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The biological effects of peroxynitrite have been recently considered to be largely dependent on its reaction with carbon dioxide, which is present in high concentrations in intra-and extracellular compartments. Peroxynitrite anion (ONOO ؊ ) reacts rapidly with carbon dioxide, forming an adduct, nitrosoperoxocarboxylate (ONOOCO 2 ؊ ), whose decomposition has been proposed to produce reactive intermediates such as the carbonate radical (CO 3 . ). Here, by the use of rapid mixing continuous flow electron paramagnetic resonance (EPR), we directly detected the carbonate radical in flow mixtures of peroxynitrite with bicarbonate-carbon dioxide over the pH range of 6 -9. The radical was unambiguously identified by its EPR parameters (g ‫؍‬ 2.0113; line width ‫؍‬ 5.5 G) and by experiments with bicarbonate labeled with 13 C. In this case, the singlet EPR signal obtained with 12 C bicarbonate splits into the expected doublet because of 13 C (a( 13 C)‫؍‬ 11.7 G). The singlet spectrum of the unlabeled radical was invariant between pH 6 and 9, confirming that in this pH range the detected radical is the carbonate radical anion (CO 3 . ).Importantly, in addition to contributing to the understanding of nitrosoperoxocarboxylate decomposition pathways, this is the first report unambiguously demonstrating the formation of the carbonate radical anion at physiological pHs by direct EPR spectroscopy.Peroxynitrite 1 is formed from the very fast reaction between nitric oxide and superoxide anion (k ϭ (6.7 Ϫ 19) ϫ 10 9 M Ϫ1 s Ϫ1 ) (see Reaction 1) (1, 2). The compound is a potent oxidant that has been receiving increasing attention as a potential pathogenic mediator in human diseases and as a cellular toxin in host defense mechanisms against invading microorganisms (3-6). At present, a significant part of the biological reactivity of peroxynitrite is ascribed to the adduct produced by its reaction with carbon dioxide (7-13). The peroxynitrite anion (ONOO Ϫ ), which is the predominant form at physiological pHs (pK a ϭ 6.8) (see reaction 2, Table II) (2, 3), reacts fast with carbon dioxide (pH-independent k ϭ 5.8 ϫ 10 4 M Ϫ1 ⅐ s Ϫ1 at 37°C) (11), producing an adduct whose structure is proposed to be ([ONOOCO 2 ] Ϫ , nitrosoperoxocarboxylate) (see reaction 3, Table II) (7). Taking into account the concentrations of carbon dioxide in equilibrium with bicarbonate present in physiological fluids, model calculations have suggested that most of the peroxynitrite that might be formed in these fluids will produce the carbon dioxide adduct before reacting with other biological targets (5, 13).Carbon dioxide modulates the reactivity of peroxynitrite by altering reaction rates, product yields, and product distribution (7-13). In these reactions, formation of the adduct nitrosoperoxocarboxylate is rate-limiting, as first proposed by Lymar and Hurst (7). This suggestion was confirmed by other authors (8 -13), and the current proposal is that in the absence of substrates, the carbon dioxide adduct decomposes to nitrate and carbon dioxide, but in ...

