Exploring Cycloreversion Reaction of Cyclobutane Pyrimidine Dimers

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A: Spectroscopy, Molecular Structure, and Quantum Chemistry

Exploring Cycloreversion Reaction of Cyclobutane Pyrimidine Dimers Quantum Mechanically Donglian Huang, Shanfeng Chen, Jingzhi Pu, Xuecai Tan, and Yan Zhou J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.8b12345 • Publication Date (Web): 18 Feb 2019 Downloaded from http://pubs.acs.org on February 18, 2019

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The Journal of Physical Chemistry

Exploring Cycloreversion Reaction of Cyclobutane Pyrimidine Dimers Quantum Mechanically Donglian Huang,† Shanfeng Chen,† Jingzhi Pu,*,‡ Xuecai Tan,† Yan Zhou*,† †

School of Chemistry and Chemical Engineering, Guangxi University for Nationalities, 188

Daxue East Road, Nanning, Guangxi 530006, China ‡

Department of Chemistry and Chemical Biology, Indiana University-Purdue University

Indianapolis, 402 N. Blackford St. Indianapolis, IN 46202, USA

ACS Paragon Plus Environment

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ABSTRACT: Cyclobutane pyrimidine dimer (CPD) is a major photoproduct of deoxyribonucleic acid (DNA) that is damaged by ultraviolet light. This DNA lesion can be repaired by DNA photolyase with the aid of UV light and two cofactors. To understand the repair mechanism of CPD and whether protonation of CPD participates in the DNA repair process, the cycloreversion reactions of four CPD models and proton transfers between the adjacent residue Glu283 and CPD models were explored through quantum mechanical method. Two-dimensional maps of potential energy surface in vacuum and in implicit water solution were calculated at the ωB97XD/6311++G(2df,2pd) level. One-dimensional potential energy profiles were computed for proton transfer reactions. Among the models that have been considered, both in vacuum and in water solution, the results indicate that the most likely repair mechanism involves CPD•2- radical anion splitting in a stepwise manner. C5-C5’ splits first, and C6-C6’ splits later. The computed free energies of activation of the two splitting steps are 0.9 and 3.1 kcal/mol, respectively. The adjacent Glu283 may stabilize CPD•2- radical anion through hydrogen bond and increase the quantum yield; however, protonating CPD radical anion by Glu283 cannot accelerate the rate of ring opening.


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1. INTRODUCTION DNA is subject to damage when exposed to far ultraviolet (far-UV, 200 - 300 nm), which causes it to form two genotoxic photoproducts: cyclobutane pyrimidine dimer (CPD) and 6-4 photolesion.1 Of the two photoproducts, CPD is the most abundant; it is formed by a [2+2] photocycloaddition reaction between two adjacent thymine nucleobases.2 CPD can affect DNA replication and transcription and is regarded as carcinogenic.3 CPD plays a crucial role in the initiation of UV-induced skin cancer, if it is unrepaired or apoptosis does not destroy the damaged cells.4 In human bodies, DNA lesion is repaired by a nucleotide excision repair mechanism.5 While in bacteria,6 fungi, plants,7 and some animals,8 a more efficient repair mechanism with 0.82 quantum yield is achieved by DNA photolyase.9 DNA photolyase is absent in human bodies. By applying photolyase-containing liposomes or sunscreen to human skin prior to exposure to UV radiation, the number of CPD was reduced.3, 10 Therefore, understanding the repair mechanism of DNA photolyase can help to improve its application in the treatment of skin cancer. The repair cycle of DNA photolyase is depicted in Scheme 1. First, DNA photolyase absorbs near-UV and visible light (300 - 500 nm) with a light-harvesting photoantenna pigment 8hydroxy-5-deazaflavin (8-HDF) or 5,10-methenyltetrahydrofolate (MTHF). Second, the antenna pigment transfers energy to excite a reduced flavin adenine dinucleotide (FADH-) coenzyme. Third, the excited FADH- promotes one electron to the neighboring CPD, and the fourmembered ring of the CPD radical anion quickly opens. Last, one electron returns to FADH• after ring opening, and then DNA is repaired.6 The repair will fail if one electron returns to FADH• before CPD splits.


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Although the cycloreversion reaction of CPD radical anion is not the rate-limiting step among the repair cycle, its competition with back-transfer of the electron and deactivation of FADH-* determines the quantum yield of DNA photolyase. Therefore, the mechanism of cycloreversion of CPD radical anion needs to be explored. Besides CPD radical anion,11-13 two other CPD species with different charge and protonation states have also been proposed as intermediates: CPD radical cation14-15 and protonated CPD radical anion.16 Experiments revealed that both radical anion and radical cation can be involved in the cycloreversion reactions in some model systems depending on whether the sensitizers are photooxidizing or photoreducing.17-19 Ab initio calculations have also shown that systems of radical anion or radical cation have considerably lower barriers than the neutral one.20-24 Experimentally, radical anion was observed when the FADH- sensitizer was employed.11, 13, 17, 25-26 Another potential intermediate is protonated radical anion, which can be formed when the neighboring residue Glu283 donates a proton to the C4 carbonyl oxygen atom O4 of the radical anion. Some research on the protonated radical anion system has been conducted. Vande Berg et al. noticed that the quantum yield was diminished by 60% in the E283A mutant.27 Rösch et al. calculated the protonated uracil dimer radical anion on the B3LYP/6-31G* and AM1 levels, and reported that protonated anion has higher barrier energies than unprotonated anion.28 Essen and Klar proposed a hypothetical repair mechanism in which the protonation of O4 of the 5’ thymine (5’ T) by Glu283 may be crucial for stabilizing CPD radical anion and avoiding non-productive back-transfer of the electron onto the FADH•.16 Masson et al. had combined quantum mechanical and molecular mechanical (QM/MM) simulations in duplex DNA and enzyme, and observed a proton hopping between Glu283 and O4 of the thymine dimer radical anion in five of seven simulations.29


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In addition to the unclear role of the Glu283 residue, the splitting mechanism of radical anion was in debate. The splitting mechanism has been proposed to be a concerted reaction mechanism,11, 23 an asynchronously concerted one-step reaction mechanism,20, 29-35 and a stepwise reaction mechanism.24, 36 For the concerted reaction mechanism, both the C5-C5’ bond and the C6-C6’ bond cleave at the same time. For the asynchronously concerted one-step reaction mechanism, one of the two bonds usually the C5-C5’ bond breaks first with a very small barrier or almost no barrier, which produces an unstable intermediate; then, the other bond breaks, and it seems as if only one barrier exists for the entire splitting process. For the stepwise reaction mechanism, the two bonds sequentially break with two distinct barriers and a stable intermediate is formed. MacFarlane’s and Liu’s experiments suggested a sequential reaction mechanism,9, 37 whereas McMordie’s experiment supported a concerted reaction mechanism.11 Quantum mechanical (QM) methods at different levels yielded different results. 20, 23, 31, 36, 38 In 2014, Barbatti had high-level QM calculations for different electrostates with different multiplicities and charge states, which indicates a sequential reaction for radical anion with no barrier for the cleavage of the C5-C5’ bond.24 An asynchronously concerted mechanism was supported by QM/MM research performed by Masson et al. in duplex DNA and enzyme29, 32 and 2D ab initio MD umbrella sampling performed by Singer et al.34-35 Either the cleavage of the C5C5’ bond 36 or the cleavage of the C6-C6’ bond20, 29, 34 has been considered to be the rate-limiting step in different studies. To investigate the mechanism of the cycloreversion reaction of CPD and the role of the Glu283 residue, we performed high-level quantum mechanical calculations in this work. This paper is organized as follows. Section 2 describes four model systems, the QM method in this study, and other computational details. Section 3 presents the computational results of cycloreversion


