JULY 2002 VOLUME 15, NUMBER 7 © Copyright 2002 by the American Chemical Society
Mapping the Reaction of Peroxynitrite with CO2: Energetics, Reactive Species, and Biological Implications Giuseppe L. Squadrito* and William A. Pryor Biodynamics Institute, Louisiana State University, Baton Rouge, Louisiana 70803-1800 Received January 24, 2002
Contents 1. Introduction 2. Thermodynamic Appraisal of the Reaction of Peroxynitrite with CO2 2.1. ∆fG° for Key Species in the Reaction of Peroxynitrite with CO2 2.1.1. ∆fG° (CO2, aq) 2.1.2. ∆fG° (ONOO-, aq) and ∆fG° (ONOOH, aq) 2.1.3. ∆fG° (CO3•-, aq) and ∆fG° (‚NO2, aq) 2.2. Energetics of the Reaction of Peroxynitrite with CO2 and Properties of Reactive Species 2.2.1. Estimation of ∆G° for Reaction 3a 2.2.2. Homolysis of ONO-OCO2(Reaction 3b) 2.2.3. pKa of ONOOCO2H 2.2.4. O2NOCO2H 2.2.5. Special Features of the Cage Mechanism of the Reaction of Peroxynitrite with CO2 3. Biological Oxidations 4. Summary and Conclusions
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1. Introduction Nitric oxide is a simple free radical with pivotal roles in cell regulation and function (1). The interaction between nitric oxide and the superoxide radical results * To whom correspondence should be addressed. Phone: (225)-3882063. Fax: (225)-388-4936. E-mail:
[email protected].
in the formation of peroxynitrite1 (2), particularly during episodes of excessive production of nitric oxide and/or superoxide. The formation of peroxynitrite, as originally proposed by Beckman et al. (3), has been linked to pathology. Research efforts directed to understand the mechanism of reaction of peroxynitrite were initially focused primarily on the reactions of peroxynitrite with substrates with zero order kinetics (e.g., dimethyl sulfoxide and deoxyribose), with substrates with relatively small second-order rate constants (e.g., methionine and ascorbate), and even a few more reactive substrates, such as thiols (3-6). Although these experiments afforded important mechanistic information, the reactions of peroxynitrite with these substrates cannot compete with the reaction of peroxynitrite with CO2 under physiological conditions. The fast reaction between peroxynitrite and bicarbonate buffers was first reported by Keith and Powell (7) but the implications of this observation were 1 The systematic name for peroxynitrite (ONOO-) is oxoperoxonitrate(1-). Naming many of the peroxides described here in a systematic manner is not a trivial task. One often finds that those names that are more widely used in the literature are not necessarily those that are recommended by IUPAC. Moreover, often the systematic names are so complex that they are of little help for the chemist that is unfamiliar with the rules for the systematic nomenclature of peroxides. Take, for example, 1-carboxylato-2-nitrosodioxidane, which is the systematic name for ONOOCO2-. Granted, chemical intuition alone would not be sufficient to associate this systematic name with the correct structure. Therefore, we choose to name ONOOCO2- throughout this article as a nitroso derivative of peroxycarbonate: nitrosoperoxycarbonate. Peroxycarbonate is not a systematic name but the connection between name and structure is almost unambiguous in this case. (Peroxycarboxylate is an alternative name for peroxycarbonate but it is used in the chemical literature about 6.5 times less frequently than peroxycarbonate; therefore, the name nitrosoperoxycarbonate is chosen over the alternative name of nitrosoperoxycarboxylate.) Peroxycarbonate is used rather than the systematic name for HOOCO2that is recommended by IUPAC: hydrogendioxoperoxocarbonate(1-). Similarly, the recommended name for CO3•- is trioxocarbonate(1-), but we simply call this species the carbonate radical.
