Cytosine

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Similarities and Differences between Thymine(6-4)Thymine/ Cytosine DNA Lesion Repairs by Photolyase Hisham M Dokainish, and Akio Kitao J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b07048 • Publication Date (Web): 20 Aug 2018 Downloaded from http://pubs.acs.org on August 24, 2018

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The Journal of Physical Chemistry

Similarities and Differences between Thymine(6-4)Thymine/Cytosine DNA Lesion Repairs by Photolyase Hisham M. Dokainish† and Akio Kitao†* †

School of Life Science and Technology, Tokyo Institute of Technology, M6-13, 2-12-1 Ookayama, Meguro, Tokyo 152-8550, Japan

AUTHOR INFORMATION *Corresponding author: Akio Kitao †

School of Life Science and Technology, Tokyo Institute of Technology, M6-13, 2-12-1

Ookayama, Meguro, Tokyo 152-8550, Japan Tel: 03-5734-3373 Fax: 03-5734-3372 e-mail: [email protected]

1

ACS Paragon Plus Environment

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 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Photolyases are ancient enzymes that harvest sunlight to repair DNA pyrimidine lesions such as pyrimidine(6-4)pyrimidone and cyclobutane dimers. Particularly, (6-4) photolyase ((6-4)PHR) plays an important role in maintaining genetic integrity by repairing thymine(6-4)thymine (T(6-4)T) and thymine(6-4)cytosine (T(6-4)C) photolesions. The majority of (6-4)PHR studies have been performed based on the former activity and assuming the equivalence of the two repair mechanisms, although the latter activity remains poorly studied. Here, we describe investigations of the repair process of T(6-4)C dimer using several computational methods from molecular dynamics (MD) simulations to large-quantum mechanical/molecular mechanical approaches. Two possible mechanisms, the historically-proposed azetidine four-member ring intermediate and the free NH3 formation pathways, were considered. The MD results predicted that important active site histidine residues employed for repair of the T(6-4)C dimer have protonation states similar to those seen in the (6-4)PHR/T(6-4)T complex. More importantly, despite chemical differences between the two substrates, a similar repair mechanism was identified: His365 protonates NH2, resulting in formation/activation mechanism of a free NH3, inducing NH2 transfer to the 5`base, and ultimately leading to pyrimidine restoration. This reaction is thermodynamically favorable with a rate-limiting barrier of 20.4 kcal mol-1. In contrast, the azetidine intermediate is unfeasible, possessing an energy barrier of 60 kcal mol-1; this barrier is similar to that predicted for the oxetane intermediate in T(6-4)T repair. Although both substrates are repaired with comparable quantum yields, the reactive complex in T(6-4)C was shown to be a 3` base radical with a lower driving force for back electron transfer combined with higher energy barrier for catalysis. These results showed the similarity in the general repair mechanisms between the two substrates, while emphasizing differences in the electron dynamics in the repair 2


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cycle.

KEYWORDS: Histidine Protonation State, Large-QM/MM, Photolyase (PHR), DNA repair, Marcus theory, DNA photo-lesion, Thymine(6-4)Cytosine.

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Introduction DNA-damage repair is an important molecular tool for the maintenance of genetic stability and ultimately the preservation of cellular life.1-3 This damage occurs naturally as a result of regular cellular processes, such as the production of reactive oxygen species during cellular metabolism.4-5 Moreover, exposure to any of several environmental factors such as radiation and genotoxic chemicals also can induce harmful damages to DNA nucleotides.6-7 Specifically, UV radiation triggers covalent modifications of adjacent pyrimidines on DNA strands, resulting in the formation

of

two

main

photoproducts:

cylcobutane

pyrimidine

dimers

(CPDs)

or

pyrimidine(6-4)pyrimidone photoproducts ((6-4)PPs) (see Scheme 1).5, 8-12 CPDs and (6-4)PPs are the most commonly formed photoproducts in the case of thymine/thymine (TT) and thymine/cytosine (TC) dimers, respectively. Their formation has been shown to disturb several cellular processes; in particular, (6-4)PPs have been linked to skin cancer.5, 13-15 Fortunately, these covalent modifications are reversible either directly via photolyase (PHR) activity or indirectly via nucleotide/base excision repair mechanisms.7-8, 16-17 PHRs are a group of flavoenzymes that harvest sunlight to repair the aforementioned lesions.9, 18-19 These protein are classified into two main groups, CPD PHRs and (6-4) PHRs, that are stereospecific for CPD and (6-4)PP repair, respectively. Unlike CPD PHRs, the repair mechanism employed by (6-4)PHRs is complicated and has been debated for many years.9, 18 Notably, the majority of (6-4)PHR studies have been performed based on the T(6-4)T lesion,20-22 assuming the equivalence of T(6-4) and T(6-4)C repair mechanisms. Generally, both substrates undergo similar main repair steps: UV light absorption by the photoantenna is followed by energy transfer and formation of the fully-reduced flavin adenine dinucleotide (FADH–) excited state, which leads to electron transfer from FADH– to (6-4)PPs and eventually to pyrimidine repair.9, 19 4


