Proton-Transfer-Steered Mechanism of Photolesion Repair by (6–4

Letter pubs.acs.org/JPCL

Proton-Transfer-Steered Mechanism of Photolesion Repair by (6−4)Photolyases Shirin Faraji and Andreas Dreuw* Interdisciplinary Center for Scientific Computing, Ruprecht-Karls University, Im Neuenheimer Feld 368, 69120 Heidelberg, Germany S Supporting Information *

ABSTRACT: DNA (6−4)-photolyases are enzymes initiating cleavage of mutagenic pyrimidine (6−4) pyrimidone photolesions by a photoinitiated electron transfer from flavin adenine dinucleotide to the lesion. Using state-of-the-art quantum chemical calculations, we present the first energetically feasible molecular repair mechanism. The initial step is electron transfer coupled to proton transfer from the protonated His345 to the N3′ nitrogen of the pyrimidone thymine of the lesion, which proceeds simultaneously with intramolecular OH transfer in a concerted reaction without formation of an oxetane or isolated water molecule intermediate. In contrast to previously suggested mechanisms, this newly identified pathway requires neither a two-photon process nor electronic excitation of the photolesion. Indeed, the recognition that the initial electron transfer is coupled to the proton transfer was critically important for clarification of the mechanism. SECTION: Biophysical Chemistry

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most recent experimental findings21 along with the geometrical arrangement of the thymine−thymine T(6−4)T-PP in the Xray structure,22 however, oxetane formation prior to the electron transfer can be ruled out. The second group18,21−23 indeed excludes the formation of an oxetane ring at all. Instead, it is suggested that spatially close histidines act as proton donors to trigger lesion repair. In this context, the formation of a transient water molecule intermediate has been suggested.22 Recent theoretical studies suggest that the repair occurs via a two-photon process,23 or proceeds via a conical intersection24 in the electronically excited state of the lesion radical anion. However, both these repair mechanisms could be ruled out as being relevant. Natural photon density is too small on the Earth’s surface to allow for an efficient two-photon process. In addition, it has been pointed out by simple thermodynamic considerations that the repair mechanism must occur in the electronic ground state of the lesion radical anion,25 since the initially absorbed photon energy is not sufficient to initiate electron transfer and to simultaneously electronically excite the radical anion of the T(6−4)T-PP. Here, we are going to demonstrate by means of quantum chemical calculations that the repair of the T(6−4)TPP definitely does not proceed via an oxetane intermediate and also that the formation of a transient, isolated water molecule does not lead to an efficient repair pathway. Instead, we identify

he most significant cellular target of UV light is DNA.1 Upon exposure to far-UV radiation (200−300 nm), adjacent pyrimidine bases, cytosine and thymine, within the same DNA strand may become covalently linked by the formation of predominantly cyclobutane pyrimidine dimers (CPDs) and, to a minor degree, of pyrimidine (6−4) pyrimidone photoproducts ((6,4)-PP).2−5 Persistence of these lesions can interfere with essential processes such as transcription, DNA/RNA polymerases, and DNA replication and may lead to mutation, nucleotide misincorporation, and cell death. To cope with these types of photodamage, organisms have developed a number of methods for their repair. Photolyases are fascinating DNA repair enzymes that again use light in the near UV/blue region themselves to eliminate these UV-derived photodamages.6,7 Upon light absorption, photoinduced electron transfer from the catalytic cofactor, reduced flavin adenine dinucleotide (FADH−), toward the lesion takes place, initiating the repair.8−12 Many biochemical and biophysical studies have been carried out to unravel the fundamentals of the subsequent electron-induced repair mechanism. In contrast to CPD13−16 photolyases, the details have remained largely elusive for (6−4) photolyases both experimentally and theoretically, and its repair mechanism is thus still a matter of ongoing debate. In general, (6−4)photolyase DNA-repair mechanisms proposed in the literature can be divided into two groups: oxetane and non-oxetane mechanisms. In the first,12,17−20 the (6−4)-PP is converted to an oxetane ring, which may occur after or before the photoinitiated electron transfer from FADH−. On the basis of © 2012 American Chemical Society

Received: November 16, 2011 Accepted: January 3, 2012 Published: January 3, 2012 227