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reactions and proton transfer reactions and the analyses based on the frontier molecular orbitals, charge distribution, bond orders, and geometries. Section 4 contains a discussions of the results. The final section presents the study’s conclusions. 2. COMPUTATIONAL DETAILS QM calculations were conducted with the G09 program.39 Four CPD models were constructed from a crystal of DNA duplex that contains a CPD lesion (PDB ID: 1N4E).40 The four models (Chart 1) include CPD anion before receiving an electron (CPD-), CPD radical anion formed after receiving an electron (CPD•2-), protonated CPD molecule (pCPD), and protonated CPD radical anion (pCPD•-). The initial structures for these four models constructed from crystal 1N4E were plotted in Figure 1S and 2S, and the Cartesian coordinates were provided below the figures. To provide a benchmark, the functional ωB97XD with long range corrections from Head-Gordon and coworkers was employed,41 and the large basis set 6-311++G(2df,2pd), including polarization and diffuse functions, was utilized. The structural optimizations at the ground state and frequency analyses were both conducted at the ωB97XD/6-311++G(2df,2pd) level. The scaling factor for the frequency analyses was 0.957. Two-dimensional maps of potential energy surface were scanned in the gas phase for these four systems. The C5-C5’ bond and the C6-C6’ bond were chosen as the two reaction coordinates. The step size for scanning was 0.3 Å. Calculations in implicit water solution were performed with the SMD model.42 The two-dimensional potential energy surfaces in water solution were obtained by single point energy calculations with the structure optimized in the gas phase. In the gas phase and water solution, stationary points were fully optimized without any constraints. Free energies of solvation of stationary structures were computed by taking the difference between the energies in water solution and the energies in the gas phase (E(SMD) – E(gas)). Proton transfers from Glu283 to


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CPD•2- or to CPD- and proton transfers from the splitting products to the deprotonated Glu283 were computed in the gas phase and water solution. The initial structure of Glu283 was obtained from the crystal structure of PDB ID 1TEZ. The initial structure for optimizing the Glu283 + CPD- system and the Glu283 + CPD•2- system is plotted in Figure 3S, and the Cartesian coordinates of the initial structure is provided below the figure. In the calculations that involve Glu283, the C3 and C3’ atoms of the two sugar rings and Cα, C, and N atoms in the backbone of Glu283 were fixed in the space during optimization. The reaction coordinate for the forward proton transfer from Glu283 to CPD models is the difference between the distance R(O-H) of Glu283 and the distance R(O4-H) , where atoms O and H are the –OH group atoms of the sidechain of Glu283, O4 is on the base of the 5’ side as shown in Chart 1. The reaction coordinate for backward proton transfer from the protonated CPD models to the deprotonated Glu283 is the opposite of that of the forward proton transfer. The interaction energy between Glu283 and CPD- or CPD•2- in water solution was calculated by subtracting the energy of the isolated Glu283 and the energy of the isolated CPD- or CPD•2- from their complex. 3. RESULTS 3.1 Geometries of reactants The optimized structures of the four CPD models in the gas phase were aligned in Figure 4S. Their structures are similar. The dihedral angle C5-C6-C6’-C5’ of the four-membered ring is 18.2° in CPD- and 24.4° in CPD•2-. The four-membered ring is twisted more once an electron is injected into CPD-. This dihedral angle is 17.6° in pCPD and 18.5° in pCPD•-. Adding a proton releases the tension of the four-membered ring of CPD-, while adding an electron achieves the opposite effect. The electron affinity of CPD- is -44.1 kcal/mol, which indicates that adding an


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extra electron to CPD- is endothermic and CPD•2- is relatively unstable in the gas phase. In DNA photolyase, the CPD- can obtain an electron with the aid of the excited FADH-. When the CPD models are solvated, the dihedral angle C5-C6-C6’-C5’ is 14.9° in CPD-, 15.2° in CPD•2-, 17.7° in pCPD, and 20.4° in pCPD•-. Unlike the different effects in vacuum, the addition of an electron or a proton amplifies the strain of the four-membered ring. The C5-C5’ bond is 1.59 Å in CPD-, elongated to 1.63 Å in CPD•2- and 1.60 Å in the other two models. The C5-C5’ bond is weakened in the subsequent three models. In vacuum and water solution, the C5-C5’ bond is usually slightly longer than C6-C6’ (Tables 1 and 2), which can be attributed to the repulsion between the two methyl groups on the C5 and C5’ atoms. The repulsion of the two methyl groups can be visualized by the NCIPLOT program.43 Figure 1 shows partial noncovalent interactions in CPD•2-. In Figures 1(a) and 1(b), the light orange color of the isosurface represents weak repulsion between the two carbon atoms of the methyl groups and the green color represents weak attraction between hydrogen atoms of the methyl groups. Therefore, adding either an electron or a proton changes the strain of the four-membered ring, and the C5-C5’ may fracture preferentially. 3.2 Cycloreversion reactions To determine whether the cycloreversion reaction is concerted or stepwise, two-dimensional maps of potential energy surface of the four systems in the gas phase and water solution were computed by scanning the C5-C5’ and C6-C6’ bonds. Maps of the potential energy surface are displayed in Figures 2 and 3. In the maps, R, P, Int, and TS represent reactant before ring opening, product after ring opening, intermediate state, and transition state, respectively. The geometrical parameters of the stationary structures and the corresponding relative changes of


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potential energy, enthalpy, and free energy are provided in Tables 1 and 2. Comparisons of the free energy profiles of the stationary structures in the gas phase and water solution are plotted in Figures 4 and 5. 3.2.1 CPDGas phase: In the gas phase, only one transition state (TS) exists in the map of the potential energy surface (Figure 2(a)); thus the cycloreversion reaction is concerted. The bond distances of C5-C5’ and C6-C6’ are 2.26 and 2.09 Å, respectively, in TS. The free energy of activation (∆Ga ) is the largest ∆Ga (68.6 kcal/mol) among the four systems (Figure 4). Similar to the forward [2+2] cycloaddition process, the reverse cycloreversion reaction is not thermally allowed, as indicated by the high free energy of activation. The HOMO of CPD- (Figure 6(a)) is in the phosphate group, which is not helpful for ring opening. The LUMO of CPD- (Figure 6(b)) is an extended orbital off the methyl group, which enables CPD- to capture an excess electron via a diffuse dipole-bound state and transfer it to a valence-bound state. The HOMO of TS (Figure 6(c)) shows that the thymine on the 5’ side (labeled as 5’ T) adopts π2a mode and the thymine on the 3’ side (labeled as 3’ T) adopts π2s mode for ring opening or forming. The dihedral angle C(methyl)-C5-C6-C4 of 5’ T is 126.0° and 146.7° in R and TS, respectively. The corresponding dihedral angle C’(methyl)-C5’-C6’-C4’ in 3’ T is -127.9° and 158.4° in R and TS, respectively. In R and TS, the absolute value of the dihedral angle C(methyl)-C5-C6-C4 in 5’ T is smaller than that of the dihedral angle C’(methyl)-C5’-C6’-C4’ in 3’ T by 1.9° and 11.7°, respectively. The smaller dihedral angle indicates that the plane defined by the four points C(methyl), C5, C6, and C4 in 5’ T before the formation of CPD is