10.1021/tx020004c CCC: $22.00 © 2002 American Chemical Society Published on Web 06/14/2002
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Scheme 1. Mechanism of the Reaction of Peroxynitrite with Carbon Dioxide
not recognized until 1993 when Radi et al. noted the biological relevance of this reaction 8. This association by Radi et al. represents a landmark and turning point in our understanding of the physiological reactions of peroxynitrite. In fact, it is now widely believed that (1) the reaction of peroxynitrite with CO2 is one of the main physiological reactions of peroxynitrite; (2) CO2 modulates the reactivity of peroxynitrite; and (3) the reaction of peroxynitrite plays a pivotal role in the oxidation of biological target molecules, including the nitration of tyrosine residues. The thermochemistry and mechanism for the decomposition of peroxynitrite in the absence of CO2 had received much attention in recent years and has been studied in detail (9, 10); but strikingly, the impact that the reaction of peroxynitrite with CO2 has in biology (2, 11-14) is not widely recognized. The elucidation of the mechanism of the reaction of peroxynitrite with CO2 began in 1995 (2, 11-14) and continues to be a matter of intense study (15, 16). It is now firmly established that the reaction of peroxynitrite with CO2 occurs between the peroxynitrite anion (ONOO-) and dissolved CO2 (14, 17-19) and forms the carbonate radical (CO3•-) and nitrogen dioxide (‚NO2), as shown in Scheme 1.
2. Thermodynamic Appraisal of the Reaction of Peroxynitrite with CO2 In this section, we present a thermodynamic evaluation of the mechanism of the reaction of peroxynitrite with CO2. 2.1. ∆fG° for Key Species in the Reaction of Peroxynitrite with CO2. A brief commentary regarding the ∆fG° for key species in Scheme 1 is important before we proceed: 2.1.1. ∆fG° (CO2, aq). Dissolved CO2 establishes an equilibrium with H2CO3, HCO3-, and CO32-. It is important to recognize that as CO2 (aq) we refer here only to the species CO2 and not to other species involved in the carbonation equilibria.2 According to Alberty (20), ∆fG° (CO2, aq) ) -385.97 kJ/mol. Similarly, we refer to the sum of carbonated species as total CO2 (TotCO2). It is
important to make this distinction and avoid the ambiguities that can lead to many related errors in the chemical and biochemical literature, as has been reviewed recently (21). 2.1.2. ∆fG° (ONOO-, aq) and ∆fG° (ONOOH, aq). Values of ∆fG° (ONOO-, aq) ) 68.6 ( 0.8 kJ/mol and ∆fG° (ONOOH, aq) ) 31.4 ( 0.8 kJ/mol had been obtained recently (9, 10) by measuring the forward and reverse rate constants and determining the standard Gibbs energy of reaction for the following equilibria (reactions 1 and 2), and using the literature values for the ∆fG° for the reaction products:
ONOO- (aq) ) •NO (aq) + O2•- (aq)
(1)
ONOOH (aq) ) •NO2 (aq) + HO• (aq)
(2)
A pKa,ONOOH ) 6.6 can be calculated from the ∆fG° (ONOOH, aq) and ∆fG° (ONOO-, aq) values given above. An apparent pKa,ONOOH ) 6.8 was previously measured using phosphate buffers of relatively high concentrations (23); however, there is some indication that pKa,ONOOH is slightly lower in dilute phosphate buffers (24, 25). This trend is striking because activity coefficients calculated using common models, such as the extended DebyeHuckel or Davies models, predict the pKa of a weak acid will increase with decreasing ionic strength (e.g., with decreasing buffer concentration). In view of this peculiar effect, we studied the changes on the rate constant of decay of peroxynitrite (kobs) caused by varying the phosphate buffer concentration while fixing the ionic strength and pH. Figure 1 shows the effect of varying the phosphate buffer concentration up to 500 mM on the observed rate constant for the decomposition of peroxynitrite, while maintaining the pH and the ionic strength (I) fixed at 7.1 and 1.0 M, respectively. Values of kobs 2 To indicate thermodynamic physical states, we follow the notation used in the NBS tables, namely aq (aqueous solution, concentration not specified) and ao (aqueous solution, ion for which no further ionization is considered, standard state m ) 1 mol/kg). All kinetic and thermodynamic values correspond to 25 °C.
