with Guanine - ACS Publications - American Chemical Society

J. Phys. Chem. B 2008, 112, 4779-4789

4779

Catalytic Involvement of CO2 in the Mutagenesis Caused by Reactions of ONOO- with Guanine P. K. Shukla and P. C. Mishra* Department of Physics, Banaras Hindu UniVersity, Varanasi 221 005, India ReceiVed: October 29, 2007; In Final Form: December 29, 2007

The catalytic role of CO2 in reactions of ONOO- with guanine, leading to the formation of the mutagenic species 8-oxoguanine (8-oxoG) and 8-nitroguanine anion (8-nitroG-), was investigated by considering the reactions of nitrosoperoxycarbonate anion (ONOOCO2-), an adduct of ONOO- and CO2, with guanine at the B3LYP/6-31G** and B3LYP/AUG-cc-pVDZ levels of density functional theory in gas phase. In order to study bulk solvent effect, single-point energy calculations in aqueous media were carried out for all the species occurring in the reactions at the B3LYP/AUG-cc-pVDZ level of theory, by use of the polarizable continuum model (PCM). Vibrational frequency analysis was performed, and zero-point-energy (ZPE)-corrected total energies and Gibbs free energy changes at 298.15 K were obtained. The genuineness of the calculated transition states was confirmed by visually examining the vibrational modes and also by intrinsic reaction coordinate (IRC) calculations. The reaction between ONOOCO2- and guanine occurring through four different mechanisms leads to the formation of 8-oxoG or its anion, while the reaction between the same two species occurring through a different scheme leads to the formation of 8-nitroG-. It has been shown that the presence of a water molecule along with ONOOCO2- would not affect the reaction mechanisms significantly. Structures of the reactant complexes, product complexes and barrier energies involved in the reactions reveal that CO2 acts as a catalyst for the reaction between ONOO- and guanine. The cause of the catalytic action of CO2 is mainly due to intermediacy of the CO3 radical anion and NO2 radical into which ONOOCO2- is fragmented while reacting with guanine. The relative stabilities of the different product complexes suggest that the mutation caused by ONOO- in the presence of CO2 would mainly involve 8-oxoG.

1. Introduction Peroxynitrite anion (ONOO-) is a reactive nitrogen oxide species (RNOS) that plays a prominent role in oxidation and nitration reactions involving biomolecules.1-3 The reaction between two fairly unreactive species, nitric oxide radical and superoxide radical anion, produced by the inflammatory cells, macrophages, and neutrophils, leads to the formation of peroxynitrite in vivo.4,5 Due to its oxidizing and nitrating capability, peroxynitrite has been shown to be a powerful cytotoxic and mutagenic agent in several bacterial and mammalian cell culture systems.2,3 Peroxynitrite has also been implicated in an increasing number of diseases including atherosclerosis, inflammation, and neurodegenerative diseases.6,7 Peroxynitrite is capable of reacting with almost all classes of biomolecules including DNA, proteins, and lipids under physiological conditions.3,8-10 In DNA, ONOO- primarily reacts with guanine, leading to the formation of 8-oxoguanine (8-oxoG) and 8-nitroguanine (8nitroG).11-16 Recently in our laboratory, the reactions of ONOO- with guanine leading to the formation of 8-oxoG and 8-nitroG were theoretically investigated and several barrier energies were found to be quite high.17 Formation of 8-oxoG in vivo leads to various deleterious consequences on cell functioning including mutation, aging, and cancer.18 8-OxoG readily mispairs with adenine during DNA replication, which causes GC f AT transversion mutation.19 Also, 8-oxoG is quite susceptible to further reactions with ONOO- that produce other highly mutagenic and carcinogenic * Corresponding author: e-mail [email protected] or pcmishra_in@ yahoo.com.

products.2,20 Formation of 8-nitroG can initiate different types of mutation, inflammation, and cancer.21 It has been observed that rate constants, product yields, and product distribution of the reactions of ONOO- with different biomolecules including DNA are strongly affected by the presence of carbon dioxide or bicarbonate.22-26 In this context, it is an important fact that the concentration of CO2 is very high in vivo.1 The reaction of ONOO- with CO2 is faster than any other reaction involving the former species.23-30 This reaction has been extensively studied and thus it is established that it leads to the formation of an intermediate species called nitrosoperoxycarbonate anion (ONOOCO2-) that has a very high reactivity with biological materials.26-32 The reaction leading to the formation of ONOOCO2- is found to be barrierless in gas phase, but it has a significant barrier energy (∼12 kcal/ mol) in aqueous media.32-34 The species ONOOCO2- is a powerful oxidizing and nitrating agent and can damage a wide variety of biomolecules including DNA and proteins.22-30,35,36 It has been suggested that ONOOCO2- can be a source for several reactive intermediates, including the carbonate radical anion and nitrogen dioxide radical, that may be formed via homolysis of the peroxo O-O bond of ONOOCO2-.1,22,27,30-32,37 The carbonate radical anion produced by the reaction between peroxynitrite and carbon dioxide has been detected in electron paramagnetic resonance (EPR) experiments.38 The carbonate radical anion and nitrogen dioxide radical are believed to be involved in oxidation and nitration of DNA when it is exposed to ONOO- in the presence of CO2.1,22,27,30-32,37-39 In view of the fact that both ONOO- and CO2 may be present in biological systems where these can readily combine to form ONOOCO2-, a study of the reaction of ONOOCO2- with

