Understanding the Reactivity of CO3Â·â and NO2Â· Radicals toward S

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Understanding the Reactivity of CO and NO Radicals Towards S-Containing and Aromatic Amino Acids Sharmistha Karmakar, and Ayan Datta J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b05186 • Publication Date (Web): 19 Jul 2017 Downloaded from http://pubs.acs.org on July 22, 2017

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The Journal of Physical Chemistry

Understanding the Reactivity of CO •3− and NO •2 Radicals towards SContaining and Aromatic Amino Acids Sharmistha Karmakar and Ayan Datta* Department of Spectroscopy, Indian Association for the Cultivation of Science, 2A and 2B Raja S. C. Mullick Road, Jadavpur – 700032, Kolkata, West Bengal, India. *Email:[email protected]

Abstract: The reactivity of CO3•− and NO2• radicals towards six amino acid side chains namely, cysteine (Cys), methionine (Met), phenylalanine (Phe), tyrosine (Tyr), histidine (His) and tryptophan (Trp), has been explored using state-of-art density functional theory (DFT) and transition state theory (TST). Three reaction mechanisms, namely hydrogen atom abstraction (HAT), radical adduct formation (RAF) and single electron transfer (SET) have been considered for detailed study. While CO3•− radical is highly reactive towards majority of amino acids, the reactivity of •− NO 2• radical is limited. The CO3 radical creates oxidative damage to amino acid residues

predominantly via HAT mechanism with moderate to high rate constant. Kinetic data suggest that tryptophan and tyrosine moiety possess the highest reactivity while the phenylalanine furnishes slow reaction. On the other hand, NO2• radical cannot produce direct damage towards most of the amino acids except tryptophan and histidine. The NO2• radical reacts exclusively by SET mechanism with 6.01×106 M-1s-1 and 4.69×102 M-1s-1 rate constant for Trp and His, respectively. Therefore, the CO3•− radical may cause severe damage to amino acid side chains during oxidative stress conditions whereas the NO2• radical is mostly inert. Moreover, the reaction of CO3•− and NO2• radicals with amino acid radical intermediates generate variety of oxidation and nitro products which explain the formation of different experimentally characterized bio-markers during oxidative stress.

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Introduction: Our cellular components are constantly exposed towards various exogenous and endogenous reactive intermediates namely free radicals (species with one or two unpaired electrons), molecular oxidants like, H2O2 and redox active metal ions (e.g. Fe and Cu).1 These reactive species are produced within biological systems by different internal and external mechanisms2,3 and they are designed to perform some specific tasks inside that organism. Oxidative phosphorylation, normal metabolic processes and exposure to harmful radiation are the major sources of free radicals including reactive oxygen and nitrogen species (RONS), carbon and sulfur centered radicals.4-7 Low to moderate concentration of these species is known to be beneficial for cells as they play important role in signal transduction, cellular senescence, and apoptosis and protect cells from toxic compounds.8-10 Under normal circumstances, the concentration of these oxidant species is critically regulated by in-built antioxidant defense mechanism of cell involving low-molecular-weight scavengers11,12 (e.g. vitamins, glutathione, ascorbic acid, thiols and polyphenols), enzymes such as catalase, superoxide dismutase and glutathione peroxidase,13,14 and damage repairing enzyme15 (e.g. methionine sulfoxide reductases). However, failure of defense systems or dysfunction of molecular machineries results in accumulation of these reactive species within cells causing severe damage to bio-molecules like lipids, proteins, RNA and DNA.8,16-19 This scenario is also known as oxidative stress (OS).20,21 Therefore, OS is associated with numerous life threatening diseases such as Alzheimer’s and Parkinson’s disease,22,23 atherosclerosis,24,25 cardiovascular disorders,26,27 cancer,28,29 diabetes and some mental disorders.30,31 The extent of oxidative damage to bio-molecules depends on several factors namely, (i) the nature of oxidant, (ii) the availability of target molecules, (iii) the location of target molecule with respect to oxidant generation centre and (iv) the possible occurrence of secondary damaging events.19 •OH radical being the most injurious reactive species oxidizes almost every biological target with a diffusion controlled rate constant of ~109 M-1s-1.16 Other two important RONS species are carbonate radical anion ( CO3 ) and nitrogen di-oxide ( NO 2• ). CO3 is a strong one•−

•−

electron oxidant with a reduction potential of 1.78 V at pH=7.0, whereas the NO 2• radical is moderately oxidizing (E0= 0.99 V at pH=7) and is present in a monomeric form at physiological condition.32,33 They are known to cause severe damage to bio-macromolecules including 2


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biothiols, guanine residues of DNA and RNA, metalloproteins, lipids and protein residues by hydrogen-atom abstraction (HAT), electron transfer (ET) and/or radical adduct formation (RAF) •− mechanisms.34-38 These radicals ( CO3 and NO 2• )

play vital role in protein oxidation and

nitration process and modify some selective amino acids namely, tryptophan (Trp), tyrosine (Tyr), cysteine (Cys), methionine (Met), histidine (His) and phenyl alanine (Phe) during oxidative and nitrosative stress condition.32,36,39-41 Nitrogen dioxide is present in large excess in polluted air and inside cells it is generated from the reaction of nitric oxide (NO) with superoxide radical anion under inflammatory condition.32,37,42 Carbonate radical anion can be produced within biological systems by one-electron oxidation of physiological buffer i.e. CO32-/ HCO3- or from the homolytic cleavage of nitrosoperoxycarbonate (ONOOCO2-) adduct which is formed by the reaction of CO2 and ONOO-.43,44 Nitrocysteine, nitrohistidine, 5- and 6-nitrotryptophan, 3nitrotyrosine and nitrophenylalanine are few distinct biomarkers for NO 2• induced protein •− damage. 45 On the other-hand there is no specific biomarker for CO3 radical, as a result the •− damage caused by this radical has largely been underestimated. Hence, CO3 and NO 2• radicals

could also be involved in the formation of disulfides (cystine), sulfenic acid, methionine sulfoxide, oxo-histidine, dityrosine, 3-hydroxytyrosine (DOPA) and hydroxytryptophan which are known to be the common outcome of OS. In this manuscript, we have investigated the mechanistic and kinetic details of the reaction •− between CO3 and NO 2• radicals with six different amino acids namely, Trp, Tyr, Cys, Met,

His and Phe using state-of-art density functional theory (DFT) in aqueous phase. We have also tried to characterize the possible oxidative products formed by these radicals at various conditions. We have considered all possible reaction channels and only the thermodynamically favorable pathways were pursued for further study. This study provides a detailed molecular level understanding for the possible routes of oxidative/nitrosative damage in protein caused by •− these two radicals. The results indicate that the CO3 radical is highly reactive towards majority

of amino acids studied here, whereas the reactivity of NO 2• •− tryptophan and histidine. The reaction of CO3 and NO 2•

radicals with amino acid radical

3

radical is restricted to only



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intermediates produces variety of oxidation/nitration products which are by-products of oxidative stress inside cell.