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electron goes to the protonated HIS365, since the positive charge of the proton strongly attracts the electron in our model calculations. This discrepancy may well originate from limitations in our molecular model. Regardless, our static model calculations do not allow one to conclude whether the electron or proton is transferred first, only that if one is transferred the other follows instantaneously. Hence these seemingly independent events are in fact strongly coupled. From the experimental data it appears most likely that the electron is transferred to the lesion first and the proton from His365 follows simultaneously, since the excited state lifetime of FADH− is substantially longer when no lesion is bound to the (6−4) photolyase.21 However, the question of whether the proton or electron is transferred first is irrelevant for further discussion of the repair mechanism. Starting from this initial situation, the oxetane and nonoxetane reaction pathways described above have been calculated at the level of DFT/B3LYP with and without dispersion correction, as well as using RIMP2/6-31G*. Benchmark calculations on an even smaller model system still capturing the essential features of the investigated oxetane and non-oxetane repair mechanisms revealed the RIMP2 results to be most accurate, since they were found in agreement with results obtained at the high level of coupled cluster singles plus doubles (CCSD) (Supporting Information). To the best of our knowledge, this is the first investigation going beyond the DFT level. The geometries of all identified minima and transition state structures have been individually optimized at all levels of theory including the geometric constraints mentioned above. Furthermore, the nature of the transition states has been confirmed using the intrinsic reaction coordinate (IRC) algorithm, which is essentially a series of steepest descent steps going downhill from the transition state to the adjacent educt and product states. For all calculations, the Q-Chem 3.2 package28 of quantum chemistry programs has been used. In Figure 2, the computed reaction energy profiles as well as the corresponding structures of the studied oxetane and nonoxetane pathways are compiled. The initial structure (I) of all pathways is the optimized equilibrium geometry of the T(6− 4)T-PP and the hydrogen-bonded protonated HIS365 plus the additional electron, which is present after the initial photoinitiated electron transfer from FADH−. As mentioned above, the additional electron is in our calculation first located on the protonated HIS365. The energy of this initial structure is set to zero. Formation of the oxetane intermediate (III) requires the attack of the O4′ oxygen at the C4′ carbon atom, which proceeds via the transition state (II) (Figure 2). The energy barrier for this process amounts to 55, 39, and 33 kcal/mol at the theoretical levels of DFT/B3LYP, DFT/B3LYP-D,26 and RIMP2, respectively. Within this oxetane mechanism, HIS365 is not involved except that it is hydrogen-bonded to the lesion. During the whole process, the transferred electron stays at the HIS365, and is thus also not involved in the process. Since, however, the electron transfer is crucial for the function of (6− 4)-photolyase, taking all these facts into account, oxetane formation can be excluded from being relevant in the repair mechanism. Nonoxetane intermediate mechanisms also require a transfer of the O4′ oxygen to the C4′ carbon atom to restore the original two thymine bases. It is generally possible to accomplish this via a concerted OH transfer, or the formation of an intermediate water molecule. The latter requires a proton transfer from the

an alternative energetically feasible pathway that occurs in the electronic ground state of the lesion, in which a proton is transferred from protonated His365 to N3′ of the 3′ thymine in concert with OH transfer and subsequent splitting and repair of the base pair. In order to allow for quantum chemical calculations at a higher level than density functional theory (DFT), and, due to the focus on the fundamental chemistry involved in the cleavage of the T(6−4)T-PP, we chose to study only the covalently linked thymine bases of the T(6−4)T-PP plus the spatially close, protonated histidine HIS365, whose central role in the mechanism is currently established. For this objective, the structure of the T(6−4)T-PP of the damaged DNA has been extracted from the X-ray structure22 (3CVU.pdb) from the Protein Data Bank. Due to the fact that HIS365 is part of the photolyase and is held in a fixed position by the protein, the relative position of the lesion and HIS365 as found in the crystal structure was conserved by imposing constraints (Supporting Information) during geometry optimizations (see Figure 1) to mimic the protein environmental effects, which are

Figure 1. X-ray structure of the complex of the photodamaged DNA and (6−4)-photolyase, and a reduced molecular model consisting of the covalently linked thymine bases of the T(6−4)T-photoproduct and protonated HIS365.

otherwise missing in the current gas phase study. However, the validity of this approach has been confirmed by quantum mechanics/molecular mechanics (QM/MM) optimization of the crystal structure at the DFT/B3LYP level, where the structural effects of the protein environment are taken into account. Indeed, the obtained equilibrium structure agrees with the one obtained from our constraint gas phase optimization. The first general mechanistic question to address is to which site the electron is transferred from the FADH− upon photoexcitation and where the radical is actually located. Quantum chemical calculations at the level of DFT/B3LYP/631G* and Møller−Plesset perturbation theory of second order (MP2) exploiting the resolution-of-the-identity (RI) approximation on the T(6−4)T-PP radical anion and the protonated HIS365 in the point charge field of the protein derived from the Amber99 force field show that the electron is transferred to the protonated HIS365. This contrasts the experimental finding that the electron is initially transferred to the T(6−4)T-PP,21 which was also assumed in all previous theoretical investigations. However, generally it is not surprising that the 228