10.1021/jp710418b CCC: $40.75 © 2008 American Chemical Society Published on Web 03/27/2008

4780 J. Phys. Chem. B, Vol. 112, No. 15, 2008 different biomolecules, including the DNA bases, is of great importance. For this reason, we have studied theoretically the reaction of ONOOCO2-at the C8 site of guanine leading to the formation of 8-oxoG and 8-nitroG. The results of this work are compared with those obtained from the study of the reaction of guanine with ONOO-. It brings out the importance of CO2 in the above-mentioned reactions. To the best of our knowledge, no previous theoretical study of these aspects has been reported. 2. Computational Details Geometries of the cis and trans conformers of ONOOCO2and guanine were fully optimized in gas phase at the B3LYP/ 6-31G** and B3LYP/AUG-cc-pVDZ levels of density functional theory (DFT).40,41 Reactant, intermediate, and product complexes as well as transition states involved in the reactions of ONOOCO2- with guanine were also fully optimized in gas phase at the above-mentioned levels of theory. However, transition states TS2, TS3, and TS8 could be optimized only at the B3LYP/6-31G** level of theory. Therefore, single-point energy calculations at the B3LYP/AUG-cc-pVDZ level of theory were performed for these transition states by use of the B3LYP/ 6-31G** optimized geometries. Single-point energy calculations in aqueous media were carried out for all the optimized species at the B3LYP/AUG-cc-pVDZ level of theory by use of the polarizable continuum model (PCM) of the self-consistent reaction field (SCRF) theory,42,43 as implemented in the Gaussian98 program,44 in order to study bulk solvent effect. It was considered possible that a water molecule might compete with ONOOCO2- in hydrogen bonding with guanine at the reactant complex and transition states, thus affecting the overall reaction mechanisms. To study this aspect, both ONOOCO2- and a water molecule were considered to be interacting with guanine at the reactant complex. Further, two main reaction stepssone corresponding to oxidation and the other corresponding to nitration of guanineswere also studied, including the presence of a water molecule. Electrostatic potential-fitted point charges located at the atomic sites were calculated by use of the CHelpG algorithm45 at the B3LYP/AUG-cc-pVDZ level of theory in aqueous media. Vibrational frequency analysis was performed for all the optimized structures to ensure that each total energy minimum had all real frequencies and each transition state had only one imaginary frequency. The genuineness of each calculated transition state was confirmed by visually examining the vibrational mode corresponding to the imaginary frequency and applying the condition that it connected the reactant and product complexes properly. Intrinsic reaction coordinate (IRC)46 calculations were also performed at the B3LYP/6-31G** level of theory in gas phase to further test the genuineness of all the optimized transition states. Zero-point-energy (ZPE)-corrected total energies and Gibbs free energies at 298.15 K were obtained for all the optimized species at the B3LYP/6-31G** and B3LYP/AUG-cc-pVDZ levels of theory in gas phase. Zero-point energy correction to the total energy and thermal energy correction to Gibbs free energy obtained at the B3LYP/AUGcc-pVDZ level of theory in gas phase were also considered to be valid for the corresponding results obtained in aqueous media. All the calculations were performed with the Windows versions of the Gaussian98 (G98W)44 and Gaussian03 (G03W)47 programs. The GaussView program48 was used for visualization of structures and vibrational modes. 3. Results and Discussion 3.1. Structures of ONOOCO2- and Its Complexes with Guanine. As we are interested only in the reactions occurring