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bended more to form CPD. The degrees of bending of the dihedrals imply that 5’ T adopts antarafacial bond topology and 3’ T adopts suprafacial bond topology. Both dihedral angles are flattened from R to TS, which elucidates the torsion requirement for a [π2s+π2a] cycloaddition reaction. Water solution: In water solution, the reaction mechanism is also concerted, as illustrated by the single TS in Figure 3(a). The bond distances of C5-C5’ and C6-C6’ are 2.40 and 1.93 Å, respectively. ∆Ga slightly decreases to 65.3 kcal/mol compared with that in the gas phase but remains the greatest ∆Ga among the four systems (Figure 5). Different from the gas phase, the HOMO of CPD- does not locate in the phosphate group; instead, it partially locates in the four-membered ring, especially in the C6-C6’ bond with a bonding feature (Figure 6(d)). If CPD- loses one electron and is converted into CPD• radical, then the C6-C6’ bond preferentially cleaves, which coincides with the experimental observations that CPD radical cation can be produced by photooxidizing sensitizers and the splitting of C6-C6’ was considered as the starting point. The LUMO of CPD- in Figure 6(e) appears as antibonding between C5 and C5’. When CPDacquires an electron and becomes CPD•2-, the repulsion between C5 and C5’ will increase and the C5-C5’ bond may cleave first. The HOMO and LUMO of CPD- explain why cycloreversion of the cyclobutane pyrimidine dimer can proceed either as radical anion or as radical cation when different sensitizers are employed. 3.2.2 CPD•2-


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Gas phase: Two transition states exist in the map of the potential energy (Figure 2(b)); thus, the reaction is stepwise. With slight elongation of C5-C5’ from 1.59 Å to 1.65 Å, CPD•2- attains its first transition state (TS1) and then the C5-C5’ bond immediately breaks. The C6-C6’ bond is 1.64 Å in the intermediate (Int), and 0.07 Å is longer than in R, which implies that C6-C6’ is weakened after the cleavage of C5-C5’. The C6-C6’ bond stretches to 1.98 Å in the second transition state (TS2). ∆Ga of TS1 is only 0.5 kcal/mol, which indicates that the breaking of C5C5’ is almost barrierless. The intermediate (Int) is 21.7 kcal/mol below R in free energy. ∆Ga of TS2 relative to Int is 2.2 kcal/mol. The free energy of activation of the CPD•2- system is the smallest energy of activation among the four systems. The splitting of C6-C6’ is the rate-limiting step. The reaction is an exergonic reaction. As predicted by the LUMO of CPD- in water solution, the HOMO of CPD•2- is antibonding between C5 and C5’ (Figure 7(a)); thus, the C5-C5’ bond tends to separate first. The unpaired electron is approximately evenly distributed between 5’ T and 3’ T in R, TS1, and TS2 (Figure 8(a), 8(b), and 8(d)). In Int, the unpaired electron primarily locates in 5’ T (Figure 8(c)), which is different from the location of the HOMO in Int (Figure 7(c)). This conflict reflects that the α

singly-occupied molecular orbital (α-SOMO) of Int is not the α-HOMO. Figure 9 displays the α and β molecular orbitals (MOs) and MO energies of Int in the gas phase. The α-HOMO is

similar to the β-HOMO and the 143th α-MO (α-HOMO – 1) is similar to the β-LUMO (Figure

9), which implies that the electrons of the α-HOMO and the β-HOMO have bonding interaction in a two-electron orbital picture and the bonding MO for the 143th α-MO is the β-LUMO.

Therefore, the 143th α-MO is the α-SOMO and is lower than α-HOMO in energy. This SOMOHOMO level inversion is a phenomenon that commonly occurs in radical species.44 In P, the unpaired electron locates in 3’ T (Figures 7(e) and 8(e)).


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The distance between C4 in 5’ T and C2’ in 3’ T is 2.35 Å in R; they are sufficiently close to form a conjugation with their p orbitals as demonstrated in Figure 7(a), to disperse the unpaired electron between 5’ T and 3’ T to stabilize the radical. The attraction between C4 and C2’ can also be illustrated by the blue color of the isosurface in Figure 1(c). The conjugation between C4 and C2’ also exists in TS1, as shown in Figure 7(b). In Int, C2’ is 3.47 Å from C4, which is too far to form effective conjugation; thus, the unpaired electron prefers to remain in 5’ T in Int (Figure 8(c)). The unpaired electron prefers to remain in 5’ T because only when the unpaired electron is in 5’ T and the unit negative charge is in 3’ T (Chart 2), can Int be stable; otherwise, the C6-C6’ bond has to be broken to translocate the unpaired electron to 3’ T. Without the conjugation between C4 and C2’ in TS2, the unpaired electron spans over 5’ T and 3’ T via the fracturing C6-C6’ bond (Figures 7(d) and 8(d)). The bond orders of C5-C5’ and C6-C6’ are close to 0.95 in R. After C5-C5’ breaks, the bond order of C6-C6’ decreases to 0.85 in Int, which signifies that C6-C6’ is weaker. The bond orders of C4-C5 and C4’-C5’ increases from approximately 0.95 to 1.2 and 1.4, respectively, which shows the partial formation of double bonds, as indicated in Chart 2. The bond orders of C5-C6 and C5’-C6’ also increase from approximately 0.95 to 1.1, respectively. In P, the bond orders of C5-C6 and C5’-C6’ are 1.7 and 1.3, respectively, and double bonds are finally formed. Water solution: In water solution, the reaction is stepwise with the same splitting sequence as in the gas phase (Figure 3(b)). ∆Ga of TS1 and TS2 are 0.9 and 3.1 kcal/mol, respectively; ∆Ga in the gas phase is 0.5 and 2.2 kcal/mol, respectively. The solvation effect elevates the barriers. Free energies of solvation (∆Gs ) were computed for the stationary structures of this system

(Table 1S). R is better solvated by 0.7 kcal/mol compared with TS1 and Int is better solvated than TS2 by 0.9 kcal/mol. ∆Gs of Int and P is 7.0 and 4.9 kcal/mol larger than that of R, which ACS Paragon Plus Environment

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means that R has the greatest solvation effect and Int has the least solvation effect among these three species. The rate-limiting step comprises the breaking of C6-C6’, whose ∆Ga remains the smallest ∆Ga among the four systems in water solution. Comparing CPD•2- with CPD-, ∆Ga is dramatically decreased upon the uptake of one electron. The reaction in water solution is also an automatic reaction but it is not as exergonic as in the gas phase. The C5-C5’ bond is 1.81 Å in TS1 (Table 2), which is longer than that in the gas phase (1.65 Å). TS1 is stabilized by the solvent. Different from the HOMO in the gas phase, the HOMO and the unpaired electron of R in water solution are primarily situated in 3’ T (Figure 7(f)) because the four-membered ring is less twisted in water solution, which produces long distance (3.22 Å) between C4 and C2’ and prevent conjugation between their p orbitals. The main resonance structures of R are the first two structures shown on the first line of Chart 2. The blue color indicating strong attraction between C4 and C2’ in the gas phase (Figure 1(c)) is replaced by orange color indicating weak repulsion in water solution (Figure 1(d)). Another possible reason for the localization of spin of R is that the radical anion can be better solvated when the unpaired electron is more concentrated. HOMO orbitals and distributions of the unpaired electron in the other stationary points in water solution are similar to those in the gas phase. The SOMO-HOMO level reversion also occurs in Int in water solution. Figure 5S shows that the (α-HOMO – 1) MO is the α-SOMO. 3.2.3 pCPD Gas phase: pCPD splits concertedly in the gas phase (Figure 2(c)). ∆Ga of TS is 36 kcal/mol. Compared with CPD-, the uptake of one proton can substantially reduce the free energy of activation; however, the decrease is not as significant as that caused by the uptake of an electron.