Computational Details: All geometry optimizations were performed in the gas phase using hybrid meta-GGA functional MPWB1K46-49 within density functional theory along-with 6-31+G (d, p) basis set.50 The MPWB1K functional has been shown to provide accurate results for kinetic calculations.51,52 Harmonic frequencies were calculated by analytical differentiation of gradients to ensure that the optimized reactants /products are local minima and transition states are first order saddle points. Intrinsic reaction coordinate (IRC) calculations were performed to obtain the minimum energy paths (MEP). Solvent effects were incorporated by performing single-point calculations on the gas-phase optimized geometries using universal solute electron density based SMD model.53 We have chosen only aqueous environments (mimicked by water in SMD model) for overall study. The Gibbs free energy at solution for a particular species can be obtained by combining the free energy correction at gas phase to the electronic energy in solution phase. As we have considered infinitely separated reactants and products during all calculations, this will lead to an overestimation of entropic effects. Hence, the relative Gibbs free energies were corrected by using the solvent cage effect proposed by Okuno54 taking into account the free volume theory55 to get more accurate estimation of activation barriers and reaction energies. The Gaussian 0956 software package was employed for all electronic structure and frequency calculations with a standard state of 1 atm. Hence, a change of standard state from 1 atm to 1 M has been applied to all species in solution. The rate constants (k) were calculated using conventional transition state theory (CTST)55,57,58 following the QM-ORSA (quantum mechanics-based test for overall free radical scavenging activity) protocol.59 The tunneling corrections were incorporated by Eckart method which is special case of zero curvature tunneling (ZCT) method.60,61 The activation barriers for the electron transfer reactions were estimated using the Marcus theory62 as mentioned in QM-ORSA methodology. In the case of rate constants close to the diffusion limit, the Collins–Kimball theory63 was employed as pointed out in QM-ORSA method. The branching ratio for a particular 4


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pathway can be described as the ratio of individual rate constant to the total rate constant. For reaction with multiple product channels (A and B), the branching ratio for path A is calculated as, ΓA = kA/(kA+kB) where kA is the rate constant for path A and kA+ kB corresponds to the total rate constant. For kinetic study, we have considered only the exergonic and isoergonic (∆G ≤ 0) pathways; however in case of electron transfer reactions, mildly endergonic paths can also be included for overall rate calculations.59 This is because even if the endergonic reactions take place in significant rate they would be reversible and hence the formed product will not be detected. However, if the formed products undergo facile follow-up reaction in a highly exergonic fashion producing stable intermediates then these steps might be important in overall kinetic calculations.

Results and Discussion: •− The CO3 and NO 2• radicals produce variety of oxidation/nitration products in biological

systems during oxidative/nitrosative stress.16,32,33,45,64 Herein we have studied the possible mechanism of the oxidation/nitration processes involving both radicals for six amino acid side chains namely, cysteine, methionine, tyrosine, phenylalanine, histidine and tryptophan. For this, •− we have primarily considered the attack of CO3 / NO 2• radical onto the amino acid side chains

to generate radical intermediates and then in the next step we have investigated the reaction of •− these radical intermediates with CO3 / NO 2• radical resulting non-radical species. The •− preliminary attack of CO3 and NO 2• radicals on amino acid side chains can take place through

three mechanism: hydrogen transfer process from amino acid side chain to the oxidant (Eq 1), radical adduct formation (Eq 2) and electron transfer process (Eq 3): .

.

Hydrogen Atom Transfer (HAT):

AH + R → A + R-H

Radical Adduct Formation (RAF):

AH + R → (R-AH)

Single Electron Transfer (SET):

.

.

.+

.

…………… (1) …………… (2)

-

AH + R → (AH) + R

…………… (3)

The primary radical attack on amino acid side chains will produce highly reactive radical intermediates which will further react with another molecule (may be radical, H-donor molecule, oxidant or scavenger molecule) via HAT or RAF pathway resulting stable biomarkers. The 5



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radical can attack at various position of the amino acid side chain depending upon its thermodynamic feasibility. We have considered all possible reaction channels for each site, however only the thermodynamically viable pathways will prevail over other channels. The reactivity of different thermodynamically feasible pathways will be governed by their kinetic parameter. Low activation barrier along with high stability makes the formation of that particular radical more probable under cellular condition and the highest exergonic pathway is generally associated with lowest barrier (though not always). The model structures of six studied amino acids (see Scheme 1) show that the side chain of amino acid is flanked by two peptide bonds that are terminated at neighboring alpha carbon. The tripeptide model used in this work has been shown to provide satisfactory results for similar reactions involving other radicals51,52 and it has also been demonstrated that the conformation of the backbone has little effect on the reaction thermodynamics and kinetics.52

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Scheme 1: Atom numbering of the six amino acids namely, cysteine, methionine, tyrosine, phenylalanine, histidine and tryptophan studied here. •− In the next step we have studied the reaction of radical intermediates with CO3 / NO 2• radical to

furnish various non-radical products. For this purpose we have chosen two most thermodynamically stable radical intermediates for detailed study. As cysteine yields only two radical intermediates, we have considered both of them and for rest of the amino acids however, we have chosen the two most thermodynamically stable radical intermediates for this step (in some cases it can be one also). It should be kept in mind that in biological systems the amino acid side chain is exposed to numerous reactive oxygen and nitrogen species which could in fact •− form the most stable radical intermediate. Hence, it is highly probable that a CO3 / NO 2• radical

encounters a radical intermediate produced by other oxidant. Therefore, the later reaction yields variety of oxidized/nitration products which we have characterized accordingly for each amino acid. For simplicity, in this manuscript we have considered only those radical intermediates •− whose formation is most plausible thermodynamically and kinetically involving CO3 radical

and we believe that the same trend will be followed for the other radical also. Hence the overall •− •− process can be described as: sequential attack of two CO3 radicals or attack of CO3 radical •− followed by NO 2• radical. This situation can arise if the local concentration of CO3 radical is

sufficiently high or by the action of nitrosoperoxycarbonate adduct. As the second step involves a radical-radical reaction, it is expected to be a barrier-less process. •− Therefore, reaction kinetics is governed by the first step i.e. reaction of CO3 / NO 2• radical with

amino acids. All the reaction free energy (∆G) and free energy of activation (∆G‡) reported in this manuscript corresponds to aqueous phase values whether mentioned or not. Cysteine: Among sulfur containing amino acids cysteine possess the smallest side chain, namely a – CH2SH group. Disulfide (RSSR), sulfenic acid (RSOH), sulfinic acid (RSO2H), sulfonic acid (RSO3H), nitrocysteine (RSNO2), nitrosocysteine (RSNO) and disulfone are few well characterized oxidation/nitration products of Cys where the oxidation state of sulfur varies from 7