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Chem. Res. Toxicol., Vol. 15, No. 7, 2002 887 Table 1. Standard Gibbs Energies of Formation in Aqueous Solution
Figure 1. Reaction rates measurements were conducted as described elsewhere (5, 51, 78, 79) on an On-Line Instrument Systems (Bogart, GA) stopped-flow instrument equipped with a rapid scan spectrophotometer and a 2 cm cell thermostated at 25.0 ( 0.1 °C. The ionic strength was adjusted to I ) 1.0 M by adding the appropriate amount of sodium chloride. Briefly, peroxynitrite solutions (0.1-0.3 mM, pH 11.5-12.0) were mixed with equal volumes of sodium phosphate buffer (50-500 mM, pH 7.0). The pH was measured both before and after mixing of reactants. A small pH jump (ca. 0.1) was usually observed on mixing, and the final pH was taken as the prevalent reaction pH. Peroxynitrite was synthesized by ozonation of an aqueous solution of sodium azide using a Sander 200 ozonizer (UetzeEltze, Germany) (80, 81).
obtained at 25 °C vary from 0.330 s-1, the value obtained by extrapolation to zero buffer concentration, to 0.392 s-1 at 500 mM phosphate buffer. kobs varies with pH according to kobs ) kONOOH × [H+]/([H+] + Ka,ONOOH) (26); assuming kONOOH is relatively insensitive to changes in buffer concentration at constant ionic strength, kONOOH ) 1.2 s-1 can be calculated using kobs ) 0.392 s-1, pH 7.1, and pKa,ONOOH ) 6.8. Using kONOOH ) 1.2 s-1, one can calculate pKa,ONOOH ) 6.6 for pH 7.1 and kobs ) 0.310 s-1. The results indicate that the decrease in kobs obtained by extrapolation to zero buffer concentration is consistent with pKa,ONOOH ) 6.6. Thus, it appears that the phosphate ions form weak complexes with peroxynitrite, thereby slightly raising pKa,ONOOH to 6.8. 2.1.3. ∆fG° (CO3•-, aq) and ∆fG° (‚NO2, aq). ∆fG° (CO3•-) ) -374 kJ/mol can be calculated from E° (CO3•-/ CO32-) ) 1.59 V (27).3 This new value of ∆fG° (CO3•-) replaces a provisional value of ∆fG° (CO3•-) ) -383 kJ/ mol that was recommended by Stanbury (28) before better experimental data became available. One must also note that the value for pKa,HCO3• has been revised recently and it is now known that HCO3• is a strong acid with pKa < 0 (29). Thus, HCO3• is essentially fully ionized to CO3•- at and near physiological pH, and we need not concern ourselves with the chemistry of HCO3•. We use ∆fG° (‚NO2, aq) ) 63 kJ/mol, as recommended by Stanbury (28). Selected values of ∆fG° for key species are given in Table 1. 2.2. Energetics of the Reaction of Peroxynitrite with CO2 and Properties of Reactive Species. A key 3 Huie et al. reported ∆ G° (CO •-) ) -371 kJ/mol in ref 27; however, f 3 this value they reported for ∆fG° (CO3•-) is not consistent with values for E° (CO3•-/CO32-) ) 1.59 V and ∆fG° (CO32-) ) -527.81 kJ/mol from which ∆fG° (CO3•-) should have been derived. The correct value for ∆fG° (CO3•-) is ∆fG° (CO3•-) ) nFE° (CO3•-/CO32-) + ∆fG° (CO32-) ) (1)(96.485 kC/mol)(1.59 V) + (-527.81 kJ/mol) ) -374 kJ/mol.
species (aq)
∆fG° kJ/mol (kcal/mol)
refs
CO2 ONOOONOOH ‚NO2 CO3•NO3CO32ONOOCO2ONOOCO2H O2NOCO2O2NOCO2H
-385.97 (-92.25) 68.6 (16.4) 31.4 (7.5) 63 (15.1) -374 (-89.4) -111.25 (-26.59) -527.81 (-126.15) -307.7 (-73.5) -321.4 (-76.8) -442 (-105.6) -449 (-107.3)
20 9, this work 9, this work 28 28, this work 74 20 this work this work this work this work
feature of the reaction mechanism depicted in Scheme 1 is the formation of ‚NO2 and CO3•- according to reaction 3.