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The uptake of a proton does not change the reaction mechanism. The free energy of reaction (∆r G) relative to R in this system is the lowest among the four systems. Water solution: The splitting of pCPD in water undergoes splitting in a concerted manner as in the gas phase (Figure 3(c)). ∆Ga of TS is 44.6 kcal/mol, which is higher than that in the gas phase. R may be better solvated than TS; thus, ∆Ga in water solution is higher. The product is the most stable product among the four systems in the water solution probably due to the zwitterion nature of pCPD. 3.2.4 pCPD•Gas phase: The two transition states in the potential energy surface (Figure 2(d)) are evidence of stepwise reaction mechanism. ∆Ga of TS1 and TS2 are 7.0 and 9.4 kcal/mol, respectively, which are greater than those in the CPD•2- system. The uptake of one electron is the key reason for the change in the reaction mechanism and the remarkable decrease in the free energy of activation, and protonating CPD•2- will decelerate the reaction. The unpaired electron is approximately evenly distributed between 5’ T and 3’ T in TS1 and TS2 (Figure 6S(b) and 6S(d)). Unlike the CPD•2- system in the gas phase, the unpaired electron primarily resides in 5’ T in R and P (Figures 6S(a) and 6S(e)) and resides in 3’ T in Int (Figure 6S(c)). The major resonance structures of the pCPD•- are plotted in Chart 1S. The paths of the location change of the unpaired electron in CPD•2- in water solution and pCPD•- in the gas phase have one thing in common. From R to Int and to P, the unpaired electron moves in a right-leftright pattern or a left-right-left pattern (Charts 2 and 1S), which implies that the splitting of the C5-C5’ and C6-C6’ bonds is homolytic. Figure 7S shows that the α-SOMO of Int is the (αHOMO – 3) MO.


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Water solution: The reaction mechanism is stepwise (Figure 3(d)). ∆Ga of TS1 and TS2 is 4.9 and 9.2 kcal/mol, respectively. The solvation effect decreases the barrier heights in the pCPD•system but increases the barrier heights in the CPD•2- system. The free energies of activation of the pCPD•- system in water solution are larger than those of the CPD•2- system, which confirms that adding a proton to CPD•2- will not accelerate the reaction rate. The bond distance of C5-C5’ is 1.63 Å in CPD•2- and 1.60 Å in pCPD•- (Table 2). Protonating CPD•2- can stabilize the reactant. The comparison between pCPD•- and CPD•2- shows that the splitting mechanism of CPD•2- is favorable. The distribution of spin density is similar to that in the gas phase, and the SOMO of Int is the (α-HOMO – 1) MO (Figure 8S). 3.3 Proton transfer To further understand the role of the neighboring Glu283 and its effect on the cycloreversion reaction, calculations of proton transfers were performed, including transfers from Glu283 to CPD- and CPD•2-, and the transfers from their products to the deprotonated Glu283 (Glu283-). The optimized structure of the complex of Glu283 and CPD•2- in water solution is illustrated in Figure 9S. The potential energy profiles of the proton transfer reactions between Glu283 and CPD models are presented in Figure 10 and 11. The geometrical parameters of the stationary structures and the relative changes in the potential energy and free energy are tabulated in Table 3. 3.3.1 CPD- + Glu283 In the gas phase and water solution, the proton transfer from Glu283 to CPD- is not automatic (Figures 10(a) and 10(c)). The proton return from the product pTpT to Glu283- in both phases is automatic (Figures 10(b) and 10(d)). The length of the O-H bond (R(O-H)) in Glu283 is 0.99 Å


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in both phases when it forms a complex with CPD-. The distance between H of Glu283 and O4 of CPD- (R(O4-H)) is 1.69 and 1.74 Å in both phases. Glu283 stabilizes CPD- with hydrogen bond and the interaction energy between Glu283 and CPD- in water solution is -5.0 kcal/mol. The unfavorable proton transfer from Glu283 to CPD- and the relatively large free energy of activation in the pCPD system can eliminate the possibility of the pCPD splitting mechanism. 3.3.2 CPD•2- + Glu283 Gas phase: The proton transfer from Glu283 to CPD•2- is automatic (Figure 11(a)), whereas the proton return from the product pTpT•- to Glu283- is not automatic (Figure 11(b)). The negatively charged CPD•2- and TpT•2- are willing to accommodate a proton. In the gas phase, once the electron transfer from FADH-* to CPD- occurs, the proton transfer from Glu283 to CPD•2- would immediately occur, then ring opening proceeds, and finally the proton return is not spontaneous unless an electron returns first (Figure 10(b)). Water solution: The proton transfer from Glu283 to CPD•2- needs to overcome a barrier of 1.2 kcal/mol in free energy, and the free energy of the system is reduced by 2.8 kcal/mol (Figure 11(c) and Table 3). The proton that returns from pTpT•- to Glu283- needs to conquer a barrier of 0.3 kcal/mol in free energy, and the free energy of the system is decreased by 1.3 kcal/mol (Figure 11(d) and Table 3). Both proton transfer processes are automatic in water solution. The length of the O-H bond (R(O-H)) in Glu283 is 1.05 or 1.06 Å in the complex of Glu283 and CPD•2- or TpT•2- (Table 3). In these two complexes, the distance between H of Glu283 and O4 of 5’ T (R(O4-H)) is 1.44 and 1.42 Å. Compared with the lengths of R(O-H) and R(O4-H) in the complex of Glu283 and CPD-, R(O-H) is longer and R(O4-H) is shorter in the complex of Glu283 and CPD•2-, which implies that the hydrogen bond between Glu283 and CPD•2- is


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stronger. The interaction energy between Glu283 and CPD•2- is -8.3 kcal/mol, which is 3.3 kcal/mol lower than the interaction energy between Glu283 and CPD-. The stabilization of CPD•2- by Glu283 may decelerate the rate of back-transfer of an electron from CPD•2- to FADH• and produce a higher quantum yield. Although Glu283 can stabilize the reactant and the product, the total reaction rate of the pCPD•- system is slower than that of the CPD•2- system. Therefore, Glu283 cannot accelerate the reaction rate by protonating CPD•2- can promote the quantum yield by stabilizing the reactant with hydrogen bonding. 4. DISCUSSION Based on the divergences on the cycloreversion mechanism of the cyclobutane pyrimidine dimer in an impaired DNA caused by UV light and the unclear role of Glu283, we performed highlevel density functional theory calculations to explore the mechanisms. The calculations were performed in the gas phase to understand the intrinsic paths and properties and were performed in water solution to evaluate the solvation effects. Proton transfer reactions between the Glu283 and CPD models were conducted to investigate the possibility of protonation during ring opening. The accuracy of the computational results, the conclusions inferred by the results, and the defect of this study should be addressed. In water solution, the computed free energy change of ring opening (∆r G) of CPD•2- is -23.5 kcal/mol and the enthalpy change (∆r H) is -19.5 kcal/mol (Table 2). The experimental values of ∆r G and ∆r H of opening the dimer radical anion of 1,3-dimethylthymine in CH3CN are

approximately -20 kcal/mol.45 Our computed results coincide with the experimental results. In water solution, the computed ∆r H of opening CPD- is -17.2 kcal/mol. The experimental ∆r H of