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1 to +6 in different scenarios.45,65 The oxidative damage to Cys by CO3•− and NO2• radicals can follow two mechanisms such as, H-atom abstraction from either C1 or S2 atom and electron transfer from sulfur lone pairs to the oxidant. The reaction free energy (∆G) for all these possible pathways has been calculated and tabulated in Table 1. For NO2• radical, it can be found that all three pathways are endergonic with ∆G value ranging from +1.6 to +21.8 kcal/mol. Thus, NO2• radical cannot produce direct damage to Cys moiety under cellular environment. On the contrary, for CO3•− radical the situation is quite different. In this case, both the HT reactions from C1 and S2 are predicted to be thermodynamically favorable with ∆G value of -7.1 and -16.7 kcal/mol, respectively. However, the single electron transfer (SET) mechanism involving CO3•− radical is .

largely endergonic (∆G= +26.2 kcal/mol) and follow similar trend with OH radical. Table 1: Reaction free energy (∆G in kcal/mol), free energy of activation (∆G‡ in kcal/mol), rate •− constant (k in M-1s-1) and branching ratio (Γ) at 298.15 K for the attack of first CO3 and NO 2•

radical onto cysteine.

Cysteine NO 2•

CO3•−

∆G

∆G‡

k

Γ

∆G

∆G‡

k

Γ

SET HT

+26.2

-

-

-

+21.8

-

-

-

C1

-7.1

+14.6

1.47×104

42.0

+11.2

-

-

-

+12.0

4

58.0

+1.6

-

-

-

S2

-16.7

2.02×10

Therefore, the HT channels are primarily responsible for

CO3•− radical induced oxidative

damage involving cysteine side chain. As the stability of S-centered radical is higher compared to the C-centered one, its formation is also more likely from thermodynamic as well as kinetic viewpoints. Evaluation of reaction barrier (∆G‡) clearly depicts that the hydrogen abstraction from S2 site is favored by +2.6 kcal/mol compared to the C1 site. Hence, H-abstraction from S2 site of Cysteine is the predominant pathway for CO3•− attack with a branching ratio of 58 %. 8


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Detailed kinetic calculation reveals that the overall rate constant for CO3•− attack on Cysteine side chain is 3.5×104 M-1s-1. In the following section, we have investigated the possible role of CO3•− and NO2• radicals for the formation of various oxidation/nitration products of Cys. For this purpose, we have studied the reaction of CO3•− / NO2• radical with Cys radical intermediates (Cys-IntC1 and Cys-IntS2). As this step involves the reaction of two radical species, it is a barrier-less process. The attack of CO3•− / NO 2• radical to Cys radical intermediates can proceed through two mechanism i) addition to the

radical center resulting adduct formation and ii) abstraction of neighboring H-atom to give unsaturated product. The thermodynamics and kinetics results show that for Cys the formation of S radical intermediate (Cys-IntS2) is more probable compared to C1 by a factor of 1.4, hence, products obtained from this pathway will be the predominant one and the Cys-IntC1 radical will follow the minor pathway. Three products have been characterized for the attack of CO3•− / NO2• radical to Cys-IntS2 namely, thioketone ( Cys − PC1/ S 2,C ), sulfur based adduct ( Cys − PSX2, A ) and cystine ( Cys − PS 2,B ). Abstraction of C1 H-atom by CO3•− / NO2• radical produces thioketone whereas addition of radical species to sulfur radical center results in sulfenic acid/nitrocysteine. -

Addition of CO3•− to sulfur radical primarily produce –SOCO2 which eventually decomposes by releasing CO2 to give sulfenic acid in a exergonic manner. Apart from this, if two Cys-IntS2 radicals are nearby in space and the concentration of reactive species is limited in that region, they rapidly dimerize by an S-S linkage resulting cystine. For CO3•− radical, the formation of thioketone (∆G = -48.1 kcal/mol) is favored over the addition and dimerization route by 16.6 and 7.1 kcal/mol, respectively. However for

NO 2•

radical, cystine furnishes the most

thermodynamically stable product followed by thioketone and nitrocysteine. Therefore, Figure 1 clearly depicts that the formation of thioketone, sulfenic acid, nitrocysteine and cystine is associated with large negative ∆G value and they can easily be formed by the attack of CO3•− / S2

NO 2• radical to Cys-Int

radical intermediate under biological condition.

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Figure 1: Reaction pathways and Gibbs free energy of reaction (∆G in kcal/mol) for the attack of

CO3•− / NO2• radical to cysteine radicals. The values outside and inside the parenthesis correspond to CO3•− and NO2• radical, respectively. On other hand, the Cys-IntC1 radical also shows similar reactivity with Cys-IntS2 and produces three products: i) addition of CO3•− / NO2• radical onto the C1 radical furnishes Cys − PCX1, A , ii) abstraction of H-atom forms α-C give Cys − PC1,B and iii) abstraction of S2 H-atom results thioketone. All of these reactions are exothermic and possess similar ∆G value with a little preference for the abstraction products in case of CO3•− radical. Although, the Cys-IntC1 radical reacts rapidly with CO3•− / NO2• radical forming a set of stable products, their experimental detection is limited due to lower concentration of Cys-IntC1 compared to Cys-IntS2 radical; as a result these products are also formed in trace amount under cellular environment.

Methionine:

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Methionine sulfoxide is the most abundant oxidized product of methionine (Met) formed during OS condition which reversibly gets back to Met by methionine sulfoxide reductase repair enzyme;15 however, another important oxidation product containing a double bond in the side chain (between C2 and C1) was also identified.66 Unlike Cys, Met offers four possible reaction sites for radical attack in its side chain. Three of them follow H-abs pathway resulting carbon radical intermediates and the remaining sulfur site produces radical cation ( S •+ ) intermediate via electron transfer mechanism. The occurrence of e- transfer event in Met strongly depends on the stability of newly formed sulfur radical cation which in term is governed by the local environment. Presence of electron donor atom in the backbone or neighboring amino acid side chains with strong electron donating group stabilize the

S •+ intermediate via 3e-2c bond

formation.52 We have evaluated the reaction free energies and barrier heights of SET and HAT pathways for both CO3•− and NO2• radicals (see Table 2) at 298.15 K in aqueous media. As can be seen from Table 2 that, the C2 site furnish the most stable radical and the stability trend follow the order: C2 > C4 > C1 > S3. Presence of sulfur lone pairs and α-Hs in neighboring carbon atom are the key factors for greater stabilization of C2 radical. Table 2: Reaction free energy (∆G in kcal/mol), free energy of activation (∆G‡ in kcal/mol), rate •− constant (k in M-1s-1) and branching ratio (Γ) at 298.15 K for the attack of first CO3 and NO 2•

radical onto methionine.