ONOO-(aq) + CO2 (aq) ) ‚NO2 (aq) + CO3•-(aq) (3) As we will review below, these free radicals have important implications in biological oxidation and nitration, for example, in the nitration of tyrosine residues in proteins and of guanine residues in DNA (30, 31). Using the data in Table 1, one can calculate ∆G° (reaction 3) ) 6.4 kJ/mol. Thus, this thermodynamic calculation indicates reaction 3 is only slightly endergonic, suggesting the facile formation of the free radicals ‚NO2 and CO3•-. The mechanism shown in Scheme 1 has been proposed by leading research groups in this field (14, 15, 19, 32) and is consistent with these thermodynamic calculations as well as other data (vide infra). Reaction 3 occurs in two steps. The first step forms nitrosoperoxycarbonate (reaction 3a) and the second step (reaction 3b) yields the free radicals ‚NO2 and CO3•- from the homolysis of the peroxide bond in nitrosoperoxycarbonate (ONO-OCO2-).
CO2(aq) + ONOO-(aq) ) ONOOCO2- (aq) (3a) ONOOCO2- (aq) ) ‚NO2 (aq) + CO3•- (aq) (3b) Nitrosoperoxycarbonate is the first intermediate in the reaction of peroxynitrite with CO2 and reaction 3a is believed to be the rate-determining step. The Gibbs energy of activation for reaction 3a (50 kJ/mol) can be calculated by applying a minor correction4 to data reported by Denicola et al. (33) in order to compensate for the acid dissociation of ONOOH [∆dissH° ) 11 ( 4 kJ/ mol (34, 35)] because these authors assumed ∆dissH° ) 0 when ∆dissH° was not known with sufficient accuracy and precision. 2.2.1. Estimation of ∆G° for Reaction 3a. One can estimate ∆G° (reaction 3a) using linear free energy relationships with related chemical reactions. A group of reactions that are related to reaction 3a is the formation of alkyl monocarbonates (ROCO2-) from the reaction of alkoxides with CO2 (reaction 4). 4 This correction is necessary because the apparent second-order rate constant for the reaction of peroxynitrite with bicarbonate depends on the pKas of CO2 and ONOOH and both of these acid/base equilibria vary with temperature (the enthalpies of the acid-base equilibria are different than zero). The correction was applied to data shown in ref 33, Figure 2A in order to extract pH-independent second-order rate constants at various temperatures and an Eyring plot of these rate constants afforded the free energy of activation (50 kJ/mol).
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RO-+ CO2 ) ROCO2-
Squadrito and Pryor
(4)
Sauers et al. (36) conducted a systematic study of the formation of ROCO2- in alcohol-water-bicarbonate systems. This study measured K4 for several alcohols by 1H NMR by integrating the resonances for the R-methylene protons in the alcohols and alkyl monocarbonates and observed a good correlation between log K4 and the pKa the corresponding alcohol (a Bronsted plot). Recently, Richardson et al. (37) studied the formation of peroxycarbonate (HOOCO2-) and alkyl monocarbonates in aqueous solutions containing hydrogen peroxide, [13C]bicarbonate and alcohols by 13C NMR (equilibrium constants were calculated by integration of the 13C resonances of the carbonate carbon atoms). Richardson et al. (37) report equilibrium constants for reaction 5 (R ) OH or C2H5). -
-
ROH + HCO3 ) ROCO2 + H2O
(5)
Using data reported by Richardson et al., one can calculate K4 for ethanol and hydrogen peroxide with knowledge of the pKa values for ethanol and hydrogen peroxide (16 and 11.65, respectively) and the equilibrium constant for reaction 6 (K6 ) 4.3 × 10-7 M).