Shukla and Mishra at the C8 site of guanine, only those reactant complexes were considered where ONOOCO2- was located near the C8 and N9 sites of guanine. Thus, we obtained seven different reactant complexes (RCi, i ) 1-7) at the B3LYP/AUG-cc-pVDZ level of theory in gas phase. Optimized structures of cis and trans conformers of ONOOCO2- and its reactant complexes with guanine (RCi, i ) 1-7) along with their ZPE-corrected total energies and values of certain optimized geometrical parameters, obtained at the B3LYP/AUG-cc-pVDZ level of theory in gas phase, are presented in Figure 1. The atomic numbering scheme adopted here is also shown in this figure. The cis conformer of ONOOCO2- was found to be more stable at the B3LYP/AUGcc-pVDZ level of theory in gas phase than the trans conformer by ∼1 kcal/mol (Figure 1a,b), the ZPE-corrected binding energy of the former in gas phase being -15.5 kcal/mol. The reactant complexes (RCi, i ) 1-7) were stabilized by hydrogen bonding between the H9 atom of guanine and the O10, O13, or O16 atom of ONOOCO2- (Figure 1). The hydrogen bonds between the H9 and O10 atoms of RCi (i ) 1-7) (Figure 1c-g) were found to be stronger (distances 1.65-1.67 Å) than those between the H9 and O13 or O16 atoms (distances ∼1.80 Å) (Figure 1h,i). This may be explained as due to a greater negative CHelpG charge (in the unit of magnitude of electronic charge) on the O10 atom (∼ -0.7) than those on the O13 atom (∼ -0.3) or O16 atom (∼ -0.1) of ONOOCO2-. Of the seven reactant complexes, RC1 (Figure 1c) is most stable and RC6 (Figure 1i) is least stable: the difference between their ZPE-corrected total energies in gas phase at the B3LYP/AUG-cc-pVDZ level of theory is ∼10.7 kcal/mol. Therefore, we will consider only the reactant complex RC1 for further study of the reactions leading to the formation of 8-oxoG and 8-nitroG. 3.2. Formation of 8-Oxoguanine and Its Anionic Tautomers. Starting with the reactant complex (RC1) of guanine with ONOOCO2- (Figure 1c), 8-oxoG and its anionic tautomers [8-oxoG-(-H7), 8-oxoG-(-H9)] complexed with other species can be obtained in four different ways as shown in Figures 2-5. ZPE-corrected barrier and released energies (in kilocalories per mole) and net CHelpG charges in aqueous media, along with certain optimized geometrical parameters of the transition states, obtained at the B3LYP/AUG-cc-pVDZ level of theory in gas phase are presented in the different figures. The barrier and released energies and the corresponding Gibbs free energy changes (in kilocalories per mole) involved in the various reaction steps of Figures 2-6 obtained at the different levels of theory in gas phase and aqueous media are presented in Tables 1-5. Figure 2 involves the reactant complex RC1, intermediate complex IC1, transition states TS1 and TS2, and the product complex PC1. PC1 is a complex of 8-oxoG- anion with the proton absent from the N7 site [8-oxoG-(-H7)], HNO2, and CO2 (Figure 2). The CHelpG charges associated with 8-oxoG-(-H7), HNO2, and CO2 moieties of PC1 were found to be -0.7, -0.3, and 0.0, respectively. Figure 3 shows how the final product complex PC2 (8-oxoG + NO2- + CO2) can be obtained, starting from the intermediate complex IC1 found in Figure 2 through two other transition states TS3 and TS4 and intermediate complexes IC2 and IC3. The intermediate complex IC3 is a complex of 8-oxoG- where the H9 proton is absent from its site [8-oxoG-(-H9)], HNO2, and CO2, and it was found to be about 4.8 kcal/mol less stable than PC2 at the B3LYP/ AUG-cc-pVDZ level of theory in aqueous media (Figure 3). Also, no energy barrier could be located between IC3 and PC2. Thus, IC3 would be readily converted to PC2. The CHelpG charges associated with 8-oxoG-(-H9), HNO2, and CO2

Catalytic Role of CO2 in ONOO- Reaction with Guanine

J. Phys. Chem. B, Vol. 112, No. 15, 2008 4781

Figure 1. Structures of (a) cis- and (b) trans-nitrosoperoxycarbonate anion (ONOOCO2-) with atomic numbering scheme and (c-i) different reactant complexes obtained in the reaction of ONOOCO2- with guanine at the B3LYP/AUG-cc-pVDZ level of theory in gas phase. ZPE-corrected total energies (au) of the different species or complexes are given in parentheses. Certain optimized geometrical parameters (bond lengths in angstroms, bond angles in degrees) are also given.

moieties of IC3 were found to be -0.9, -0.1, and 0.0, respectively. Formation of the product complex PC2 starting from the intermediate complex IC2 found in Figure 3 can also take place via two other transition states TS5 and TS6 and intermediate complexes IC3 and IC4 as shown in Figure 4). The intermediate complex IC2 can also be obtained directly from RC1 through a single transition state TS7 as shown in Figure 5). As RC1 and TS1 are common to Figures 2 and 3, these are shown only in Figure 2). Similarly, as IC1 and TS3 are common to Figures 3 and 4, these are shown only in Figure 3. In RC1, a small negative charge (∼ -0.1) is transferred from ONOOCO2- to guanine (Figure 2). In going from RC1 to TS1, the following structural changes take place. The O13-O14 bond of ONOOCO2- is almost dissociated, the distance between the two oxygen atoms being 2.23 Å. Thus at TS1, CO3 and NO2 groups are ready to be formed, and the O12 oxygen atom of the CO3 group has moved close to the C8 site of guanine. The

CHelpG charges associated with the CO3, NO2, and guanine moieties at TS1 are found to be -1.0, -0.5, and 0.5, respectively (Figure 2). Thus at TS1, ONOOCO2- dissociates into the CO3 radical anion and NO2 radical-like species. This finding supports the previous theoretical and experimental proposals, according to which homolysis of the OO bond of ONOOCO2- leads to formation of CO3 radical anion and NO2 radical.1,22,27,30-32,37,38 The barrier energy involved at this step of the reaction of Figure 2 (∆Eb1) was found to be 35.5 kcal/mol at the B3LYP/AUGcc-pVDZ level of theory in gas phase, while the corresponding ∆Gb1 value was found to be 35.9 kcal/mol (Table 1). The values of ∆Eb1 and ∆Gb1 are drastically lowered down to 18.9 and 19.3 kcal/mol, respectively, in aqueous media (Figure 2, Table 1). The minimum ZPE-corrected barrier energy involved in the dissociation of ONOO- while reacting with guanine to produce 8-oxoG (first step of the reaction) at the B3LYP/AUG-cc-pVDZ