Methionine

NO 2•

CO3•−

∆G

∆G‡

k

Γ

∆G

∆G‡

k

Γ

SET HT

+16.7

-

-

-

+12.3

-

-

-

C1

-4.5

+17.5

1.97×102

0.01

+13.8

-

-

-

+ 9.1

2.63×10

6

78.34

+6.2

-

-

-

7.26×10

5

21.65

+11.6

-

-

-

C2 C4

-12.1 -6.7

+11.7

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The thermochemical data suggest that the SET from central Met is not feasible for both CO3•− and NO2• radicals and possess large positive reaction free energy, ∆G= +16.7 and +12.3 kcal/mol respectively, in aqueous media. While, in case of terminal Methionine the situation is quite different which we will discuss in the following section. Central methionine describes a methionine fragment situated at the middle position of a peptide chain and in terminal methionine it resides at the end position. Alternatively, all the HAT pathways are exergonic for

CO3•− with C2 being the most favorable one for radical attack with ∆G value of -12.1 kcal/mol. Hence, the CO3•− radical can induce damage to Met side chain by any of three possible HAT mechanisms and the SET pathway has been excluded due to large endergonicity. The relative contribution of various pathways in oxidative damage can be obtained by kinetic calculations. The free energy of activation (∆G‡) is minimum for C2 site with ∆G‡ value of +9.1 kcal/mol and the corresponding transition states for C1 and C4 lie 8.4 kcal/mol and 2.6 kcal/mol above compared to C2. Rate constant calculations also reveal that HAT from C2 is the most reactive channel with branching ratio (Γ) of 78.3%, followed by C4 (21.6%) and C1 (0.01%). The overall rate constant for CO3•− attack on central Meth side chain is 3.35×106 M-1s-1. On the other hand, for NO 2• radical, all four reaction channels comprising both SET and HAT mechanisms are found to

be endergonic with ∆G value ranging from +6.2 to +13.8 kcal/mol. Hence, for central Meth the possibility of NO2• induced oxidative damage has been ruled out. In case of terminal methionine, stabilization of S •+ intermediate through electron donating – NH2 or –COO- group facilitates the electron transfer event for both radicals. The effect of –NH2 group is more prominent compared to –COO- group. Therefore, SET involving CO3•− / NO2• radical for terminal Met is another viable pathway for radical attack. Low activation barrier along with suitable reaction energy makes the SET mechanism more plausible compared to other reaction channels for terminal methionine residue.

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Figure 2: Mechanistics details and reaction free energy (∆G in kcal/mol) for the attack of CO3•− / NO 2• radical to methionine radicals. The values outside and inside the parenthesis correspond to

CO3•− and NO2• radical, respectively.

In the following section, we have investigated the possible product formation from the attack of

CO3•− / NO2• radical to methionine radicals. We have considered two most thermodynamically stable as well as kinetically favorable radical intermediates namely, Met-IntC2 and Met-IntC4 for detailed study. As under biological circumstances, formation of Met-IntC2 is preferred over MetIntC4 by roughly a factor of 4, hence products obtained from Met-IntC2 radical will be the major one and they can be detected experimentally (see Figure 2). The Met-IntC2 radical can undergo

Met − PC 2, A and

abstraction or addition reaction forming

Met − PCX2,B set of products,

respectively. On the other hand, addition at Met-IntC4 radical produces the most stable product Met − PCX4 for both CO3•− and NO 2• radicals with ∆G value of -61.4 and -51.6 kcal/mol,

respectively. For CO3•− radical, the C2 abstraction product Met − PC 2, A (∆G= -58.0 kcal/mol) lie •−

3 very close in energy with Met − PCCO and the reaction exergonicity increases in the following 4 •−

3 3 order: Met − PCCO > Met − PC 2, A > Met − PCCO . Whereas the same for NO2• radical is as follows: 4 2, B

•

•−

X NO2 2 Met − PCNO 4 > Met − PC 2,B > Met − PC 2 , A . Although, Met − PC 4 is the most stable product obtained •

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from both radicals its experimental detection is limited because it is formed by minor pathway. Hence, Met − PC 2, A and

Met − PCX2,B are two important modifications of methionine which

could be obtained during oxidative/nitrosative stress condition. Apart from this, we have also considered the reaction of CO3•− / NO2• radical with Met-IntS2 intermediate. In spite of lower stability of the sulfur radical cation (Met-IntS2), it is produced within biological systems by variety of oxidant molecules with a moderate concentration. Interaction of CO3•− radical with Met-IntS2 generates methionine sulfoxide whereas NO2• radical does not form any stable adduct. .

Therefore, methionine sulfoxide is not only the outcome of OH induced oxidative damage, it can be also be produced by the reaction of CO3•− radical with S •+ intermediate. Tyrosine: Moving from aliphatic to aromatic amino acid opens up numerous possible sites for radical attack and the phenol side chain of tyrosine (Tyr) act as the main target for various oxidant molecules. Apart from abstraction and SET pathways, addition mechanism to the aromatic ring carbon could also be observed for Tyr. The side chain of tyrosine offers six different abstraction sites namely C1, C3, C4, C6, C7 and O8 and six addition centers comprising the aromatic ring carbons (C2, C3, C4, C5, C6 and C7). The reaction free energy for all possible reaction channels including the SET mechanism has been evaluated for both radicals in order to determine the thermodynamically feasible pathways (see Table S1 in SI). The Gibbs free energy of reaction (∆G), Gibbs free energy of activation (∆G‡), rate constants (k) and relative branching ratios (Γ) for the exergonic channels and SET mechanism are provided in Table 3 at 298.15 K. In the case of NO2• radical, all the reaction channels involving HAT, RAF and SET mechanisms are found to be endergonic irrespective of the reaction site. Hence, it cannot produce direct damage to tyrosine moiety. However, the CO3•− radical shows slightly different reactivity pattern. Thermochemical data reveal that for CO3•− radical, all the RAF channels and SET mechanism are endergonic with ∆G ranging from +7.7 to +17.6 kcal/mol. Detailed thermodynamic analysis for the HAT pathways shows that only the H-abstraction from the phenolic group (site O8) and aliphatic carbon atom (site C1) of Tyr are thermodynamically feasible with reaction free energy 14