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opening the neutral dimer of 1,3-dimethylthymine in CH3CN is -19 kcal/mol.46 Our computational result is consistent with the experimental result. Based on Figures 4 and 5, the CPD•2- system has the lowest free energy of activation for the ratelimiting step; therefore, the CPD•2- splitting mechanism can be rationalized as the most possible reaction mechanism. In this mechanism, C5-C5’ breaks first followed by the breaking of C6-C6’. In water solution, the computed ∆a G of the two cleavages are 0.9 and 3.1 kcal/mol. According to the transition state theory, the computed reaction rates are 1.4 and 0.033 ps-1. The lifetimes derived from the computational results are 0.74 and 30 ps. In 2011, the two experimental lifetimes obtained by Liu and coworkers were 10 and 90 ps.9 The computed lifetime for C5-C5’ breaking is considerably shorter than the experimental results but the computed lifetime for C6C6’ breaking is on the same order as the experimental value. The free energies of activation derived from the experimental lifetime are 2.4 and 3.7 kcal/mol. The computed free energy of activation for C6-C6’ breaking shows agreement with the experimental value (3.1 vs. 3.7 kcal/mol). The rate of proton transfer from Glu283 to CPD•2- is comparable to that of the first bond breaking of CPD•2- (1.2 vs. 0.9 kcal/mol for ∆Ga , respectively); thus, proton hopping to CPD•2before it begins to break is possible in a complex environment like enzyme. If the rate-limiting steps are used to estimate the reaction rate constants (k) for the pCPD•- and CPD•2- systems in water solution, the ratio k(pCPD•-)/k(CPD•2-) is 3×10-4, which shows that the effect of the pCPD•reaction mechanism is minimal. Therefore, protonating CPD•2- by Glu283 cannot increase the reaction rate but Glu283 can stabilize CPD•2- with hydrogen bond and may decelerate the rate of back electron transfer, which produces a higher quantum yield and may explain why E283A has a 60% reduction in quantum yield.27


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The potential energy surface and the free energy profile of the CPD•2- system in water solution confirm that the reaction mechanism is stepwise, in which C5-C5’ breaks almost immediately after the uptake of an electron, the intermediate is relatively stable, and the rate-limiting step is the cleavage of C6-C6’. The HOMO of CPD•2- is antibonding between C5 and C5’. Consistent with the Möbius concept of Zimmerman,47-48 the HOMO of the first transition state indicates that 5’ T adopt π2a mode and 3’ T adopts π2s mode, which exhibits more strain on the C5-C5’ bond. These two factors and the steric effect caused by the two methyl groups on the C5 and C5’ atoms cause the priority of the C5-C5’ bond to break. The HOMO of CPD- shows that CPD- can also open by losing one electron. When CPD• is generated, the C6-C6’ bond is predicted to break first by the HOMO of CPD-, which is consistent with the experimental observations11, 15 and other theoretical calculations.22, 28, 49 Clinically, proper photooxidizing or photoreducing photosensitizers may be added to liposomes to apply to skin to treat sunburn. In the CPD•2- system, the solvation effect increases the barrier heights by better solvation of the reactant and the intermediate state compared with their subsequent transition states. The unpaired electron in the reactant and the intermediate state primarily locates in one thymine ring, while the unpaired electron in the two transition states is approximately evenly distributed between the two thymine rings. The spin delocalization may decrease the solvation effect. In the CPD•2- system in water solution, the unpaired electron and the unit negative charge in the reactant and the product reside in the same ring and the same atoms. For example, the unpaired electron and the unit negative charge of R both reside in atoms C4’ and O4’ of 3’ T in water solution, which is displayed by Figure 7(f) or schematically shown by the first two resonance


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structures of the first line of Chart 2. While in the intermediate state the unpaired electron and the unit negative charge reside in different thymine rings as indicated by Chart 2. This differential polarization of spin and charge was also found in other radicals.50 The differential polarization of Int can be caused by the SOMO-HOMO level inversion. As displayed in Figure 5S, α-HOMO and β-HOMO mainly locate in atoms C4’, O4’, and C5’ of 3’ T, which coincides with the distribution of the unit negative charge as shown in the first three main resonance structures of Int in Chart 2; thus, the unit negative charge is correlated with the α-HOMO or β-HOMO. The α-SOMO containing the unpaired electron locates in 5’ T. Consequently, it appears that the spin and the charge are differentially polarized in Int. In R and P, the SOMO-HOMO level inversion does not occurs and the differential polarization does not exist. In previous quantum mechanical studies, the models that have been employed included uracil dimer,31 thymidine dimer with a methylene bridge (-CH2-),36 thymidine dimer with phosphate group calculated in the gas phase not in water solution,51 thymine dimer,24, 31 and thymidine dimer without phosphate group.24 The models in this study are thymidine dimers with a phosphate group connecting the two thymidine monomers. The models in this study are closer to the CPD in a damaged DNA and can produce the parallel stacking conformation for the products by preventing free movement of the monomers after the cleavage. The employment of the implicit water model in this study provides closer environment to the enzyme than in the gas phase. However, the implicit water model differs from the explicit water solvent and true enzyme environment. To obtain a more accurate picture of the repair mechanism that is catalyzed by the DNA photolyase, calculations for enzymes with explicit water solvent and DNA photolyase are needed, which will be included in our next study. 5. CONCLUSIONS


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To investigate the mechanism of repairing photoinduced product CPD catalyzed by DNA photolyase and determine whether protonation of CPD participates in the DNA repair process, high-level density functional theory calculations were performed to study the cycloreversion reactions of CPD-, CPD•2-, pCPD, and pCPD•- systems and the processes of proton transfer between Glu283 and CPD models in the gas phase and water solution. The CPD•2- system has the smallest free energy of activation for the rate-limiting step via a stepwise reaction mechanism which is considered to be the most possible reaction mechanism. This stepwise reaction mechanism differs from the already proposed concerted mechanism and the asynchronously concerted one-step reaction mechanism. In the concerted reaction mechanism, only one transition state with two breaking bonds exists and there is no intermediate state. In the asynchronously concerted one-step reaction mechanism, the two bonds break sequentially but only one transition state usually with breaking C6-C6’ bond exits and there is no stable intermediate state. The stepwise reaction mechanism proposed in this study has two transition states with breaking C5-C5’ bond and breaking C6-C6’ bond, respectively, and one stable intermediate state with broken C5-C5’ bond and unbroken C6-C6’ bond. The cleavages of the C5-C5’ bond and the C6-C6’ bond are homolytic. The computed free energies of activation for C5-C5’ and C6-C6’ breaking in water solution are 0.9 and 3.1 kcal/mol, respectively. The breaking of the C6-C6’ bond is the rate-limiting step. The steric effects of the methyl groups and the repulsion between C5 and C5’ caused by the unpaired electron render the C5-C5’ bond fragile. The SOMO-HOMO level reversion occurs in the intermediate state. Protonation of CPD•2- by Glu283 will reduce the reaction rate but Glu283 can stabilize CPD•2- via hydrogen bond and increase the quantum yield. The solvation effects increases the free energies of activation.


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ASSOCIATED CONTENT Supporting Information. Initial structures of the models. Optimized structures of four CPD models in the gas phase. Molecular orbitals and orbital energies of the CPD•2- system in water solution and the pCPD•- system in both phases. Spin density of the pCPD•- system in the gas phase. Major resonance structures of the pCPD•2- system. Optimized structure of the complex of CPD•2- and Glu283 in water solution. Free energies of solvation of the CPD•2- system. Cartesian coordinates of all stationary points. AUTHOR INFORMATION *Corresponding author. E-mail: [email protected], [email protected]. ACKNOWLEDGMENT This study was supported by the National Natural Science Foundation of China (Grant 21503043), the Hundred-Talent Program for Introduction of Overseas High-level Talents by Universities in Guangxi (GJR2015-57), the Natural Science Foundation of Guangxi Province (Grant 2015GXNSFCA139020), the Scientific Research Foundation of Guangxi University for Nationalities (Grant 2015MDQD013), the Special Subsidies for Local Science and Technology Development Guided by the Central Committee (ZY18076005), and the Innovation Project of Guangxi Graduate Education (gxun-chxzs201719). This study was also supported by the Network and Information Management Center of Guangxi University for Nationalities. REFERENCES 1.