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Figure 2. Energy profiles and corresponding structures along the oxetane (solid line) versus the non-oxetane repair mechanisms comprising water cleavage (long dashed line) and the concerted OH-transfer (dotted line) at the theoretical levels of DFT using B3LYP and B3LYP-D as well as MP2. (Energies (in kcal mol−1) are given relative to the initial structure (I)).

respectively. In structure (VIII), the additional electron, i.e., the radical, is now located at C5. From structure (VIII), the covalently linked thymine bases can now be split via transition state (IX) passing an energy barrier of only 10.2 and 10.0 kcal/ mol at the DFT/B3LYP-D and RIMP2 levels of theory. This results in the separated thymine bases, which eventually backtransfer the proton to the histidine and retransfer the electron to FADH•. This pathway is energetically feasible, as it exhibits no energy barriers larger than 13 kcal/mol at RIMP2, the most accurate level of theory employed. Our purely theoretical findings are in nice agreement with recent ultrafast time-resolved spectroscopic data.21 It has been shown that upon photoinitiated electron transfer, a proton transfer from the protonated HIS365 to the N3′ of the pyrimidine thymine is realized within 425 ps, which initiates repair, i.e., transfer, of the hydroxy group and splitting of the covalently linked thymines. On the basis of our calculations, the spectroscopically identified features can be ascribed to structures (I), (VIII), and (X). (6−4)-Photolyases repair (6−4) photolesions, which can occur between two thymine bases as described above and between a thymine and a cytosine. It is clear that the identified mechanism must be universal for both kinds of lesions. For that objective, we have also studied the repair of a T(6−4)C photoproduct.27 Our results demonstrate that analogous protonation of N3′ of 3′ cytosine by HIS365 initiates concerted NH2 transfer from 5′ thymine to the C4 position of the 3′ cytosine. Our proposed mechanism is thus universal, as it operates for both T(6−4)T and T(6−4)C photolesions. With the help of elaborate quantum chemical calculations, we have for the first time identified a molecular repair mechanism of (6−4) photolyases, which is energetically feasible and does not require an unlikely two-photon process or an electronic excitation of the photolesion. It is now clear that a photoinitiated electron transfer from FADH− and a coupled

HIS365 to the O4′ and concomitant electron transfer to the attached pyrimidin thymine. This proceeds via transition state (IV) in Figure 2 leading to the intermediate (V) with one free water molecule. The energy barrier for this process is found to be 16.5, 15, and 9.3 kcal/mol at DFT/B3LYP, DFT/B3LYP-D, and RIMP2 levels of theory. Also, the stable intermediate (V) is lower in energy than the educt, which is clearly due to the high stability of the isolated water molecule. However, successful restoration of the TT base pair requires the attack of the water at the C4′ carbon of the pyrimidone thymine and subsequent proton transfer from the water molecule to N3. However, the energy barrier connected with the corresponding addition of the water molecule is as large as 80 kcal/mol, which again is most likely due to the large stability of the isolated water molecule. Although the barrier for the initial proton transfer is quite low, this large barrier practically excludes this pathway as an efficient repair mechanism. In addition, in the crystal structure, a conserved water molecule has been identified (Wat697), which is initially hydrogen bonded to the OH group at the 5′ thymine. If this additional water molecule is taken into account, a water dimer will be formed, in which the charge density on the oxygen of the formed water is even lower due to the hydrogen bond making the addition of the water molecule at the C4′ carbon even more unfavorable. In contrast to protonation of the O4′ oxygen, an alternative is the protonation of the N3′ by HIS365 in concert with the electron transfer to the pyrimidone thymine, where it is mostly located on C4′. As a consequence, O4′ also approaches simultaneously to the proton-coupled electron transfer C4′ and forms a four-membered oxetane-like transition state (VII). This transition state leads directly to the intermediate structure (VIII), in which the OH group is now transferred from the pyrimidine to the pyrimidine thymine. The energy barrier for this concerted reaction is 21.1, 18.2, and 13.4 kcal/mol at the theoretical levels of DFT/B3LYP, DFT/B3LYP-D, and RIMP2, 229