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of -16.6 and -13.6 kcal/mol, respectively in aqueous media. All other H-abstraction pathways, comprising the aromatic H atoms, were found to be largely endergonic. As mentioned before, we have excluded all endergonic reaction channels from further study as they do not contribute effectively to overall rate constant. Thus, H-abstraction from O8 and C1 site is primarily responsible for the initiation of CO3•− radical induced oxidative damage to Tyr moiety. In order to determine the relative contribution of different HAT pathways, we have characterized the corresponding transition state structures. Evaluation of activation barrier reveal that the O8 site possess very low reaction barrier of 4.9 kcal/mol which is ~10.4 kcal/mol lower compared to the C1 site. As in the TS for O8 site, H atom is shared unequally between two O atoms and lies closer to the carbonate oxygen and hence, significant radical character is developed over O8 which is distributed over the aromatic ring carbons resulting in enhanced stabilization of the corresponding TS. However for the other TS involving C1 site, the H-atom is shared almost equally between two atoms and the radical centre is mostly localized on the C1 atom. Therefore, greater stability of phenoxyl radical compared to carbon radical lowers the activation barrier for O8 site selectively. Rate constant calculations for the thermodynamically viable reaction channels show that the HAT from O8 site is not only the major pathway but also it accounts for almost the whole reactivity of CO3•− radical towards Tyr moiety with a branching ratio of 99.99%. The calculated overall rate constant between CO3•− radical and Tyr is 1.32×109 M-1s-1 which corresponds to diffusion controlled regime.

Table 3: Reaction free energy (∆G in kcal/mol), free energy of activation (∆G‡ in kcal/mol), rate •− constant (k in M-1s-1) and branching ratio (Γ) at 298.15 K for the attack of first CO3 / NO 2•

radical onto tyrosine.

15



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Page 16 of 32

Tyrosine

CO3•−

NO 2•

∆G

∆G‡

k

Γ

∆G

∆G‡

k

Γ

SET HT

+17.6

-

-

-

+13.2

-

-

-

C1

-13.6

+15.3

4.53×103

0.01

+4.7

-

-

-

O8

-16.6

+4.9

1.32×109

99.99

+1.7

-

-

-

In this subsection, we have tried to elucidate the possible mechanism for the formation of various oxidized/nitration products of Tyr within biological systems. 3-Nitrotyrosine, 3,5-dinitrotyrosine, 3-hydroxytyrosine, DOPA and dityrosine are few well-characterized oxidation/nitration products of Tyr formed within the cell during inflammatory condition.16,45,67 For this, we have studied the reaction of tyrosyl radical with different reactive oxygen and nitrogen species e.g. CO3•− and NO 2• radicals (see Figure 3a). As from the previous section, it is well established that H-

abstraction from Tyr yields phenoxyl radical (Tyr-IntO8) exclusively, in this section we will solely focus on the reaction of

CO3•− / NO2• radical with Tyr-IntO8 radical. The radical

intermediate, Tyr-IntO8, can only escape through addition mechanism by forming non-radical species. Depending on the site of attack and nature of reactive species, a variety of products can be obtained. Dimerization of two tyrosyl radicals through O-C or C-C(C4-C4) linkage produce dityrosine, with ∆G value of -28.5 and -46.7 kcal/mol, respectively. Hence, formation of C-C cross-linked tyrosine dimer is thermodynamically more viable. Additionally, it (Tyr-IntO8) can also undergo radical addition reaction at C4/C6 via a non-aromatic intermediate which eventually aromatize to give substituted tyrosine derivatives. For NO2• radical, nitro-tyrosine (substituted at C4/C6 i.e. 3 or 5 NO2-Tyr) is formed with reaction free energy of -42.2 and -43.6 kcal/mol. However, the

CO3•−

radical primarily furnishes aryl carbonate intermediates,

−

2 Tyr − POOCO 8, A / B , which gradually decarboxylates to form hydroxy-tyrosine (3 or 5 substituted) in a

highly exothermic manner. Herein, it can be seen that all the pathways producing dityrosine, 16


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nitrotyrosine and hydroxytyrosine are energetically favorable with large exergonicity. Hence, we are able to explain the formation of different biomarkers of tyrosine detected experimentally during oxidative stress condition from the reaction of CO3•− / NO2• radical with tyrosyl radical.

Figure 3: Reaction mechanism and reaction Gibbs free energy (∆G in kcal/mol) for the attack of

CO3•− / NO2• radical to (a) tyrosine radical and (b) phenylalanine radical. The values outside and inside the parenthesis correspond to CO3•− and NO2• radical, respectively. Phenylalanine: The phenylalanine moiety shows close structural resemblance with tyrosine side chain, substitution of C5-hydroxyl group in Tyr by H-atom yields phenylalanine (Phe). Nitrophenylalanine, dihydroxyphenylalanine (DOPA) and ortho/ meta / para-tyrosine act as specific biomarkers for Phe.16,45 The phenylalanine moiety follow similar reactivity pattern with tyrosine. Likewise, the radical attack in Phe side chain can take place through three reaction mechanisms namely, HAT, RAF and SET. The reaction free energy for all possible pathways at 17



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Page 18 of 32

each reaction site was calculated to obtain the thermodynamically viable pathways (see Table S2 in SI). Table 4 reports the Gibbs free energy of reaction (∆G), Gibbs free energy of activation (∆G‡), rate constants (k) and relative branching ratios (Γ) for the exergonic channels and SET mechanism at 298.15 K. The thermochemical data suggests that the NO2• radical is unable to produce direct damage to Phe because all the reaction channels comprising HAT, RAF and SET mechanisms are endergonic in nature (see Table S2 in SI). Furthermore for CO3•− radical, it can be found that all the RAF and SET pathways are largely endergonic as was computed for Tyr. In case of HAT mechanism, all the aromatic H atoms possess positive ∆G value. However, Habstraction from the aliphatic C (site C1) was found to be exergonic with ∆G value of -13.6 kcal/mol. Hence, HAT from the C1 site is the only viable reaction pathway that can be observed for CO3•− induced damage on Phe. Additionally, the activation barrier for C1 site was computed to be +15.3 kcal/mol which is quite high compared to other amino acids. Large reaction barrier lowers the overall rate for CO3•− radical to 1.47×104 M-1s-1 implying that Phe side chain is comparatively less reactive to oxidative damage involving carbonate radical anion. Table 4: Reaction free energy (∆G in kcal/mol), Free energy of activation (∆G‡ in kcal/mol), •− Rate constant (k in M-1s-1) and Branching ratio (Γ) at 298.15 K for the attack of first CO3 / NO 2•

radical onto phenylalanine.