Faraji, S.; Dreuw, A., Physicochemical Mechanism of Light-Driven DNA Repair by (6-4)

Photolyases. Annu. Rev. Phys. Chem. 2014, 65, 275-292.


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Page 23 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2.

Cadet, J.; Vigny, P., Bioorganic Photochemistry, volume 1: The Photochemistry of Nucleic

Acids. Morrison, Harry ed.; Wiley-Interscience: New York, 1990; p 1-272. 3.

Stege, H.; Roza, L.; Vink, A. A.; Grewe, M.; Ruzicka, T.; Grether-Beck, S.; Krutmann, J.,

Enzyme Plus Light Therapy to Repair DNA Damage in Ultraviolet-B-Irradiated Human Skin. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 1790-1795. 4.

Cadet, J.; Douki, T., Formation of UV-induced DNA Damage Contributing to Skin Cancer

Development. Photochem. Photobiol. Sci. 2018, 17, 1816-1841. 5.

Fuss, J. O.; Cooper, P. K., DNA Repair: Dynamic Defenders against Cancer and Aging.

PloS Biol. 2006, 4, e203. 6.

Sancar, A., Structure and Function of DNA Photolyase. Biochemistry 1994, 33, 2-9.

7.

Teranishi, M.; Nakamura, K.; Morioka, H.; Yamamoto, K.; Hidema, J., The Native

Cyclobutane Pyrimidine Dimer Photolyase of Rice Is Phosphorylated. Plant Physiol. 2008, 146, 1941-1951. 8.

Selby, C. P.; Sancar, A., A Cryptochrome/Photolyase Class of Enzymes with Single-

Stranded DNA-Specific Photolyase Activity. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 1769617700. 9.

Liu, Z.; Tan, C.; Guo, X.; Kao, Y. T.; Li, J.; Wang, L.; Sancar, A.; Zhong, D., Dynamics

and Mechanism of Cyclobutane Pyrimidine Dimer Repair by DNA Photolyase. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 14831-14836. 10. Berardesca, E.; Bertona, M.; Altabas, K.; Altabas, V.; Emanuele, E., Reduced UltravioletInduced DNA Damage and Apoptosis in Human Skin with Topical Application of a Photolyase-


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Page 24 of 47

Containing DNA Repair Enzyme Cream: Clues to Skin Cancer Prevention. Molecular Medicine Reports 2012, 5, 570-574. 11. McMordie, R. A. S.; Begley, T. P., Mechanistic Studies on DNA Photolyase. 5. Secondary Deuterium Isotope Effects on the Cleavage of the Uracil Photodimer Radical Cation and Anion. J. Am. Chem. Soc. 1992, 114, 1886-1887. 12. Kim, S.-T.; Sancar, A.; Essenmacher, C.; Babcock, G. T., Evidence from Photoinduced EPR for a Radical Intermediate during Photolysis of Cyclobutane Thymine Dimer by DNA Photolyase. J. Am. Chem. Soc. 1992, 114, 4442-4443. 13. Yeh, S.-R.; Falvey, D. E., Model Studies of DNA Photorepair: Energetic Requirements for the Radical Anion Mechanism Determined by Fluorescence Quenching. J. Am. Chem. Soc. 1992, 114, 7313-7314. 14. Rokita, S. E.; Walsh, C. T., Flavin and 5-Deazaflavin Photosensitized Cleavage of Thymine Dimer: A Model of in Vivo Light-Requiring DNA Repair. J. Am. Chem. Soc. 1984, 106, 4589-4595. 15. Burdi, D.; Begley, Mechanistic Studies on DNA Photolyase. 3. The Trapping of the OneBond-Cleaved Intermediate from a Photodimer Radical Cation Model System. J. Am. Chem. Soc. 1991, 113, 7768-7770. 16. Essen, L.-O.; Klar, T., Light-driven DNA repair by photolyases. Cell. Mol. Life Sci 2006, 63, 1266-1277. 17. Begley, T. P., Photoenzymes: A Novel Class of Biological Catalysts. Acc. Chem. Res. 1994, 27, 394-401.


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Page 25 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


18. Pouwels, P. J.; Hartman, R. F.; Rose, S. D.; Kaptein, R., Photo-CIDNP Study of Pyrimidine Dimer Splitting I: Reactions Involving Pyrimidine Radical Canion Intermediates. Photochem. Photobiol. 1995, 61, 563-574. 19. Pouwels, P. J.; Hartman, R. F.; Rose, S. D.; Kaptein, R., Photo-CIDNP Study of Pyrimidine Dimer Splitting II: Reactions Involving Pyrimidine Radical Anion Intermediates. Photochem. Photobiol. 1995, 61, 575-583. 20. Durbeej, B.; Eriksson, L. A., Thermodynamics of the Photoenzymic Repair Mechanism Studied by Density Functional Theory. J. Am. Chem. Soc. 2000, 122, 10126-10132. 21. Voityuk, A. A.; Michel-Beyerle, M. E.; Rösch, N., A Quantum Chemical Study of Photoinduced DNA Repair: On the Splitting of Pyrimidine Model Dimers Initiated by Electron Transfer. J. Am. Chem. Soc. 1996, 118, 9750-9758. 22. Aida, M.; Kaneko, M.; Dupuis, M., An Ab Initio MO Study on the Thymine Dimer and Its Radical Cation. Int. J. Quantum Chem. 1996, 57, 949-957. 23. Voityuk, A. A.; Rösch, N., Ab Initio Study on the Structure and Splitting of the Uracil Dimer Anion Radical. J. Phys. Chem. A 1997, 101, 8335-8338. 24. Barbatti, M., Computational Reference Data for the Photochemistry of Cyclobutane Pyrimidine Dimers. Chem. Phys. Chem. 2014, 15 (15), 3342-3354. 25. Chinnapen, D. J.-F.; Sen, D., A Deoxyribozyme that Harnesses Light to Repair Thymine Dimers in DNA. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 65-69.


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26. Thiagarajan, V.; Byrdin, M.; Eker, A. P. M.; Muller, P.; Brettel, K., Kinetics of Cyclobutane Thymine Dimer Splitting by DNA Photolyase Directly Monitored in the UV. Proc. Natl. Acad. Sci. U. S. A. 2011, 108 (23), 9402-9407. 27. Vande Berg, B. J.; Sancar, G. B., Evidence for Dinucleotide Flipping by DNA Photolyase. J. Biol. Chem. 1998, 273, 20276-20284. 28. Rak, J.; Voityuk, A. A.; Michel-Beyerle, M. E.; Rösch, N., Effect of Proton Transfer on the Anionic and Cationic Pathways of Pyrimidine Photodimer Cleavage. A Computational Study. J. Phys. Chem. A 1999, 103, 3569-3574. 29. Masson, F.; Laino, T.; Rothlisberger, U.; Hutter, J., A QM/MM Investigation of Thymine Dimer Radical Anion Splitting Catalyzed by DNA Photolyase. Chem. Phys. Chem. 2009, 10 (2), 400-410. 30. Saettel, N. J.; Wiest, O., DFT Study of the [2 + 2] Cycloreversion of Uracil Dimer Anion Radical: Waters Matter. J. Am. Chem. Soc. 2001, 123, 2693-2694. 31. Harrison, C. B.; O'Neil, L. L.; Wiest, O., Computational Studies of DNA Photolyase. J. Phys. Chem. A 2005, 109 (32), 7001-7012. 32. Masson, F.; Laino, T.; Tavernelli, I.; Rothlisberger, U.; Hutter, J., Computational Study of Thymine Dimer Radical Anion Splitting in the Self-Repair Process of Duplex DNA. J. Am. Chem. Soc. 2008, 130, 3443-3450. 33. Tachikawa, H.; Kawabata, H., Interaction between Thymine Dimer and Flavin-Adenine Dinucleotide: A DFT and Direct Ab Initio Molecular Dynamics Study. J. Phys. Chem. B 2008, 112, 7315-7319.


