 × 10 9 M -1 s -1 was previously reported by Huie and Padmaja. 9 Strikingly, no attempt has been made to understand this reaction within the theoretical field of diffusion-controlled/diffusion-influenced re- actions. 14 Contrary to what is found under physiologic conditions, ONOO - is fairly stable in the absence of H 3 O + , CO 2 , and other Lewis acids. 3 Thanks to this fact, the reverse reaction (reaction 1B) rate constant has been accurately studied in strongly alkaline aqueous media in the presence of sufficiently high concentrations of • NO and O 2 •- scavengers that allow the continuous monitoring of the irreversible advance of the thermohomolytic reaction. 15-18 In this way, Sturzbecher and co-workers could establish the temperature (15-55 °C) and pressure (5-175 MPa) depend- encies of the rate constant of reaction 1B. 18 Thus, an equilibrium state between ONOO - and • NO, O 2 •- can be assumed to occur in strongly alkaline solutions (pH > 12) (eqs 2 and 3) * To whom correspondence should be addressed. H.B.: phone, 0598- 25220910 ext. 143; e-mail, hbotti@pasteur.edu.uy. R.R.: phone, 0598- 29249562; e-mail: rradi@fmed.edu.uy. † Instituto Pasteur de Montevideo. ‡ Center for Free Radical and Biomedical Research. § Facultad de Ciencias, Universidad de la Repu ´blica. | Vanderbilt University. # ETH Zu ¨rich. ∇ Facultad de Medicina, Universidad de la Repu ´blica. ⊥ Current Address: Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66045, United States. • NO + O 2 •- f ONOO - k f (1A) ONOO - f • NO + O 2 •- k r irr (1B) J. Phys. Chem. B XXXX, xxx, 000 A 10.1021/jp105606b  XXXX American Chemical Society