TABLE 1.

Kinetic aspects of peroxynitrite-mediated oxidations: selected reactions of biochemical relevance

The majority of the information highlights the reactivity of peroxynitrite with biomolecules and provides considerations about its quantitative relevance in biological systems. The last three listed compounds are examples of synthetic molecules utilized to either decompose or detect peroxynitrite. Prx, peroxiredoxin.

Reactant	ka	Process	Commentary
	m−1s−1
Tyrosine	0	Tyr oxidation and nitration	There is no direct reaction (40). Tyr oxidation and nitration is accomplished by peroxynitrite-derived radicals (23).
Tryptophan	40	Trp oxidation and nitration	The direct reaction is rather slow and can cause Trp nitration (96).
Methionine	360	Methionine sulfoxide formation	It can account for enzyme inactivation (97). It is sometimes used to scavenge peroxynitrite in biochemical systems.
Uric acid	500	A variety of oxidation products can be formed, including allantoin, alloxan, and triuret (83, 85). The intermediate formation of uric acid-derived radicals may promote secondary oxidation reactions and products such as urate hydroperoxide (83, 98).	It is good inhibitor of peroxynitrite-dependent processes in vitro and in vivo. The direct reaction is relatively slow, so protection is ascribed to reaction with peroxynitrite-derived radicals. Uric acid is also a physiological substrate of myeloperoxidase (98) and may therefore interfere in heme peroxidase-dependent nitration reactions as well.
Glutathioneb	1400	It evolves mainly to glutathione disulfide through the intermediacy of glutathione sulfenic acid (13). Glutathionyl radicals can be formed from peroxynitrite-derived radicals.	It is an endogenous compound that decomposes peroxynitrite. Considering a 5 mm intracellular concentration, the k[GSH]c product results in a value of 7 s−1, significantly faster that the rate constant of homolysis (0.9 s−1)d but much less than that of other direct reactions, so its direct reaction with peroxynitrite in biological systems is modest.
Cysteineb	5900	It evolves to cysteine disulfide (cystine) through the intermediacy of cysteine sulfenic acid (13, 24).	This was the first determination of a second-order rate constant of peroxynitrite reaction with a biomolecule. It provided the concept that direct reactions of peroxynitrite may be more relevant in biology than homolysis.
Human serum albumin	9700	About 40% of the direct reactivity is due to the reaction with the single thiol group (Cys-34) (40), leading to the sulfenic acid derivative.	A highly abundant plasma protein, it consumes a fraction of intravascular peroxynitrite but cannot outcompete the reaction with CO2.
Oxyhemoglobin	2.3 × 104	It isomerizes peroxynitrite to nitrate (99).	It is relevant for peroxynitrite detoxification in red blood cells. At a concentration of 5 mm, k[oxy-Hb] = 340 s−1, a remarkable velocity. However, peroxiredoxin-2 outcompetes oxyhemoglobin in peroxynitrite detoxification in the erythrocyte (13).
Mn-SOD	>104	The reaction of peroxynitrite anion with the Mn2+ atom produces enzyme nitration at Tyr-34 (43).	The nitration of the critical Tyr residue leads to enzyme inactivation. This process is largely observed in vivo under inflammatory conditions.
CO2	5.8 × 104	Nucleophilic addition of peroxynitrite anion to CO2 yields an unstable intermediate that undergoes homolysis (35, 36, 38).	This is a central reaction controlling peroxynitrite reactivity in biological system. A k[CO2] value of ∼60–100 s−1 has been established as a desirable starting range for a peroxynitrite scavenger to be competitive (13).
Aconitasee	1.4 × 105	Oxidation and disruption of the iron-sulfur cluster (57, 58)	A key reaction in mitochondria, aconitase inactivation slows down the Krebs cycle and causes iron release.
Peroxiredoxins	106–107	Fast reaction with the peroxidatic cysteine residue (30, 81)	Microbial and mammalian peroxiredoxins constitute a central catalytic mechanism for the detoxification peroxynitrite. The k[Prx] value ranges from >102 to 103 s−1 depending on cell types.
Ebselen	4.6 × 106	A synthetic seleno-containing compound that in the reduced state (selenol) undergoes two-electron oxidation, a reaction chemistry similar to that of thiols (100)	Ebselen readily decomposes peroxides and can create catalytic redox cycles at the expense of reducing compounds such as glutathione. It can be used pharmacologically to neutralize peroxynitrite (54).
MnP	>107	Mn2+ reduces peroxynitrite to nitrite and is catalytically recycled by endogenous reductants and the electron transport chain (89).	These compounds are used pharmacologically and can achieve 5–10 μm concentrations in vivo. Thus, with k[MnP] > 100 s−1, they can effectively eliminate part of peroxynitrite (13).
Boronate-based compounds	>106	Peroxynitrite anion reacts directly via two-electron oxidation with boronate-based compounds to yield their corresponding hydroxyl derivatives (95). In the case of aryl boronates, the corresponding phenols are the major final products.	These compounds are a novel class of probes that can be utilized for peroxynitrite detection. They react with peroxynitrite at rates ∼106 faster than hydrogen peroxide. The high rate constant and the lack of formation of probe-derived radical intermediates minimize secondary reactions and confounding results.

^a Stopped-flow spectrophotometry has been utilized to determine the rate constants of peroxynitrite reaction with most compounds, taking advantage of the distinctive optical absorption of ONOO⁻ at 302 nm (ϵ = 1670 m⁻¹ cm⁻¹) as originally reported (24). Alternative approaches have been also used, with the application of competition kinetics with reference reactions of known rate constants (81).

^b The actual reactants are peroxynitrous acid and the thiolate anion (Equation 7) (27); thus, the observed apparent reaction rate is strongly pH-dependent (24), with the thiol pK_SH representing a relevant variable. The table shows k_app, which is on the order of 10³ m⁻¹ s⁻¹; however, the actual (pH-independent) rate constant of the reaction is on the order of 10⁵ m⁻¹ s¹ (27, 30).

^c The product of the second-order rate constant times the concentration of the reactant provides a pseudo-first-order rate constant in s⁻¹ that allows ready comparison of the kinetic biological relevance among different peroxynitrite targets.

^d In fact, the homolytic yields of ^•NO₂ and ^•OH are ∼30% of ONOOH due to “in cage” recombination of nascent radicals to nitrate (NO₃⁻) before their diffusion to the bulk aqueous phase.

^e Peroxynitrite also causes aconitase tyrosine nitration, but this is not related to the loss of activity, which is exclusively due to the oxidation of the [4Fe-4S] cluster (59).