Phenylalanine CO3•−

NO 2•

∆G

∆G‡

k

Γ

∆G

∆G‡

k

Γ

SET

+30.8

-

-

-

+26.4

-

-

-

-13.3

+14.0

1.47×104

100.0

+5.0

-

-

-

HT C1

In the following section, we have investigated the possible outcomes for the reaction of CO3•− / C1

NO 2• radical with phenylalanine radical (Phe-Int ). In this manuscript we are considering only

18


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the C1 radical intermediate as its formation is more feasible for Phe compared to other carbon radicals involving aromatic ring. The Phe-IntC1 radical can undergo two types of follow up reaction as shown in Figure 3b: i) radical addition at C1 site producing stable adduct (

Phe − PCX1,B ) and ii) abstraction of α-H to give α, β unsaturated product ( Phe − PC1, A ). For NO2• radical, adduct formation (∆G= -41.9 kcal/mol) is more favorable compared to the abstraction pathway by 6.4 kcal/mol. However in case of CO3•− radical, both of these paths possess similar reaction free energies which are ~15 kcal/mol lower than the NO2• radical. Although, the reaction of Phe radical with CO3•− / NO2• radical produces stable products e.g. Phe − PCX1,B and Phe − PC1, A , but there exist hardly experimental reports for them. The reason may be following i) these species are not enough stable to act as biomarker or ii) the concentration of phenylalanine radical (Phe-IntC1) in biological systems is significantly less compared to other adduct species of phenylalanine and hence, products obtained from Phe-IntC1 intermediate are of too small concentration to be detected experimentally. Histidine: Histidine is another important building block of cellular constituents that gets modified by variety of one or two e- oxidants. Formation of oxohistidne, nitrohistidine and histidinyl radical, have been detected experimentally during oxidative stress condition.16,45 As seen from Scheme 1, histidine contains an imidazole ring in its side chain and possesses four different sites for Habstraction by CO3•− / NO2• radical namely, C1, C3, C5 and N6. It can also undergo addition reaction onto the double bond of aromatic ring (site: C2, C3 and C5) and SET mechanism. Here, we have excluded the possibility of radical addition at N center as it is known to produce unstable products. The Gibbs free energies for all paths for both radicals were estimated and shown in Table S3 in SI. Thermodynamics data for CO3•− radical reveal that HAT from C1 site is more plausible in terms of energetics, followed by N6 site with ∆G value of -16.2 kcal/mol. All other HAT paths (C3 and C5) have positive values of ∆G ranging from +17.7 to +19.3 kcal/mol. Moreover, the addition reaction to the imidazole ring is more favorable compared to other amino acids and even makes adduct formation at C5 thermodynamically feasible for CO3•− radical. The other RAF pathways and the SET mechanisms were also found to be endergonic. Therefore, it is 19



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expected that the oxidative damage to His involving CO3•− radical can occur through three pathways namely, H-abstraction from C1 and N6 sites and RAF at C5 site. Table 5 shows the activation barrier, reaction free energy, rate constant and branching ratios for exergonic pathways and SET mechanism. The C1 site possesses the lowest barrier of +9.5 kcal/mol among the three viable pathways. The addition TS lies in between two abstraction TSs with ∆G‡ value of +10.4 kcal/mol and the corresponding TS for N6 H-abstraction is +1.8 kcal/mol higher than the addition TS. Therefore, H abstraction from C1 site is the most feasible pathway thermodynamically as well as kinetically for histidine. Kinetic calculations reveal that, the C1 site shows maximum contribution to the overall rate followed by C5 and N6 site.

The

corresponding branching ratios are 95.1 %, 4.7 % and 0.2 % respectively and the overall rate constant for CO3•− attack to His is 1.08×107 M-1s-1. Hence for histidine side chain, HAT from C1 site is the predominant pathway for the initiation of CO3•− radical induced oxidative damage. Table 5: Reaction free energy (∆G in kcal/mol), Free energy of activation (∆G‡ in kcal/mol), •− Rate constant (k in M-1s-1) and Branching ratio (Γ) at 298.15 K for the attack of first CO3 / NO 2•

radical onto histidine.

Histidine

CO3•−

∆G

∆G‡

NO 2•

k

Γ

SET HT

+13.0

-

-

C1

-16.2

+9.5

1.03×107

N6

∆G

∆G‡

k

-

+8.5

+13.8

4.69×10

Γ 2

100.0

95.11

+2.1

-

-

-

4

0.13

+9.2

-

-

-

4.75

+9.3

-

-

-

-9.1

+12.2

1.44×10

-2.1

+10.4

5.14×105

RAF C5

In the following section we have investigated the possible role of CO3•− / NO2• radical behind the formation of various biomarkers for His. We have chosen two most thermodynamically stable 20


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histidine radical namely, His-IntC1 and His-IntN6, for radical attack. Table 6 reports the reaction free energy for the attack of CO3•− / NO2• radical to histidine radicals. The His-IntN6 radical intermediate can not only be formed by H abstraction from N6 site, it can also be produced by SET pathway. Variety of oxidant molecules furnish histidine radical cation via SET mechanism which ultimately loss a proton to give His-IntN6 intermediate.45 The His-IntC1 radical yields two kind of products similar to other amino acid radicals namely, the abstraction product ( His − PC1, A ) and the addition product ( His − PCX1,B ). When the attacking species is CO3•− radical, formation −

2 of His − PC1, A is favored over His − PCOCO by 4.4 kcal/mol. However the NO2• radical shows 1,B 2 reverse trend with His − PCNO being more stable. 1, B

The N6 histidine radical shows similar

reactivity pattern with tyrosyl radical and form different substituted histidine species in a highly exergonic fashion. The imidazole ring offers two addition sites namely, C3 and C5. The NO2• radical produces nitro-histidine with ∆G ranging from -47.0 to -51.2 kcal/mol. On the other hand, −

2 the CO3•− radical primarily gives aryl carbonates, His − PNOCO , which eventually loss a CO2 6, A / B

molecule to generate hydroxy-histidine with reaction free energy of ~ -62 kcal/mol. The 2hydroxyhistidine can tautomerise to give 2-oxohistidine. Formation of addition products from His-IntN6 radical is more thermodynamically feasible by ~15 kcal/mol compared to His − PC1, A and His − PCX1,B . Therefore, we have explored the possible pathways for the formation of oxohistidine and nitro-histidine from histidine radical (His-IntN6) involving the CO3•− / NO2• radical. Table 6: Reaction free energy (∆G in kcal/mol) for the attack of CO3•− / NO2• radical onto histidine radicals.