4782 J. Phys. Chem. B, Vol. 112, No. 15, 2008

Shukla and Mishra

Figure 2. Reactant complex (RC1), intermediate complex (IC1), product complex (PC1), and transition states (TS1 and TS2) involved in the reaction of ONOOCO2- with guanine. ZPE-corrected barrier and released energies (kilocalories per mole) and CHelpG charges associated with different moieties (in brackets) (in the unit of magnitude of electronic charge) obtained at the B3LYP/AUG-cc-pVDZ level of theory in aqueous media are given. Certain optimized geometrical parameters (bond lengths in angstroms, bond angles in degrees) of transition states are also given. The locations of different structures in terms of energy values are not to scale.

level of theory was found to be 37.00 kcal/mol in aqueous media.17 The corresponding barrier energy of the reaction between ONOOCO2- and guanine in aqueous media is almost half (18.9 kcal/mol) that between ONOO- and guanine. Thus, ONOOCO2- is much more reactive with guanine than ONOO-. At IC1, the O12 atom of the CO3 group has moved and is attached to the C8 site of guanine, producing Fapyguanine (FapyG) complexed with the CO2 and NO2 moieties. The CHelpG charges located on the FapyG, CO2, and NO2 moieties of IC1 were found to be -0.1, 0.0, and -0.9, respectively. Thus,

here the NO2 group looks like a closed-shell anion. In going from IC1 to TS2, the H8 atom that was bonded to the C8 site of FapyG moves and becomes attached to the N15 atom of the NO2 group, thus producing the product complex PC1. ZPEcorrected barrier energy for this step of the reaction (∆Eb3) and the corresponding ∆Gb3 value, at the B3LYP/AUG-cc-pVDZ level of theory in gas phase, were found to be 4.6 and 7.1 kcal/ mol, respectively (Table 1). However, in aqueous media, the values of ∆Eb3 and ∆Gb3 were found to be appreciably enhanced to 10.2 and 12.9 kcal/mol, respectively (Table 1). The barrier



Figure 3. Formation of product complex (PC2) after the intermediate complex (IC1) of Figure 2 through two other transition states (TS3 and TS4) and intermediate complexes IC2 and IC3. ZPE-corrected barrier and released energies (kilocalories per mole) and CHelpG charges associated with the different moieties (in brackets) (in the unit of magnitude of electronic charge) obtained at the B3LYP/AUG-cc-pVDZ level of theory in aqueous media are given. Certain optimized geometrical parameters (bond lengths in angstroms, bond angles in degrees) of transition states are also given. The locations of different structures in terms of energy values are not to scale.

and released energies obtained at the different levels of theory in gas phase and aqueous media (Table 1) show that the reaction that leads to the formation of PC1 (Figure 2) is highly exothermic. In Figure 3, in going from IC1 to IC2 through TS3, the following structural changes take place. The H9 atom bonded to the N9 site of FapyG in IC1 moves and becomes attached to the O16 atom of the NO2 moiety, the N9-C8 distance is reduced by 0.58 Å to 2.04 Å, and the CO2 moiety moves close to the O12 atom. The barrier energy involved in this process (∆Eb5) at the B3LYP/AUG-cc-pVDZ level of theory in gas phase was found to be 8.8 kcal/mol, the corresponding ∆Gb5 value being 11.6 kcal/mol (Table 2). In aqueous media, the values of ∆Eb5 and ∆Gb5 are enhanced to 17.4 and 20.2 kcal/mol, respectively (Table 2). In IC2, the CO2 moiety is weakly bonded to the O12 atom that is already attached to the C8 site of guanine, the C11O12 distance being 1.52 Å (Figure 3). Further in this case, the

N9-H9 hydrogen-bonding distance is 1.72 Å (Figure 3). Figures 3 and 4 are different only in terms of how the H8 atom attached to the C8 site of guanine moves and becomes bonded to the N7 site, leading to the formation of IC3. In going from IC2 to TS4, the H8 atom moves away from the C8 site to near the N7 site and the C11-O12 distance is increased to 2.49 Å, thus producing the intermediate complex IC3 (Figure 3). The barrier energy for this reaction step (∆Eb7) at the B3LYP/AUG-ccpVDZ level of theory in gas phase was calculated to be 8.8 kcal/mol (Table 2). Its value was found to be significantly enhanced in aqueous media to 18.5 kcal/mol. The corresponding ∆Gb7 value was found to be lower than that of ∆Eb7 by ∼2.1 kcal/mol in both gas phase and aqueous media (Table 2). In IC3, the H9 atom of the HNO2 moiety is involved in a strong hydrogen bond with the N9 site of guanine, the hydrogenbonding distance being 1.51 Å, and the O16-H9 bond of the HNO2 moiety is elongated to 1.09 Å. It appears that IC3 would


Shukla and Mishra

Figure 4. Formation of product complex PC2 after the intermediate complex (IC2) of Figure 3 through two other transition states (TS5 and TS6) and other intermediate complexes IC3 and IC4. ZPE-corrected barrier and released energies (kilocalories per mole) and CHelpG charges associated with the different moieties (in brackets) (in the unit of magnitude of electronic charge) obtained at the B3LYP/AUG-cc-pVDZ level of theory in aqueous media are given. Certain optimized geometrical parameters (bond lengths in angstroms, bond angles in degrees) of transition states are also given. The locations of different structures in terms of energy values are not to scale.