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Historically, repair of the T(6-4)C dimer was postulated to occur via the formation of a short-lived four-member ring intermediate (aztidine; AZT),23-24 a structure that is similar to the previously-proposed oxetane intermediate used in repair of the T(6-4)T dimer.18 However, oxetane formation in T(6-4)T has been shown to be unfeasible due to a high-energy barrier in several studies25-26. Although a recent experimental study investigated the formation of the AZT intermediate using lesions, the molecule used in the study was in fact a CPD analog that does not reflect the actual chemistry in T(6-4)C.24

O O

O CH 3

H

CH 3

H

N

O

5'

5

6'

6

N

H CH

N

O

N

4'

N

3' N

5'

5'

O

N

5

4'

N

4' 3' N

O

5'

3'

(6-4)PP

T(6-4)T O

NH 3 Formation CH3

H

NH 3

N

N

O

CH

N

O

O

H O

6

T(6-4)C

CPD

CH 3

6

3' 3'

N

5

4'

N

CH 3

H

H

H N

O

CH 3

O

O

O

N

NH 2 H N

5'

CH 3 N

O

3'

NH 2 O

O

3'

5'

H

NH N

NH

N

3'

NH O

N CH

CH3

N CH

N

O

N

5'

CH 3

H

N CH

O

N

5'

CH

O

N

3'

AZT Formation

Scheme 1. Top: UV-induced adjacent pyrimidine photolesions, CPD and (6-4)PP. Bottom: 5



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Schematic representation of the two proposed pathways for T(6-4)C repair via NH3 or AZT formation.

Despite the chemical difference between T(6-4)T and T(6-4)C photolesions, the X-ray structure of the complex of (6-4)PHR and a DNA duplex including a T(6-4)C photoproduct27 has active site interactions similar to that of a complex incorporating T(6-4)T, as indicated by shared hydrogen-bonding

pairs,

including

His365/FADH–,

His365/(6-4)PP,

His369/Tyr423,

His365/His369 (Fig. 1). Based on these similarities, Glas et al.27 proposed a non AZT mechanism in which electron transfer leads to departure of the NH3 group, NH2 transfer to the 3`base, and C– C bond breaking, resulting in pyrimidine repair (NH3 formation pathway in Scheme 1).

FADH– H365

H369

Y423

(6-4)PP

Figure 1. Superposition of the (6-4) PHR active site of the (6-4)PHR/T(6-4)C (PDB:2WB2; carbons shown in magenta) and (6-4)PHR/T(6-4)T (PDB:3CVU; carbons shown in cyan)

6


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complexes. Dotted lines show key hydrogen bonds.

Dokainish et al. recently proposed a water formation/activation mechanism for the repair of T(6-4)T,28 a mechanism that is similar to that of the NH3 formation pathway. Dokainish et al. also proposed a previously unrecognized role for an active site lysine (Lys246) in the mechanism.28 In addition to Lys, the active site of (6-4)PHR includes several residues (e.g., His365 and His369) that are important to the repair.15 The actual protonation states of these His residues were recently studied by long Molecular Dynamics (MD) simulations of all possible combinations of the two His; these simulations suggested that the combination of HIP365 (two nitrogens protonated) and HID369 (Nδ protonated) is the most probable protonation state in the T(6-4)T repair process.29 Unfortunately, the protonation states of these histidine residues have not yet (to our knowledge) been investigated in the T(6-4)C complex. Notably, the mechanism proposed by Dokainish et al.28 is in agreement with the previously-proposed mechanism, which was based on the X-ray structure of the (6-4)PHR/T(6-4)C complex.27 In the present study, we examined the transferability of the T(6-4)T repair mechanism to T(6-4)C repair, and investigated the feasibility of the AZT mechanism, by using several computational approaches including MD, quantum mechanics/molecular mechanics (QM/MM), and a large-QM/MM calculations. We showed that the repair mechanism via the NH3 formation is feasible whereas repair via the AZT formation is unfeasible due to a high-energy barrier

Methods MD simulation. The MD starting structure for the (6-4)PHR/DNA complex, including a T(6-4)C lesion and FADH, was modeled based on the X-ray crystallographic structure of D. melanogaster 7