∆G( CO3•− )

∆G( NO2• )

His − PNX6, A

-61.3

-51.2

X N 6, B

His − P

-63.1

-47.0

His − PC1, A

-53.0

-34.6

21



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His − PCX1,B

-48.6

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-38.0

Tryptophan: The side chain of tryptophan differs significantly from other amino acids and contains a bicyclic indole group where a six-membered benzene ring is fused with five-membered pyrrole ring. The exposure of tryptophan to reactive oxygen or nitrogen species produces variety of oxidation/nitration products namely, 5- and 6-nitrotryptophan, N-formylkynurenine, oxindole, hydroxytryptophan, tryptophanyl radical and hydropyrroloindole.16,45 In this subsection, we have explored the reactivity of tryptophan side chain towards CO3•− / NO2• radical. All possible reaction mechanisms namely, addition, abstraction and electron transfer, were considered for detailed study. Tryptophan offers total six addition centers (C2, C4, C5, C6, C7 and C10) and seven abstraction sites (C1, C4, C5, C6, C7, N9 and C10) for radical attack in its side chain. The reaction free energies for all possible pathways were estimated and shown in Table S4 in SI. In the following section we will mostly focus on the results of

CO3•− radical and in the next

paragraph we will discuss about NO2• radical. For the addition reaction, all the C atoms, except the C10 of pyrrole ring, follow similar trend with phenylalanine and tyrosine and show positive ∆G value of +11.4 to +19.4 kcal/mol. Hence, CO3•− radical addition to the C10 site yields the most stable adduct with Gibbs free energy of -6.4 kcal/mol and makes it thermodynamically viable reaction channel. Focusing on the thermodynamics data for HAT channel shows that Habstraction from benzene ring is highly endergonic as was with phenylalanine and tyrosine. On the other hand, H-abstraction from N9 and C1 sites produces the most stable radical intermediates, Trp-IntN9 and Trp-IntC1, with ∆G value of -12.9 and -15.0 kcal/mol, respectively. The ∆G value of SET mechanism for CO3•− radical is +4.8 kcal/mol. Although, the electron transfer is mildly endergonic for CO3•− radical, still it can play important role in overall reactivity. As the SET from tryptophan produces tryptophan radical cation, which is highly acidic and instantly deprotonates yielding a more stable tryptophanyl radical (Trp-IntN9). Therefore, formation of highly stable Trp-IntN9 radical intermediate from tryptophan radical cation makes the overall process irreversible resulting the SET mechanism viable. Hence, the 22


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primary reaction of CO3•− radical with tryptophan side chain can occur through four thermodynamically viable pathways namely, HAT from C1 and N9 site, addition reaction at C10 and SET mechanism. In order to get insight into the relative contribution of various pathways to the overall reactivity, corresponding transition states were characterized. Table 7 tabulates the Gibbs free energy of activation, reaction free energy, rate constants and branching ratios for the viable reaction paths. In can be seen from the ∆G‡ values that the H-abstraction from N9 site possesses the lowest barrier of 6.2 kcal/mol. Moreover, the activation barrier for the electron transfer process is similar to that of H-abstraction from N9 site. The ∆G‡ values follow the increasing order as- C1 > C10 > SET ≈ N9. The Gibbs free energy of activation for C1 and C10 site lie 7.3 kcal/mol and 2.4 kcal/mol higher than the N9 site, respectively. The N9 abs TS shows analogous characteristics with tyrosine O8 abstraction TS and gets enhanced stabilization from delocalization of radical character over the aromatic rings and carbonate radical. However for C1 site the radical character is mostly localized on the attacking O atom of carbonate radical and the aliphatic C atom. In the addition TS, the CO3•− radical approaches the pyrrole ring C (C10) from the perpendicular plane resulting a O-C distance of 1.95 Å. Table 7: Reaction free energy (∆G in kcal/mol), Free energy of activation (∆G‡ in kcal/mol), •− Rate constant (k in M-1s-1) and Branching ratio (Γ) at 298.15 K for the attack of first CO3 / NO 2•

radical onto tryptophan.

Tryptophan

CO3•−

∆G SET HT C1 N9 RAF C10

∆G‡

NO 2•

k

Γ 8

+4.8

+6.4

1.26×10

-15.0

+13.5

7.30×104 8

-12.9

+6.2

3.52×10

-6.4

+8.6

6.11×106

∆G

∆G‡

k

Γ 6

25.95

+0.4

+8.2

6.01×10

100.00

0.01

+3.3

-

-

-

72.77

+5.4

-

-

-

1.26

+5.1

-

-

-

Rate constant calculations show that HAT from N9 site and SET pathway contribute almost entirely to the overall reactivity of CO3•− radical with branching ratios of 72.8 % and 25.9%, 23



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Page 24 of 32

respectively. The RAF from C10 site comprises the minor pathway with Γ value of 1.2 %. Large activation barrier for C1 site lowers its reactivity resulting negligible branching ratio of 0.01 %. Therefore, the action of

CO3•− radical on tryptophan produces tryptophanyl radical (Trp-IntN9)

predominantly via HAT or SET pathway. Table 7 shows that the NO2• radical follows similar reactivity pattern with phenylalanine and tyrosine and makes all reaction channels except the SET pathway endergonic. The SET mechanism for NO2• radical produces isothermic (∆G≈0) reaction channel. As discussed above, we have now considered the electron transfer from tryptophan to NO2• radical as a viable process and the rate of SET turns out to be 6.01×106 M-1s-1. Hence, NO2• radical can directly damage the tryptophan moiety by the formation of tryptophan radical cation. As can be concluded from previous section that, the formation of tryptophanyl radical (Trp-IntN9) is most plausible in terms of thermodynamics and kinetics point of view under biological condition. Therefore, it is highly probable that a CO3•− / NO2• radical encounter a tryptophanyl radical within biological systems producing variety of different products. Here, we will mostly focus on this radical-radical recombination reaction which will help to explain the formation of different oxidation/nitration products of tryptophan.