be converted barrierlessly into the product complex PC2, as no transition state could be located between IC3 and PC2 at any level of theory. The product complex PC2 at the B3LYP/AUGcc-pVDZ level of theory was found to be more stable than IC3 by about 0.5 and 4.8 kcal/mol in gas phase and aqueous media, respectively. Figure 4 shows how, in going from IC2 to IC4 through TS5, the H8 atom moves from the C8 site of guanine toward the O10 atom of the CO2 group, eventually becoming bonded to it. In IC4, an HCO3 group is attached to the C8 site of guanine(-H9) radical while the H9 atom of the HNO2 moiety is involved in a strong hydrogen bond with the N9 site of it, the hydrogen-bonding distance being 1.63 Å. The calculated barrier energy between IC2 and IC4 corresponding to transition state

TS5 at the B3LYP/AUG-cc-pVDZ level of theory in gas phase was found to be negative (∆Eb9) -0.5 kcal/mol), while a low barrier energy (3.4 kcal/mol) was found for the same step in aqueous media (Table 3). A negative barrier energy, in general, would indicate a barrierless reaction. The changes in Gibbs free energy (∆Gb9) were found to be greater by ∼1.0 kcal/mol in both gas phase and aqueous media than those of ∆Eb9 (Table 3). The intermediate complex IC4 is converted barrierlessly into the intermediate complex IC3 by transferring the H8 atom attached to the O10 atom to the N7 site of guanine (Figure 4), the calculated barrier energy for this reaction step (∆Eb11) being -0.8 kcal/mol in gas phase and -0.6 kcal/mol in aqueous media (Table 3). The ∆Gb11 magnitudes obtained in both gas phase and aqueous media corresponding to those of the barrier energy


J. Phys. Chem. B, Vol. 112, No. 15, 2008 4785 TABLE 2: Energies Involved in the Reaction of ONOOCO2- with Guanine According to Figure 3 gas phase

aqueous media

energies,a kcal/mol

B3LYP/ 6-31G**

B3LYP/ AUG-cc-pVDZ

B3LYP/ AUG-cc-pVDZ

∆Eb5 ∆Gb5 ∆Er6 ∆Gr6 ∆Eb7 ∆Gb7 ∆Er8 ∆Gr8

4.7 6.1 -9.6 -7.4 11.4 9.9 -65.0 -65.4

8.8 11.6 -9.3 -7.1 8.8 6.7 -65.2 -65.0

17.4 20.2 -20.6 -18.4 18.5 16.4 -57.8 -57.6

a ∆Eb, ZPE-corrected barrier energy; ∆Er, ZPE-corrected released energy; ∆Gb and ∆Gr, corresponding Gibbs free energy changes at 298.15 K. See Figure 3 for definition of the different energies.

TABLE 3: Energies Involved in the Reaction of ONOOCO2- with Guanine According to Figure 4 gas phase

aqueous media

energies,a kcal/mol

B3LYP/ 6-31G**

B3LYP/ AUG-cc-pVDZ

B3LYP/ AUG-cc-pVDZ


0.3 1.4 -43.4 -44.0 -0.7 -0.7 -9.8 -12.2

-0.5 0.5 -42.1 -42.5 -0.8 -0.6 -13.0 -15.7

3.4 4.4 -26.4 -26.9 -0.6 -0.4 -15.6 -18.4


Figure 5. Formation of the intermediate complex (IC2) involved in the reaction of ONOOCO2- with guanine from the reactant complex (RC1) in a single step through the transition states (TS7). ZPE-corrected barrier and released energies (kilocalories per mole) and CHelpG charges associated with the different moieties (in brackets) (in the unit of magnitude of electronic charge) obtained at the B3LYP/AUG-ccpVDZ level of theory in aqueous media are given. Certain optimized geometrical parameters (bond lengths in angstroms, bond angles in degrees) of transition states are also given. The locations of different structures in terms of energy values are not to scale.

TABLE 1: Energies Involved in the Reaction of ONOOCO2- with Guanine According to Figure 2 gas phase energies,a

aqueous media

kcal/mol

B3LYP/ 6-31G**

B3LYP/ AUG-cc-pVDZ

B3LYP/ AUG-cc-pVDZ


37.9 38.3 -43.2 -47.6 1.4 3.4 -52.1 -51.4

35.5 35.9 -46.4 -51.8 4.6 7.1 -53.7 -52.9

18.9 19.3 -42.8 -48.3 10.2 12.9 -50.1 -49.3


∆Eb11 at the B3LYP/AUG-cc-pVDZ level of theory are smaller by ∼0.2 kcal/mol each (Table 3). Figure 5 shows that the intermediate complex IC2 can also be formed from RC1 through a single transition state TS7. In going from RC1 to TS7, the O13-O14 bond of the ONOOCO2moiety is broken. Thus, the CO3 and the NO2 groups are formed