Nitric oxide (NO•; nitrogen monoxide) is known to be a critical regulator of cell and tissue function through mechanisms that utilize its unique physicochemical properties as a small and uncharged free radical with limited reactivity. Here, the basic chemistry and biochemistry of NO• are summarized through the description of its chemical reactivity, biological sources, physiological and pathophysiological levels, and cellular transport. The complexity of the interactions of NO• with biotargets, which vary from irreversible second-order reactions to reversible formation of nonreactive and reactive nitrosyl complexes, is noted. Emphasis is placed on the kinetics and physiological consequences of the reactions of NO• with its better characterized biotargets. These targets are soluble guanylate cyclase (sCG), oxyhemoglobin/hemoglobin (HbO2/Hb) and cytochrome c oxidase (CcOx), all of which are ferrous heme proteins that react with NO• with second-order rate constants approaching the diffusion limit (k on approximately 107 to 108 M–1 s–1). Likewise, the biotarget responsible for the most described pathophysiological actions of NO• is the superoxide anion radical (O2 •–), which reacts with NO• in a diffusion-controlled process (k approximately 1010 M–1 s–1). The reactions of NO• with proteins containing iron–sulfur clusters ([FeS]) remain little studied and the reported rate constants of the first steps of these reactions are considerable (k approximately 105 M–1 s–1). Not surprisingly, the interactions of proteins containing iron–sulfur clusters with NO• remain ambiguous and have been associated with both physiological and pathophysiological effects. Overall, it is emphasized that any claimed biological action of NO• should be connected with its interaction with kinetically relevant biotargets. Although reactivity toward biotargets is only one of the factors contributing to cellular and tissue responses mediated by short-lived species, such as NO• and other oxygen-derived species, it is a critical factor. Therefore, taking reactivity into account is important to advancing our knowledge on redox signaling mechanisms.

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Free radicals and oxidants are now implicated in physiological responses and in several diseases. Given the wide range of expertise of free radical researchers, application of the greater understanding of chemistry has not been uniformly applied to biological studies. We suggest that some widely used methodologies and terminologies hamper progress and need to be addressed. We make the case for abandonment and judicious use of several methods and terms and suggest practical and viable alternatives. These changes are suggested in four areas: use of fluorescent dyes to identify and quantify reactive species, methods for measurement of lipid peroxidation in complex biological systems, claims of antioxidants as radical scavengers, and use of the terms for reactive species.

Peroxynitrite mediates the oxidation of the thiol group of both cysteine and glutathione. This process is associated with oxygen consumption. At acidic pH and a cysteine/peroxynitrite molar ratio of < or = 1.2, there was a single fast phase of oxygen consumption, which increased with increasing concentrations of both cysteine and oxygen. At higher molar ratios the profile of oxygen consumption became biphasic, with a fast phase (phase I) that decreased with increasing cysteine concentration, followed by a slow phase (phase II) whose rate of oxygen consumption increased with increasing cysteine concentration. Oxygen consumption in phase I was inhibited by desferrioxamine and 5,5-dimethyl-1-pyrroline N-oxide, but not by mannitol; superoxide dismutase also inhibited oxygen consumption in phase I, while catalase added during phase II decreased the rate of oxygen consumption. For both cysteine and glutathione, oxygen consumption in phase I was maximal at neutral to acidic pH: in contrast, total thiol oxidation was maximal at alkaline pH. EPR spin-trapping studies using N-tert-butyl-alpha-phenylnitrone indicated that the yield of thiyl radical adducts had a pH profile comparable with that found for oxygen consumption. The apparent second-order rate constants for the reactions of peroxynitrite with cysteine and glutathione were 1290 +/- 30 M-1.S-1 and 281 +/- 6 M-1.S-1 respectively at pH 5.75 and 37 degrees C. These results are consistent with two different pathways participating in the reaction of peroxynitrite with low-molecular-mass thiols: (a) the reaction of the peroxynitrite anion with the protonated thiol group, in a second-order process likely to involve a two-electron oxidation, and (b) the reaction of peroxynitrous acid, or a secondary species derived from it, with the thiolate in a one-electron transfer process that yields thiyl radicals capable of initiating an oxygen-dependent radical chain reaction.

SummaryThe unequivocal demonstration that the carbonate radical (CO 3 .7 ) is produced from the reaction between the ubiquitous carbon dioxide and peroxynitrite, renewed the interest in the pathogenic roles of oxidants derived from the main physiological buffer, the bicarbonate-carbon dioxide pair. Here, we review the biochemical properties of both the carbonate radical and peroxymonocarbonate (HCO 4 7 ), and discuss the evidence of their formation under physiological conditions. Overall, the review emphasizes the recognition of the biological relevance of oxidants derived from the main physiological buffer as a crucial step into the understanding and control of numerous pathological states. IUBMB Life, 59: 255-262, 2007