CO2 + H2O ) HCO3- + H+
(6)
Notably, the value of K4 for ethanol that can be calculated from data by Richardson et al. (K4,C2H5OH ) 3.9 × 108 M-1)5 is in good agreement with the value reported by Sauers et al. (K4,C2H5OH ) 1.47 × 108 M-1). For hydrogen peroxide, Richardson et al. report K5,H2O2 ) 0.32 ( 0.02 M-1 from which one can calculate K4,H2O2 ) 6.1 × 104 M-1. Data from Sauers et al. (36) and values we calculated from data reported by Richardson et al. (37) are shown as a Bronsted plot in Figure 2. The solid line is described by the relationship: log K4,ROH ) 1.0 × pKa,ROH - 8.3 which indicates that the formation of monocarbonates is increasingly favored with increasing basicity of RO- (or higher pKa for ROH). Using pKa,ONOOH ) 6.6, one calculates log K3a ) -1.7, K3a ) 0.020 M-1 and ∆G° (reaction 3a) ) 9.7 kJ/mol. The data for this plot are given in Table 2. The value of 1.0 for the slope of the straight line in Figure 2 (which is βeq in standard Bronsted-type plots) relates to the change in formal charge on the nucleophile oxygen atom for the entire reaction 4 which is also 1.0. This indicates that substituents affect the stability of the monocarbonates and free alcohols to a similar extent. Sauers et al. (36) also measured the rate constant k5 for several alkoxides and observed that k4 varied only slightly despite the fact that the pKa of the corresponding alcohols spanned 2.55 pKa units (Table 3). These investigators interpreted this behavior as reflecting an early transition state for these reactions in which there is only slight distortion of the linear structure of carbon dioxide. Remarkably, despite the fact that the pKa,ONOOH is 6.6, 5 K 8 -1 is an approximate value we have 4,C2H5OH ) 3.9 × 10 M calculated from K5,C2H5OH ) 1.8 reported by Richardson et al. in ref 35 for [H2O] ) 19.6 M, and using the pKa values for ethanol and hydrogen peroxide (16 and 11.65, respectively), and K6 ) 4.3 × 10-7 M.
Figure 2. Bronsted plot for the addition of CO2 to alkoxides to form alkyl monocarbonates (K4). For this plot, the standard error in the slope is 0.15 (or 14%), 2.1 (or 25%) in the intercept, and r2 ) 0.89. Table 2. Values of log K4,ROH and pKa,ROH Plotted in Figure 2 ROH
log K4,ROH
pKa,ROH
refs
CH3CH2OH CH3CH2OH CH3OH CH3OCH2CH2OH HCCCH2OH H2O F3CCH2OH H2O2
8.17 8.59 8.01 6.96 5.16 7.65 3.55 4.79
16 16 15.54 14.82 13.55 15.7 12.43 11.65
36 37, this work 36 36 36 36 36 37, 55, this work
Table 3. Rate Constants (k4) for the Nucleophilic Attack by Some Alkoxides and Peroxynitrite to Carbon Dioxide nucleophile
k4 (M-1s-1)
pKa
T (°C)
CH3OCH3CH2OCH3OCH2CH2OHCCCH2OONOO-
1.68 × 105 1.27 × 105 6.01 × 104 4.35 × 104 3 × 104
15.54 16 14.82 13.55 6.6
25 25 25 25 24
refs 36 36 36 36 17
k4 for peroxynitrite (also k3a) is 3 × 104 M-1 s-1,6 a value of magnitude comparable to those observed for the alkoxides in Table 3 that have pKa values in the range 13.55-16. The values in Table 3 are plotted as a Bronsted plot in Figure 3. The slope of the straight line (βnuc in standard Bronsted plots) is 0.06, indicating that an early transition state also occurs in the reaction of peroxynitrite with carbon dioxide. 2.2.2. Homolysis of ONO-OCO2- (Reaction 3b). The ∆fG° (ONOOCO2-, aq) can be calculated from ∆fG° (ONOOCO2-, aq) ) ∆G° (reaction 3a) + ∆fG° (CO2, aq) + ∆fG° (ONOO-, aq) ) 9.7 kJ/mol + (-385.97 kJ/mol) + 68.6 kJ/mol ) -307.7 kJ/mol. ∆G° (reaction 3b) can then be calculated from ∆G° (reaction 3b) ) ∆fG° (‚NO2, aq) + ∆fG° (CO3•-, aq) - ∆fG° (ONOOCO2-, aq) ) 63 kJ/mol + (-374 kJ/mol) - (-307.7 kJ/mol) ) - 3.3 kJ/mol. The low value of ∆G° (reaction 3b) indicates that the peroxide 6 The value of 3 × 104 M-1 s-1 is actually the experimental pHindependent rate constant for the overall reaction of peroxynitrite with CO2 at 25 °C. This value is given in ref 17 and also can be obtained from ref 33, Figure 1 or Figure 2A (but see also footnote 4). This value (3 × 104 M-1 s-1) is very close to k3a, because, in this reaction, the intermediate decomposes to products much faster than it can revert to the starting materials.