TABLE 4: Energies Involved in the Reaction of ONOOCO2- with Guanine According to Figure 5 gas phase a

aqueous media

energies, kcal/mol

B3LYP/ 6-31G**

B3LYP/ AUG-cc-pVDZ

B3LYP/ AUG-cc-pVDZ

∆Eb13 ∆Gb13 ∆Er14 ∆Gr14

54.2 55.3 -64.3 -66.0

52.3 53.7 -64.1 -65.1

42.5 43.3 -69.6 -70.6


that are complexed with guanine. The CHelpG charges located on CO3, NO2, and guanine in TS7 were found to be -0.9, -0.3, and 0.2, respectively. Thus, the CO3 group at TS7 looks like the CO3 radical anion while the NO2 group looks like the NO2 radical. At TS7, the O12 atom of the CO3 group is weakly bonded to the C8 site of guanine. In going from TS7 to IC2, the O16 atom of the NO2 group abstracts the H9 atom of guanine (Figure 5). The barrier energy involved in this reaction step (∆Eb13) (52.3 kcal/mol in gas phase and 42.5 kcal/mol in aqueous media at the B3LYP/AUG-cc-pVDZ level) is highest among all the barrier energies found in this study (Tables 1-5). It shows that this would not be a favored scheme for the reaction. The Gibbs free energy changes (∆Gb13) were found to be slightly greater than those of the barrier energy ∆Eb13 (by ∼1.0 kcal/ mol) in both gas phase and aqueous media (Table 4). 3.3. Formation of 8-Nitroguanine. Figure 6 shows the mechanism of formation of 8-nitroguanine(-H9) anion complexed with H2CO3, denoted by PC3, starting from RC1 through two transition states TS8 and TS9 and a sole intermediate complex IC5. In going from RC1 to TS8, the O13-O14 bond


Shukla and Mishra

Figure 6. Reactant complex (RC1), intermediate complex (IC5), product complex (PC3), and transition states (TS8, TS9) involved in the reaction of ONOOCO2- with guanine. ZPE-corrected barrier and released energies (kilocalories per mole) and CHelpG charges associated with the different moieties (in brackets) (in the unit of magnitude of electronic charge) obtained at the B3LYP/AUG-cc-pVDZ level of theory in aqueous media are given. Certain optimized geometrical parameters (bond lengths in angstroms, bond angles in degrees) of transition states are also given. The locations of different structures in terms of energy values are not to scale.

of ONOOCO2- is broken and the NO2 moiety thus formed moves above the plane of guanine near the C8 site, leaving the CO3 moiety hydrogen-bonded to the H9 atom. After a minor geometry relaxation in TS8, the intermediate complex IC5 is formed (Figure 6). The CHelpG charges on the CO3, NO2, and guanine moieties of TS8 were found to be -1.0, -0.3, and 0.3, respectively. It reveals again the fact that ONOOCO2- is dissociated into CO3 radical anion and NO2 radical while reacting with guanine. The barrier energy for this step of the reaction (∆Eb15) was found to be 29.9 kcal/mol at the B3LYP/ AUG-cc-pVDZ level of theory in gas phase, while the corresponding barrier energy in aqueous media was found to be

significantly lowered, to 23.6 kcal/mol (Table 5). The changes in Gibbs free energy (∆Gb15) corresponding to the barrier energy ∆Eb15 were found to be smaller by 0.8 kcal/mol in both gas phase and aqueous media (Table 5). The calculated barrier energies involved in the first step of the nitration reaction of guanine with ONOO- at the B3LYP/ AUG-cc-pVDZ level of theory were found to be 49.79 and 43.39 kcal/mol in gas phase and aqueous media, respectively.17 Thus, in going from ONOO- to ONOOCO2-, the barrier energy involved in the first step of the nitration reaction of guanine is almost halved in both gas phase and aqueous media. It is clear that ONOOCO2- is a much better nitrating agent than ONOO-.



TABLE 5: Energies Involved in the Reaction of ONOOCO2- with Guanine According to Figure 6 gas phase