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(13) Kao, Y. T.; Saxena, C.; Wang, L.; Sancar, A.; Zhong, D. Direct Observation of Thymine Dimer Repair in DNA by Photolyase. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 16128−32. (14) Langenbacher, T.; Zhao, X.; Bieser, G.; Heelis, P. F.; Sancar, A.; Michel-Beyerle, M. E. Substrate and Temperature Dependence of DNA Photolyase Repair Activity Examined with Ultrafast Spectroscopy. J. Am. Chem. Soc. 1997, 119, 10532−10536. (15) Harrison, C. B.; O’Neil, L. L.; Wiest, O. Computational Study of DNA Photolyase. J. Phys. Chem. A 2005, 109, 7001−7002. (16) 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. (17) Zhao, X.; Liu, J.; Hsu, D. S.; Zhao, S.; Taylor, J. S.; Sancar, A. Reaction Mechanism of (6−4) Photolyase. J. Biol. Chem. 1997, 272, 32580−90. (18) Hitomi, K.; Nakamura, H.; Kim, S. T.; Mizukoshi, T.; Ishikawa, T.; Iwai, S.; Todo, T. Role of Two Histidines in the (6−4) Photolyase Reaction. J. Biol. Chem. 2001, 276, 10103−9. (19) Friedel, M. G.; Cichon, M. K.; Carell, T. Model Compounds for (6−4) Photolyases: A Comparative Flavin Induced Cleavage Study of Oxetanes and Thietanes. Org. Biomol. Chem. 2005, 3, 1937−1941. (20) Yamamoto, J.; Tanaka, Y.; Iwai, S. Spectroscopic Analysis of the Pyrimidine(6−4)Pyrimidone Photoproduct: Insights into the (6−4) Photolyase Reaction. Org. Biomol. Chem. 2009, 7, 161−166. (21) Li, J.; Liu, Z.; Tan, C.; Guo, X.; Wang, L.; Sancar, A.; Zhong, D. Dynamics and Mechanism of Repair of Ultraviolet-Induced (6−4) Photoproduct by Photolyase. Nature 2010, 466, 887−890. (22) Maul, M. J.; Barends, T. R. M.; Glas, A. F.; Cryle, M. J.; Domratcheva, T.; Schneider, S.; Schlichtling, I.; Carell, T. Crystal Structure and Mechanism of a DNA (6−4) Photolyase. Angew. Chem., Int. Ed. 2008, 47, 10076−10080. (23) Sadeghian, A.; Bocola, M.; Merz, T.; Schütz, M. Theoretical Study on the Repair Mechanism of the (6−4) Photolesion by the (6− 4) Photolyase. J. Am. Chem. Soc. 2010, 132, 16285−16295. (24) Domratcheva, T.; Schlichting, I. Electronic Structure of (6−4) DNA Photoproduct Repair Involving a Non-Oxetane Pathway. J. Am. Chem. Soc. 2009, 131, 17793−17799. (25) Harbach, P. H. P.; Borowka, J.; Bohnwagner, M.; Dreuw, A. DNA (6−4) Photolesion Repair Occurs in the Electronic Ground State of the TT Dinucleotide Dimer Radical Anion. J. Phys. Chem. Lett. 2010, 1, 2556−2560. (26) Grimme, S. Semiempirical GGA-Type Density Functional Constructed with a Long-Range Dispersion Correction. J. Comput. Chem. 2006, 27, 1787−1789. (27) Glas, A. F.; Schneider, S.; Maul, M. J.; Hennecke, U.; Carell, T. Crystal Structure of the T(6−4)C Lesion in Complex with a (6−4) DNA Photolyase and Repair of UV-Induced (6−4) and Dewar Photolesions. Chem.Eur. J. 2009, 15, 10387−10396. (28) Shao, Y.; Molnar, L. F.; Jung, Y.; Kussmann, J.; Ochsenfeld, C.; Brown, S.; Gilbert, A.; Slipchenko, L.; Levchenko, S.; O'Neill, D.; et al. Advances in Methods and Algorithms in a Modern Quantum Chemistry Program Package. Phys. Chem. Chem. Phys. 2006, 8, 3172−3191.

proton-transfer to the N3′ nitrogen occur concerted with OH transfer via the formation of an oxetane-like transition state in the electronic ground state. Our theoretical findings are in agreement with recent experimental findings.21 In fact, the recognition that the initial electron and proton transfer steps are strongly coupled is critically important for clarification of the mechanism. This lowers the barrier for the proton transfer and steers it toward the N3′ of the pyrimidine thymine with the additional electron located at the C4 position to make concerted OH transfer possible. In the future, we plan to perform further QM/MM simulations to shed more light onto the influence of the protein environment. Existing estimates of the influence of the geometrical contraints of the protein as well as its point charge field on the electronic structure have revealed a rather small effect on our results. Also, preliminary QM/MM results already demonstrate the validity of the here chosen theoretical methodology to perform high-level quantum chemical calculations on a reduced molecular model.

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ASSOCIATED CONTENT

S Supporting Information *

Model structure superimposed on crystal structure, list of structural constraints, and benchmark calculations. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; phone: +49-6221-548770; fax: +49-6221-548868.

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REFERENCES

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