The Trp-IntN9 intermediate can only

undergo addition reaction at various positions of aromatic rings primarily giving non-aromatic species which eventually rearranges to aromatic product. These addition reactions produce variety of substituted tryptophan derivatives in highly exergonic manner. The reaction free energies of all reaction channels for both radicals have been reported in Table 8. When the attacking radical is NO2• , different structural isomers of nitro-tryptophan is formed with ∆G in the 2 range -22.8 to -49.2 kcal/mol. The N-centered adduct ( Trp − PNNO 9, E ) shows the least stability

whereas the other reaction channels follow similar exergonicity trend. On the other hand, the −

−

OCO2 2 ) which CO3•− radical primarily produces aryl carbonate derivatives ( Trp − PNOCO 9, A Trp − PN 9,F

spontaneously decarboxylate to generate hydroxy-tryptophan isomers. The ∆G values for the formation of aryl carbonates lie in the range from -15.9 to -54.4 kcal/mol with the lowest value obtained for N9 site. We have excluded the N9 addition pathway from overall reactivity due to very low stability of the corresponding adduct species. Therefore, we have explored the possible 24


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pathway for the formation of different nitro- and hydroxy derivatives of tryptophan during oxidative stress condition. Table 8: Reaction free energy (∆G in kcal/mol) for the attack of CO3•− / NO2• radical onto tryptophanyl radical.

Site of Radical Attack

∆G( CO3•− )

∆G( NO2• )

Trp − PNX9, A

C4

-50.6

-41.2

Trp − PNX9,B

C5

-52.7

-47.7

Trp − PNX9,C

C6

-53.6

-49.2

Trp − PNX9,D

C7

-53.2

-49.0

Trp − PNX9,E

N9

-15.9

-22.8

Trp − PNX9,F

C10

-54.4

-45.4

Finally, the reactivity of six different amino acids towards CO3•− radical follow the increasing order as: Tyr > Trp > His > Met > Cys > Phe where Tyr and Trp furnish rate constant in the diffusion controlled regime. On the other hand

NO 2•

radical is mostly un-reactive towards

majority of amino acids except tryptophan for which it reacts via SET pathway producing tryptophan radical cation with appreciable rate constant (~106 M-1s-1). The NO2• radical reacts only through SET mechanism, it neither abstract H-atom nor add to double bond. Histidine also undergoes electron transfer involving NO2• radical with a small rate constant of 4.69×102 M-1s-1. However, for CO3•− radical the primary mechanism of oxidative damage involves HAT pathway. It rapidly abstract an H-atom from X-H (X=S, O, N) bond or β-C for different amino acids. For methionine H-abstraction from C2 site is the most preferred one followed by C4. The addition reactions for aromatic amino acids are nearly all endergonic with large positive ∆G values. Only addition at C5 of histidine and C10 of tryptophan furnish mildly exergonic reaction channels which are still much lower compared to the HAT pathway. Thus, CO3•− radical is unable to produce stable adduct species. In case of SET mechanism, tryptophan reacts via SET pathway 25



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with almost equal efficiency with the HAT channel and rest of the amino acids do not undergo electron transfer process involving CO3•− radical. Therefore, the reactivity of CO3•− radical towards various amino acids is largely governed by H-atom abstraction efficiency and it can induce oxidative damage to almost all amino acids studied here with moderate to high rate constant. Whereas the NO2• radical is primarily inert to most of the amino acids; and cannot create direct damage to them. The only exception is tryptophan which produces radical cation via SET pathway with significant rate constant. The second part illustrates the formation of various oxidation/nitration products from the reaction of CO3•− and NO2• radicals with amino acid radical intermediate. For sulfur containing amino acids (cysteine and methionine), formation of abstraction product is more plausible compared to addition one involving CO3•− radical. However, the NO2• radical does not show such specificity. For cysteine the important oxidized /nitration products are nitro-cysteine, cystine, thio-ketone and hydroxy-cysteine. On the other hand, the radical intermediates of aromatic amino acids predominantly undergo adduct formation with both radicals. The reaction of tyrosyl, tryptophanyl and histidyl radicals with CO3•− / NO2• radical produces different conformation of nitro-tyrosine, hydroxy-tyrosine, nitro- and hydroxy-tryptophan, nitro-histidine and oxo-histidine respectively. All these species act as stable biomarkers for them. Therefore it can be clearly seen that the nitro-products cannot be produced by the direct reaction of NO2• radical with amino acids. They are formed in a secondary process by the reaction of •− NO 2• radical with the corresponding radical intermediates. The CO3 radical also furnish variety

of oxidized products which are similar with hydroxyl radical induced damage. As a result the role of CO3•− radical in oxidative damage has largely been underestimated in spite of its high reactivity.

Conclusion: Herein, we have investigated the overall reactivity of CO3•− and NO2• radicals towards six amino acid side chains namely, Cys, Met, Phe, Tyr, His and Trp. We have also studied the reaction of these radicals with corresponding amino acid radical by density functional theory (DFT). Three 26


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reaction mechanism were considered for detailed study: hydrogen atom transfer (HAT), radical adduct formation (RAF) and single electron transfer (SET). Thermodynamic and kinetic calculations indicate that CO3•− radical reacts with almost all amino acids studied here, predominantly via HAT mechanism, with moderate to high rate constant. The tryptophan and tyrosine moiety show the highest reactivity whereas phenylalanine furnishes the lowest. On the other hand, the NO2• radical is inert towards majority of amino acids except histidine and tryptophan. It undergoes SET mechanism with both of the amino acids producing radical cation intermediates. The tryptophan side chain provides appreciable rate constant with NO2• radical; while, histidine possesses low reactivity. Therefore, it can be concluded that the CO3•− radical may cause severe damage to variety of bio-molecules during oxidative stress condition whereas the reactivity of NO2• radical is limited. The reaction of CO3•− / NO2• radical with amino acid radicals generates various oxidation/nitration products which have been characterized experimentally in presence of excess reactive oxygen/nitrogen species. This step provides a detailed mechanistic understanding of how most of the oxidized/nitro products are formed within biological systems. Although the NO2• radical produces specific bio-markers such as nitro compounds; there is no distinct bio-marker for CO3•− radical and most of the oxidized products of this radical are similar to hydroxyl radical which creates difficulty in detecting the oxidative damage caused by CO3•− radical. The action of nitrosoperoxycarbonate adduct on these amino acids will also result in analogous product formation. This study will shed light on the detrimental effect of CO3•− / NO2• radical towards biomolecules during oxidative/nitrosative stress.

SUPPORTING INFORMATION Optimized geometries for all reactants, products and transition states, table for reaction free energies for radical attack onto histidine, phenylalanine, tyrosine and tryptophan and scheme for products obtained from the reaction of CO3•− and NO2• radicals with amino acid radicals. The 27



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Understanding the Reactivity of CO3Â·â and NO2Â· Radicals toward S

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