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Page 27 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


34. Hassanali, A. A.; Zhong, D. P.; Singer, S. J., An AIMD Study of the CPD Repair Mechanism in Water: Reaction Free Energy Surface and Mechanistic Implications. J. Phys. Chem. B 2011, 115 (14), 3848-3859. 35. Hassanali, A. A.; Zhong, D. P.; Singer, S. J., An AIMD Study of CPD Repair Mechanism in Water: Role of Solvent in Ring Splitting. J. Phys. Chem. B 2011, 115 (14), 3860-3871. 36. Chatgilialoglu, C.; Guerra, M.; Kaloudis, P.; Houée-Lévin, C.; Marignier, J.-L.; Swaminathan, V. N.; Carell, T., Ring Opening of the Cyclobutane in a Thymine Dimer Radical Anion. Chem. Eur. J. 2007, 13, 8979-8984. 37. MacFarlane, A. W.; Stanley, R. J., Cis-Syn Thymidine Dimer Repair by DNA Photolyase in Real Time. Biochemistry 2003, 42, 8558-8568. 38. Tantillo, D. J.; Chen, J.; Houk, K. N., Theozymes and Compuzymes: Theoretical Models for Biological Catalysis. Curr. Opin. Chem. Biol. 1998, 2, 743-750. 39. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Petersson, G. A.; Nakatsuji, H. et al. Gaussian 09, Revision E. 01, Gaussian 09, Revision E. 01; Gaussian, Inc., Wallingford CT, 2013. 40. Park, H.; Zhang, K.; Ren, Y.; Nadji, S.; Sinha, N.; Taylor, J.-S., Crystal Structure of a DNA Decamer Containing a cis-syn Thymine Dimer. Proc. Natl. Acad. Sci. U. S. A. 2002, 99 (25), 15965-15970. 41. Chai, J.-D.; Head-Gordon, M., Long-Range Corrected Hybrid Density Functionals with Damped Atom–Atom Dispersion Corrections. Phys. Chem. Chem. Phys. 2008, 10, 6615-6620.


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42. Marenich, A. V.; Cramer, C. J.; Truhlar, D. G., Universal Solvation Model Based on Solute Electron Density and on a Continuum Model of the Solvent Defined by the Bulk Dielectric Constant and Atomic Surface Tensions. J. Phys. Chem. B 2009, 113, 6378-6396. 43. Contreras-García, J.; Johnson, E. R.; Keinan, S.; Chaudret, R.; Piquemal, J.-P.; Beratan, D. N.; Yang, W., NCIPLOT: A Program for Plotting Noncovalent Interaction Regions. J. Chem. Theory Comput. 2011, 7, 625-632. 44. Kumar, A.; Sevilla, M. D., SOMO-HOMO Level Inversion in Biologically Important Radicals. J. Phys. Chem. B 2018, 122, 98-105. 45. Scannell, M. P.; Fenick, D. J.; Yeh, S.-R.; Falvey, D. E., Model Studies of DNA Photorepair: Reduction Potentials of Thymine and Cytosine Cyclobutane Dimers Measured by Fluorescence Quenching. J. Am. Chem. Soc. 1997, 119, 1971-1977. 46. Scannell, M. P.; Yeh, S.-R.; Falvey, D. E., Model Studies of DNA Photorepair: Enthalpy of Cleavage of a Pyrimidine Dimer Measured by Photothermal Beam Deflection Calorimetry. Photochem. Photobiol. 1996, 64, 764-768. 47. Zimmerman, H. E., On Molecular Orbital Correlation Diagrams, the Occurrence of Möbius Systems in Cyclization Reactions, and Factors Controlling Ground- and Excited-State Reactions. I. J. Am. Chem. Soc. 1966, 88 (7), 1564-1565. 48. Zimmerman, H. E., Molecular Orbital Correlation Diagrams, Mobius Systems, and Factors Controlling Ground- and Excited-State Reactions. II. J. Am. Chem. Soc. 1966, 88 (7), 1566-1567.


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49. Rak, J.; Voityuk, A. A.; Rösch, N., Splitting of Cyclobutane-Type Uracil Dimer Cation Radicals. Hartree-Fock, MP2, and Density Functional Studies. J. Phys. Chem. A 1998, 102, 71687175. 50. Fehir, R. J.; McCusker, J. K., Differential Polarization of Spin and Charge Density in Substituted Phenoxy Radicals. J. Phys. Chem. A 2009, 113, 9249-9260. 51. Ando, H.; Fingerhut, B. P.; Dorfman, K. E.; Biggs, J. D.; Mukamel, S., Femtosecond Stimulated Raman Spectroscopy of the Cyclobutane Thymine Dimer Repair Mechanism: A Computational Study. J. Am. Chem. Soc. 2014, 136 (42), 14801-14810.


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Figure 1. Partial gradient isosurface (s = 0.5 au) for CPD•2- (a) among atoms of four-membered ring in the gas phase, (b) among atoms of four-membered ring in water solution, (c) between atoms C4 and C2’ in the gas phase, and (d) between atoms C4 and C2’ in water solution. The surface is colored on a blue-green-red scale according to values of sign(λ2)ρ ranging from -0.03 to 0.03 au. Blue denotes strong attractive interactions, red denotes strong repulsive interactions.


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Figure 2. Two-dimensional maps of potential energy surface of (a) CPD anion (CPD-), (b) CPD radical anion (CPD•2-), (c) protonated CPD molecule (pCPD), and (d) protonated CPD radical anion (pCPD•-) in the gas phase.


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105 90 75 60 45 30 15 0 -5 -10 -15

R (C6-C6')/Å

3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6

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3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6

50 40 30 25 20 15 10 5 1 0 -5 -8 -10 -13 -15 -20

R (C5-C5')/Å

R (C5-C5')/Å 3.0

3.0

2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6

70 60 50 40 30 20 15 10 5 0 -5 -10 -15 -20

2.8 2.6

R (C6-C6')/Å

100 80 70 60 50 40 30 20 10 0 -10 -25

2.8

R (C6-C6')/Å

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

R (C6-C6')/Å


2.4 2.2 2.0 1.8 1.6 1.4 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6

R (C5-C5')/Å

R (C5-C5')/Å

Figure 3. Two-dimensional maps of potential energy surface of (a) CPD anion (CPD-), (b) CPD radical anion (CPD•2-), (c) protonated CPD molecule (pCPD), and (d) protonated CPD radical anion (pCPD•-) in water solution. The potential energies in these four maps were obtained by single point energy calculations in SMD model with the structures optimized in the gas phase.


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Figure 4. Free energy profiles of stationary points in the gas phase. The relative free energies are given in parentheses. The sugar rings are omitted in the chemical structures.


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Figure 5. Free energy profiles of stationary points in water solution. The relative free energies are given in parentheses. The sugar rings are omitted in the chemical structures.


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Figure 6. Frontier molecular orbitals of the CPD- system in the gas phase: (a) HOMO of R, (b) LUMO of R, (c) HOMO of TS, and frontier molecular orbitals of the CPD- system in water solution: (d) HOMO of R, (e) LUMO of R. The isovalue is 0.03.