aqueous media

energies,a kcal/mol

B3LYP/ 6-31G**

B3LYP/ AUG-cc-pVDZ

B3LYP/ AUG-cc-pVDZ


32.1 30.9 -0.6 -2.1 -0.6 1.5 -81.3 -80.8

29.9 29.1 -3.1 -4.8 -0.2 2.3 -79.6 -78.9

23.6 22.8 -2.4 -4.0 -7.4 -4.9 -68.0 -67.3


In IC5, the NO2 moiety is placed above the C8 site of guanine, the C8-N15 distance being 2.66 Å, and the CO3 moiety is located in the plane of guanine and makes a strong hydrogen bond with H9, the O10-H9 hydrogen-bonding distance being 1.64 Å (Figure 6). The ∆Eb17 and ∆Gb17 values in aqueous media show that IC5 is readily converted into the product complex PC3 (8-nitroG- + H2CO3) through the transition state TS9 in a barrierless manner (Figure 6, Table 5). 3.4. Role of Specific Solvent Effect. Treatment of the bulk solvent effect by use of the polarizable continuum model42,43 does not include the possible effect of interaction of specific solvent molecules with the solute or reactant complex (specific solvent effect), which may play an important role. It may be argued that a specific water molecule may compete with ONOOCO2- in making a hydrogen bond with the H9 atom of guanine, thus weakening the hydrogen bond between the two moieties of RC1 (Figure 1c). If so, RC1 may not be properly defined and the various reaction schemes shown in the different figures may become questionable. Also the structures of the various transition states and hence the different barrier energies may be affected. For this reason, a water molecule was placed such that the H9 atom of guanine was involved in hydrogen bonding with it as well as ONOOCO2-, and then the structure of the whole complex was optimized in gas phase. We found two possible structures of RC1 in the presence of a water molecule that are denoted as RC1W and RC1W′ (Figure 7a,b). The structures of the transition states involved in the first step of each of the oxidation and nitration reactions (TS1 and TS8) were also optimized with a water molecule near the H9 atom of guanine, and the structures thus obtained are denoted as TS1W and TS8W (Figure 7c,d). The optimized structures of RC1W, RC1W′, TS1W, and TS8W were also solvated in bulk water by use of the polarizable continuum model.42,43 All these geometry optimization calculations were performed at both the B3LYP/6-31G** and B3LYP/AUG-cc-pVDZ levels of theory, while the solvation calculations were performed only at the B3LYP/AUG-cc-pVDZ level of theory. ZPE-corrected relative total energies of RC1W, RC1W′, TS1W, and TS8W in gas phase and aqueous media with respect to that of RC1W, along with the values of certain optimized interatomic distances and CHelpG charges associated with the different moieties, obtained at the B3LYP/AUG-cc-pVDZ level of theory are shown in Figure 7. The structures of the reactant complexes RC1W and RC1W′ are different only with regard to the position of the water molecule (Figure 7a,b). RC1W′ is more stable than RC1W by ∼0.2 kcal/mol in gas phase while the order of stability is reversed in aqueous media, the total energy difference being ∼1.9 kcal/mol (Figure 7a,b). Thus both

Figure 7. Optimized structures of (a, b)RC1W and RC1W′ and (c, d) transition states TS1W and TS8W at the B3LYP/AUG-cc-pVDZ level of theory in gas phase. ZPE-corrected relative total energies (kilocalories per mole) in gas phase and aqueous media (in parentheses) obtained at the B3LYP/AUG-cc-pVDZ level of theory with respect to that of reactant complex (RC1W) are given. Certain optimized interatomic distances (angstroms) in gas phase and CHelpG charges in aqueous media (in square brackets) are also given.

the structures RC1W and RC1W′ would coexist in gas phase while the former would be somewhat preferred over the latter in aqueous media. From the optimized structures, it is clear that the position of ONOOCO2- in RC1 (Figure 1c) is not affected significantly due to the presence of the water molecule (RC1W, RC1W′) (Figure 7a,b). The complex RC1W′ would not be involved in the reaction but may occur independently of it. In RC1 and RC1W, the O10-H9 hydrogen-bond distance is the same, 1.67 Å, and thus the presence of a water molecule near H9 in RC1 (Figure 1c) does not significantly affect the structure of the reactant complex. We also note that the presence of a water molecule does not affect the structures of the transition states significantly (Figures 2, 6, and 7c,d). The barrier energy involved at the first step of the reaction leading to the formation of 8-oxoG (∆Eb1) in gas phase is decreased from 35.5 to 34.5 kcal/mol, but in aqueous media it is significantly increased from 18.9 to 25.8 kcal/mol due to the presence of a water molecule (Table 1, Figure 7). Similarly, the barrier energy involved at the first step of the reaction leading to the formation of 8-nitroG- (∆Eb15) in gas phase is reduced from 29.9 to 28.5 kcal/mol, while in aqueous media it is increased from 23.6 to 25.1 kcal/mol due to the presence of a water molecule (Table 5, Figure 7). Thus, it seems that the specific solvent effect of water would affect the barrier energies to different extents in gas phase and aqueous media, but it would not affect the structure of RC1 and the different reaction mechanisms in a broad sense.


Shukla and Mishra

Figure 8. Summary of all the schemes of the reaction of ONOOCO2- with guanine studied here. ZPE-corrected relative total energies of all the stationary points occurring in the reaction obtained at the B3LYP/AUG-cc-pVDZ level of theory in aqueous media are given in parentheses with respect of that of the reactant complex RC1.

3.5. Summary of Reaction Schemes. A summary of all the schemes of reactions between guanine and ONOOCO2- discussed here is presented in Figure 8. The ZPE-corrected relative total energies (in kilocalories per mole) of all the stationary points involved in the reactions, calculated with respect to that of the reactant complex RC1 at the B3LYP/AUG-cc-pVDZ level of theory in aqueous media, are given in Figure 8. However, the relative total energy-wise locations of the different stationary points shown in this figure are only qualitative and not to scale. It is evident from Figure 8 that all the transition states, intermediate complexes, and product complexes involved in the formation of 8-oxoG, excepting TS1 and TS7, have lower energies with respect to that of RC1. The relative total energies given in Figure 8 show that the oxidation product PC2 is much more stable than the nitration product PC3. On the whole, Figure 8 shows that the yield of the oxidation product PC2 would be much more than that of nitration product PC3. 3.6. Biological Consequences. Structures of the product complexes show that free CO2 is liberated after 8-oxoG or its anion is formed, while it is liberated in the form of H2CO3 after 8-nitroG- is formed. Thus CO2 acts as a catalyst for the reactions