Perspective
Chem. Res. Toxicol., Vol. 15, No. 7, 2002 889 Table 4. pKa Values for Nitrosoperoxycarbonic Acid, Nitrocarbonic Acid, and Structurally Related Compounds
Figure 3. Bronsted plot for the addition of CO2 to alkoxides to form alkyl monocarbonates (k4). For this plot, the standard error in the slope is 0.03 (or 43%), 0.4 (or 10%) in the intercept, and r2 ) 0.63.
bond in ONO-OCO2- is extremely labile, and consequently, this species will be short-lived. For comparison, the bond dissociation energies of dialkyl peroxides is typically in the 100-130 kJ/mol range (38). Theoretical calculations had also indicated that the peroxide bond in ONOOCO2- is exceptionally weak (39). This has been confirmed experimentally; ONO-OCO2- does not accumulate during the reaction of ONOO- with CO2 and cannot be detected spectrophotometrically (2, 12, 17, 40).7 Furthermore, ONOOCO2- has not been chemically trapped or scavenged (2, 41). Having estimated K3a ) 0.020 M-1, and knowing that k3a ) 3 × 104 M-1 s-1 one can calculate k-3a from k-3a ) k3a/K3a ) (3 × 104 M-1 s-1)/(0.020 M-1) ) 1.5 × 106 s-1. Since ONOOCO2- does not accumulate during the reaction of ONOO- with CO2, k3b must be larger than k-3a. Even though ONOOCO2- cannot be observed due to its extremely low concentration during the time frame for the decay of peroxynitrite, the stopped-flow experiments provide limiting, indirect information about ONOOCO2-. An upper limit to the lifetime of ONOOCO2is imposed by the dead time (typically e 1 ms) of the stopped-flow instrument (12, 40), but the lifetime of ONOOCO2- is really much shorter than 1 ms. Additionally, when substrates such as phenol (42) or tyrosine (43) are used as probes to scavenge intermediates that are formed during the CO2-catalyzed decomposition of peroxynitrite, the formation of nitration products is always as fast as the disappearance of peroxynitrite. This suggests that the rate of homolysis of ONOOCO2- via forward reaction 3b is nearly as fast as the formation of ONOOCO2- via forward reaction 3a.8 The rate constant k-3b is likely around 5 × 108 M-1 s-1, the value that has been determined for the overall rate constant for the reaction between ‚NO2 and CO3•(27). [We will discuss this important point further in 7 Goldstein et al. recently demonstrated in this journal (ref 40) that the claim from Meli et al. (ref 44) for observing ONOOCO2- was actually due to an experimental artifact. 8 Another limit imposed from stopped-flow experiments is that the rate of decay ONOOCO2- approaches its rate of formation; however, since the formation of ONOOCO2- is bimolecular, an estimation of the lifetime of ONOOCO2- based on its rate of formation would depend on the initial conditions, and we chose to not elaborate further on this variable limitation to the lifetime.
substance
pKa
refs
HOH HOOH ONOOH O2NOOH CH3-O-CO2H CH3-CH2-CO2H CH3-CH2-CH2-CO2H CH3-CO2H HO-CH2-CO2H O2N-CH2-CH2-CO2H HCO2H CH3-O-CH2-CO2H HO-CO2H HO-O-CO2H ONO-O-CO2H O2N-CH2-CO2H O2N-O-CO2H •O-CO H 2
15.74 11.65 6.6 5.9 5.61 4.874 4.817 4.765 3.831 3.81 3.751 3.57 3.5 2.9 2.4 1.68 1.2
