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Chem. Res. Toxicol. 1998, 11, 720-721
Peroxynitrite Reactions and Diffusion in Biology Rafael Radi* Department of Biochemistry, Facultad de Medicina, Universidad de la Republica, Avda. General Flores 2125, 11800 Montevideo, Uruguay Received May 4, 1998
Peroxynitrite Reaction Pathways. There has been considerable debate in the literature on whether free •OH or an activated intermediate (ONOOH*) is formed during the unimolecular decomposition of peroxynitrite (k1 ) 1 s-1, at 37 °C and pH 7.4) (1, 2). We favor the hypothesis proposing the formation of some free •OH (3), in a caged radical pair [•NO2 •OH] (Scheme 1). However, since both ONOO- and ONOOH can directly react with biomolecules (Scheme 1, routes I-III) with significant secondorder rate constants (4) (Table 1), under biological conditions most peroxynitrite will be directly consumed instead of escaping through the slower unimolecular route that leads to the formation of ‚OH or •OH-like oxidants. The relative importance of a given secondorder reaction in a biological compartment will be a function of the rate constant and target concentration. At the concentrations of biomolecules existing in different compartments, it can be estimated that under biological conditions, >95% peroxynitrite would be consumed through direct reactions, with the •OH-like pathway accounting for 105 M-1 s-1 and lead to loss in enzyme activity. Peroxynitrite oxidizes ferrous and ferric forms of hemoproteins (cytochrome c2+, oxyhemoglobin, peroxidases) with reaction constants in the order of 104-106 M-1 s-1. Some of these reactions (e.g., myeloperoxidase) result in the formation of strong nitrating intermediates. Peroxynitrite reacts with Cu-Zn-, Mn-, and Fe-containing superoxide dismutases (SOD). The reaction of Cu-Zn SOD * Corresponding author. E-mail:
[email protected]. Fax: 5982924-9563.
Scheme 1. Peroxynitrite Reaction Pathways
Table 1. Rate Constants of Peroxynitrite Reactions with Biomolecules and Some Other Relevant Compounds at Physiological pHa ks (M-1 s-1)
reaction
Fe(III)TMPyP Mn(II)TMPyP ebselen
2.2 × 1.8 × 106 c 1.6 × 106 c
myeloperoxidase horseradish peroxidase alcohol dehydrogenase aconitase cycochrome c oxyhemoglobin
>106 d 7 × 105 c 3 × 105 e 1.4 × 105 c 1.3 × 104 c 1 × 104 c
Cu-Zn SOD CO2 bovine serum albumin cysteine glutathione methionine tryptophan ascorbate
reaction
106 b
ks (M-1 s-1) 103-105 b 4 × 104 b 6 × 103 b 5 × 103 b 1.35 × 103 b 1.8 × 102 c 1 × 102 b 1 × 102 c
a Reported rate constants were obtained from the literature and represent the apparent values (pH-dependent) in the physiological pH range (7.2-7.6) for the reactions of peroxynitrite with biomolecules and three other relevant synthetic compounds. The synthetic compounds reported at the top of the table represent molecules that have been proposed as compounds that may interact and attenuate the toxic effects promoted by peroxynitrite. b T ) 37 °C. c T ) 25 °C. d T ) 12 °C. e T ) 23 °C.
promotes nitration reactions including self-nitration, although the enzyme does not become inactivated. Peroxynitrite reactions with Mn SOD lead to nitration and inactivation of this enzyme. 2. Oxidation Reactions That Involve the •OH-like Pathway. Some processes may be initiated by the small fraction of peroxynitrite undergoing unimolecular decomposition. This can be the case for lipid peroxidation processes initiated by peroxynitrite (5). Other processes such as thiyl radical formation by peroxynitrite can also initiate an oxygen-dependent radical chain reaction. Peroxynitrite can cause DNA strand breaks (in isolated duplex DNA) with a pattern comparable to that caused by •OH. Cellular DNA strand breaks by peroxynitrite have been also observed. However, it is more likely that secondary intermediates from peroxynitrite are responsible for the observed effects at the cell level (i.e., reaction with carbon dioxide, see below). 3. Nitration Reactions. Peroxynitrite promotes nitration of tyrosine and tryptophan residues of proteins, guanine residues in DNA, and aliphatic groups of fatty acids and sugars. However, as discussed in detail in
S0893-228x(98)00096-4 CCC: $15.00 © 1998 American Chemical Society Published on Web 07/20/1998
Forum: Reactive Species of Peroxynitrite
other articles in this Forum, peroxynitrite is a poor nitrating agent. Biological nitrations are promoted by transition metals and metalloproteins and by carbon dioxide (Scheme 1). Nitration catalysts increase nitration yields and through a fast reaction with ONOO- favor nitration over other competing processes such as oxidations; thus, intracellular protein nitration is observed even in the presence of millimolar glutathione. 4. Nitrosation Reactions. Peroxynitrite is a poor nitrosating agent, but it can nitrosate thiols, sugars, and lipids in low yields. Some of these products elicit strong biological activity (i.e., vasodilation), but their formation may not represent a primary physiological regulatory mechanism. Even under optimal conditions the •NO releasable from these compounds will be a small fraction (90% of the initial reactivity of peroxynitrite. The formation of secondary radicals upon the reaction of ONOOCO2with extracellular targets (uric acid-derived, thiyl, ascorbyl, tyrosyl, and tryptophanyl radicals) has been detected. Intracellularly, the higher concentration of thiols and metal centers among other targets decreases the relative contribution of the CO2 reaction to the initial reactivity of peroxynitrite, possibly still accounting for 30-40% of it. As the role of carbon dioxide in peroxynitrite biochemistry becomes more apparent, it is important to reevaluate whether various cellular effects previously ascribed to peroxynitrite itself may in fact be due to ONOOCO2and also how the biological effects of peroxynitrite may be influenced by varying levels of bicarbonate/carbon dioxide. With this perspective, processes such as cellular DNA strand breaks by extracellular peroxynitrite may primarily rely on reactions with ONOOCO2- rather than with oxidants derived from peroxynitrite as it is highly unlikely that the •OH-like oxidant from peroxynitrite will
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be formed and react within the nuclei to any significant extent. Peroxynitrite Diffusion through Membranes. Both the protonated and anionic forms of peroxynitrite can diffuse across biological membranes (8, 9). Peroxynitrous acid can cross the lipid bilayers by passive diffusion (8) and with a permeability coefficient comparable to that of water (9). In addition, ONOO- has been shown to diffuse through erythrocyte membranes via the DIDSinhibitable anion channel which normally participates in the bicarbonate-chloride exchange (8). Both mechanisms of diffusion have biological relevance, since: (1) the pKa of peroxynitrite determines that under physiological or pathological conditions, both acid-base forms coexist and (2) although at physiological pH ONOO- is normally the predominant form, non-erythroid cells may be scarce in anion transporters, and the passive diffusion mechanism would become more relevant under those conditions. An important question is whether the presence of extracellular carbon dioxide precludes the diffusion of peroxynitrite to intracellular compartments. Evidence indicates that although carbon dioxide will limit peroxynitrite diffusion, peroxynitrite can have effects in target cells even in the presence of carbon dioxide. At distances in the order of 1-10 µm, diffusion rates can be similar to or faster than the rate of formation of the ONOOCO2-. However, once ONOOCO2- is formed, this species will mostly decay or react in the same compartment where it was formed, as its estimated half-life is