between ONOO- and guanine. The various barrier energies involved in the reactions of ONOO- with guanine, leading to the formation of 8-oxoG and 8-nitroG-, were found to be too large to allow these reactions to occur efficiently.17 However, peroxynitrite (ONOO-) anion is known to be a powerful agent that causes oxidation and nitration of guanine efficiently in biological media.1-3,11-16 These two contradictory findings can be reconciled in the present study, where it has been convincingly shown that CO2, which is present abundantly in biological media, would play an important catalytic role for reactions between ONOO- and guanine. Thus the barrier energies of the first steps of each of the reactions of ONOOCO2- with guanine leading to the formation of 8-oxoG and 8-nitroG- in aqueous media are almost half those of the corresponding steps of reactions between ONOO- and guanine. As the product complex involving 8-oxoG is appreciably more stable than that involving 8-nitroG-, ONOO- in the presence of CO2 would mainly act as an oxidizing agent for guanine. In other words, mutation would be caused by ONOO- in the presence of CO2 mainly through the formation of 8-oxoG.

Catalytic Role of CO2 in ONOO- Reaction with Guanine 4. Conclusions We arrive at the following conclusions from the present study: (i)Peroxynitrite(ONOO-)makesastablecomplex(ONOOCO2-) with CO2, the cis conformer of which is somewhat more stable than the trans conformer. Also, the cis conformer of ONOOCO2makes a more stable complex involving the H9 atom of guanine than the trans conformer. (ii) The reaction between ONOOCO2- and guanine through four different mechanisms (Figures 2-5) leads to the formation of 8-oxoG or its anion, while the reaction between the same two species through a different scheme (Figure 6) leads to the formation of 8-nitroG-. (iii) Structures of product complexes and barrier energies involved in the reactions of ONOOCO2- with guanine in the different schemes leading to the formation of 8-oxoG and 8-nitroG- reveal that CO2 acts as a catalyst for the reactions between ONOO- and guanine. While CO2 is present as such in the product complexes involving 8-oxoG, it occurs as H2CO3 in the product complexes involving 8-nitroG-. Due to dissociation of the O-O bond, ONOOCO2- is fragmented into the CO3 radical anion and NO2 radical while reacting with guanine. Thus the cause of the catalytic action of CO2 is mainly due to intermediacy of the CO3 radical anion and NO2 radical. (iv) The calculated total energies of the product complexes suggest that the yield of 8-oxoG would be much more than that of 8-nitroG-. Thus the mutation caused by ONOO- in the presence of CO2 would mainly occur through the formation of 8-oxoG. (v) The bulk solvent effect of water affects the calculated barrier energies to different extents. It reduces the first (large) barrier energy involved in the oxidation reaction drastically, while it reduces the barrier energies involved in the nitration reaction also appreciably. The presence of a water molecule along with ONOOCO2- does not seem to affect the reaction mechanisms significantly. Acknowledgment. We are thankful to the Council of Scientific and Industrial Research (New Delhi) and the University Grants Commission (New Delhi) for financial support. PKS is grateful to the University Grants Commission (New Delhi) for a research fellowship. References and Notes (1) Squadrito, G. L.; Pryor, W. A. Free Radical Biol. Med. 1998, 25, 392. (2) Dedon, P. C.; Tannenbaum, S. R. Arch. Biochem. Biophys. 2004, 423, 12. (3) Halliwell, B.; Gutteridge, J. M. C. Free Radicals In Biology and Medicine; Oxford Science Publications: Oxford, U.K., 1999. (4) Pryor, W. A.; Squadrito, G. L. Am. J. Physiol. 1995, 268, L699. (5) Huie, R. E.; Padmaja, S. Free Radical Res. Commun. 1993, 18, 195. (6) Ischiropoulos, H. Arch. Biochem. Biophys. 1998, 356, 1. (7) Greenacre, S. A.; Ischiropoulos, H. Free Radical. Res. 2001, 34, 541. (8) Radi, R.; Beckman, J. S.; Bush, K. M.; Freeman, B. A. Arch. Biochem. Biophys. 1991, 288, 481. (9) Beckman, J. S.; Beckman, T. W.; Chen, J.; Marshal, P. M.; Freeman, B. A. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 1620. (10) King, P. A.; Anderson, V. E.; Edwards, J. O.; Gustafson, G.; Plumb, R. C.; Suggs, J. W. J. Am. Chem. Soc. 1992, 114, 5430. (11) Tretyakova, N. Y.; Burney, S.; Pamir, B.; Wishnok, J. S.; Dedon, P. C.; Wogan, G. N.; Tannenbaum, S. R. Mutat. Res. 2000, 447, 287. (12) Niles, J. C.; Burney, S.; Singh, S. P.; Wishnok, J. S.; Tannenbaum, S. R. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11729. (13) Yermilov, V.; Rubio, J.; Becchi, M.; Friesen, M. D.; Pignatelli, B.; Ohshima, H. Carcinogenesis 1995, 16, 2045. (14) Gu, F.; Stillwell, W. G.; Wishnok, J. S.; Anthony, J. S.; Jones, R. A.; Tannenbaum, S. R. Biochemistry 2002, 41, 7508.

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with Guanine - ACS Publications - American Chemical Society

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