35


Page 36 of 47

Figure 7. HOMO of the CPD•2- system in the gas phase: (a) R, (b) TS1, (c) Int, (d) TS2, (e) P, and (f) the HOMO of R in water solution. The isovalue is 0.03.


36

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Figure 8. Spin density of the CPD•2- system in the gas phase: (a) R, (b) TS1, (c) Int, (d) TS2, (e) P. The isovalue is 0.003.


37


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Figure 9. α and β molecular orbitals (MOs) of Int of the CPD•2- system in the gas phase. MO energies are given in parentheses. The isovalue for plotting MOs is 0.03. The singly-occupied molecular orbital (SOMO) is the 143th α-MO (α-HOMO – 1).


38

Page 39 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 10. Potential energy profiles of proton transfer reactions: (a) Glu283 + CPD- → Glu283+ pCPD in the gas phase. (b) Glu283- + pTpT → Glu283 + TpT- in the gas phase. (c) Glu283 + CPD- → Glu283- + pCPD in water solution. (d) Glu283- + pTpT → Glu283 + TpT- in water solution. Glu283- is the deprotonated Glu283. The RC of (a) and (c) is RC = R(O-H) – R(O4-H), and the RC of (b) and (d) is RC = R(O4-H) – R(O-H). The potential energy profiles in the gas phase were optimized at the ωB97XD/6-311++G(2df,2pd) level. The potential energy profiles in water solution were the single point energies of ωB97XD/6-311++G(2df,2pd) with the structures optimized at the ωB97XD/6-31+G(d,p) level in water solution.


39


Page 40 of 47

Figure 11. Potential energy profiles of proton transfer reactions: (a) Glu283 + CPD•2- → Glu283+ pCPD•- in the gas phase. (b) Glu283- + pTpT•- → Glu283 + TpT•2- in the gas phase. (c) Glu283 + CPD•2- → Glu283- + pCPD•- in water solution. (d) Glu283- + pTpT•- → Glu283 + TpT•2- in water solution. Glu283- is the deprotonated Glu283. The RC of (a) and (c) is RC = R(O-H) – R(O4-H), and the RC of (b) and (d) is RC = R(O4-H) – R(O-H). The potential energy profiles both in the gas phase and in water solution were optimized at the ωB97XD/6-311++G(2df,2pd) level.


40

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Scheme 1. Repair cycle of DNA photolyase. TpT: thymidine deoxydinucleotide; CPD: cyclobutane

pyrimidine

dimer;

8-HDF:

8-hydroxy-5-deazaflavin;

MTHF:

5,10-

methenyltetrahydrofolate.


41

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 42 of 47

Chart 1. Chemical structures of CPD anion before electron transfer (CPD-), CPD radical anion formed after electron transfer (CPD•2-), protonated CPD molecule (pCPD), and protonated CPD radical anion (pCPD•-). O4-H

O4' CH3 CH3

C4 N2 C2

C4' C5

C5'

C6

C6'

N3' C2'

N1

O2

N1'

O2'

H H O O HO

OH

O O P

O

O


42

Page 43 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


Chart 2. Major resonance structures of the stationary structures of the CPD•2- system in the gas phase. The sugar rings are omitted. O

O

O

H C5 C6

C5'

H N

N

C5

C6'

N

O

O

O

H C5

C5'

C6

C6'

N

O

N

O

O

C6

C6'

N

O

O

C6

C6'

C5

C5'

O

C5

C5'

C6

C6'

N

N

N

O

C6

C6'

N

N

O

H N

O

O

H

N

N H H

O

O

O

H

N

N H H

H H

H C5'

O

C6'

N

H N

O

N

H H

N

O

O

C5

O

C5'

H N

H H

N

N

H

N

N

O

C6'

O

H

O

C6

C5 C6

O

H

N

H H

H C5'

C5'

N

O

O

C5

C5

H N

N

O

O

O

H

N

C6'

N

O

H N

H H

N

O

H

N

C5'

H H

O

H

C5 C6

N

O

H N

H H

O

O

H

N

C6'

N

O

H H

N

C5'

C6 N

O

H

H N

O

O

C5

C5'

C6

C6'

N

N

N

O

H H


43


Page 44 of 47

Table 1. Geometrical parameters of the stationary structures optimized in the gas phase and the corresponding relative changes of potential energy, enthalpy, and free energy.

CPD-

CPD•2-

pCPD

pCPD•-

R(C5-C5') Å

R(C6-C6') Å

ΔΕ kcal/mol

ΔH kcal/mol

ΔG kcal/mol

R

1.59

1.57

0.0

0.0

0.0

TS

2.26

2.09

71.6

67.9

68.6

P

4.02

4.07

-21.7

-22.3

-26.4

R

1.59

1.57

0.0

0.0

0.0

TS1

1.65

1.57

1.4

0.3

0.5

Int

3.12

1.64

-18.5

-18.5

-21.7

TS2

3.22

1.98

-16.1

-16.9

-19.5

P

3.43

3.32

-23.4

-24.1

-30.4

R

1.58

1.58

0.0

0.0

0.0

TS

2.41

1.73

38.3

35.8

36.0

P

4.24

3.75

-36.0

-37.0

-38.5

R

1.58

1.58

0.0

0.0

0.0

TS1

2.01

1.57

8.8

7.3

7.0

Int

3.21

1.59

-12.0

-12.8

-13.7

TS2

3.31

2.07

-1.8

-4.0

-4.3

P

4.57

4.57

-28.4

-30.1

-33.5


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Table 2. Geometrical parameters of the stationary structures optimized in water solution and the corresponding relative changes of potential energy, enthalpy, and free energy.

CPD-

CPD•2-

pCPD

pCPD•-

R(C5-C5') Å

R(C6-C6') Å

ΔΕ kcal/mol

ΔH kcal/mol

ΔG kcal/mol

R

1.59

1.56

0.0

0.0

0.0

TS

2.40

1.93

68.8

66.0

65.3

P

3.62

3.87

-16.5

-17.2

-21.6

R

1.63

1.57

0.0

0.0

0.0

TS1

1.81

1.57

2.1

0.8

0.9

Int

3.10

1.60

-11.5

-11.9

-13.9

TS2

3.17

1.95

-8.2

-9.7

-10.8

P

3.55

3.83

-18.5

-19.5

-23.5

R

1.60

1.56

0.0

0.0

0.0

TS

2.53

1.73

47.9

44.4

44.6

P

4.22

4.05

-23.8

-25.0

-28.2

R

1.60

1.57

0.0

0.0

0.0

TS1

1.99

1.56

6.6

4.9

4.9

Int

3.05

1.59

-11.1

-11.8

-13.1

TS2

3.16

2.07

-1.3

-3.2

-3.9

P

3.53

3.86

-19.4

-21.4

-25.8


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Table 3. Geometrical parameters of stationary structures of the proton transfer reactions and the relative changes of potential energy and free energy in water solution.a R(O-H) Å

R(O4-H) Å

ΔΕ kcal/mol

ΔG kcal/mol

Glu283 + CPD•2- → Glu283- + pCPD•R

1.05

1.44

0.0

0.0

TS

1.14

1.28

2.5

1.2

P

1.66

1.00

-2.2

-2.8

Glu283- + pTpT•- → Glu283 + TpT•2R

1.56

1.01

0.0

0.0

TS

1.15

1.26

2.3

0.3

P

1.06

1.42

0.4

-1.3

a

R(O-H) is the distance between carboxyl oxygen and the carboxyl hydrogen of Glu283. R(O4H) is the distance between carboxyl hydrogen of Glu283 and O4 atom in 5’ T of CPD models.


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47

Exploring Cycloreversion Reaction of Cyclobutane Pyrimidine Dimers

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