PHR (PDB ID: 2WQ7)30 complexed with a non-natural N4-methyl T(6-4)C lesion. This X-ray structure was chosen because its resolution (2.0 Å) is higher than that of the (6-4)PHR complex with the natural T(6-4)C substrate (PDB:2WB2, resolution: 2.9Å).27 The extra methyl group in the lesion was deleted to restore the natural T(6-4)C dimer. To ensure that there were no significant structural differences in the active site between the two X-ray structures, we superimposed the two structures; this analysis confirmed that here were no significant structural differences with root-mean-square deviations (RMSDs) exceeding 0.12 Å (See Fig. S1). The previously-determined protonation states of His365 and His369 (HIP365/HID369)29 were employed in the simulation. MD simulations were performed using the AMBER14 software package incorporating the ff14SB force field for the protein and DNA.31 The generalized AMBER force field (GAFF) parameters32 were used to parameterize the T(6-4)C lesion and FADH−. Partial charges were obtained using Antechamber (AMBER program) based on quantum mechanical calculations at the B3LYP/6-31G (d) level of theory and the restraint electrostatic potential fit (RESP).33 The AMBER program xLEAP was used to prepare the MD starting structures by adding missing hydrogen atoms and sodium ions (Na+) to neutralize the complexes.34 Then, the complex was solvated with the TIP3P water in a box with a minimal buffer of 10 Å from any solute atoms. The PMEMD module in Amber1431 was used to first energy-minimize the solvated structure with positional restraints on the solute atoms for a total of 10,000 steps. Then, the system was freely minimized for another 30,000 steps (10,000 steepest descent and 20,000 conjugated gradient methods). The system was first heated gradually from 0 to 300 K by MD using positional restraints on the solute with a force constant of 10 kcal mol-1 Å-2, and then equilibrated at 300 K and 1 atm for 100 ps followed by a production run for 100 ns without positional restraints with 8


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the NPT ensemble (Langevin thermostat and Berendsen barostat), using a time step of 2 fs. The SHAKE algorithm was employed for all of the bonds involving hydrogen atoms. Electrostatic interactions were calculated using the Particle Mesh Ewald (PME) method with periodic boundary condition.35 The VMD program36 was used to visualize the generated trajectories; the AMBER module CPPTRAJ37 was used to analyze and cluster all the trajectories. Based on the active site RMSD, five clusters were generated using the last 75 ns of the simulation and one representative structure (cluster centroid) of the most populated cluster was used as the starting point for the following QM/MM calculations, in which solvent atoms more than 5 Å away from any solute atoms were deleted. QM/MM calculations. All the QM/MM calculations were performed using the Gaussian 09 suite of programs (Version: g09e01)38 within the mechanical embedding ONIOM formalism. The approach used in this work was similar to that employed in our previous study on the T(6-4)T repair process.28 The unrestricted hybrid-meta-GGA density functional method (UM06-2X)39 was used for the QM-high-layer and the AMBER parm96 force field40 for the MM layer. As with the T(6-4)T study,28 geometry optimization was conducted in two steps as follows. First, the QM layer included the active site residues and excluded the FADH– isoalloxazine moiety was optimized (Fig. 2); The second optimization was performed using the QM/QM/MM approach where the isoalloxazine moiety was freely optimized as the first QM layer, and the second QM layer consisting of the previously optimized active site residues was fixed in addition to the isoalloxazine. After this two-step optimization, relative energies were obtained using the large-QM/MM approach.

9



Figure 2. The QM/MM two-step optimization procedure and the final energy evaluation step using the large-QM/MM approach. The numbers indicate the number of atoms included in each layer.

The QM optimizations were performed at the same level of theory with the 6-31G (d) basis set. The first QM layer contained (6-4)PP, Asn406, His365, His369, Lys246, Gln299, Tyr, FADH–, and one water molecule. All the transition states (TSs) were freely optimized with exceptions as follows: the rotation of Lys246 in the transition state, TSLys; the rotation of His365 in TS4; and the formation of AZT in TS2AZT (Note that these transition states will be defined in the Results section) The large-QM/MM approach utilized the 6-311G(d,p) basis set with 991 atoms, similar to the previous study on T(6-4)T.28 Relative energies also were obtained for the QM/MM and QM/QM/MM models at the same level of theory (UM06-2X/6-311G(d,p); see 10


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Table S1). Free energy corrections were obtained using the optimized QM/MM model. Furthermore, the effect of using electrostatic embedding on the relative energies also was tested, revealing over-stabilization of the post-back electron transfer (post-BET); therefore, mechanical embedding results subsequently were used as final energies (Table S2). The results shown in Tables S1 and S2 confirmed that the results presented here are robust. Also, as noted in the previous study,28 the QM/MM and QM/QM/MM approaches lead to higher energy barriers, indicating that the charged environment of the active site should be treated using the Large-QM/MM approach.

Results and discussion Transferability of histidine protonation states between T(6-4)T and T(6-4)C. The MD-generated trajectory was analyzed to examine the stability of the selected His protonation states (HIP365 and HID369), which were set to be the same as those concluded in the previous study on T(6-4)T.29 The quantities compared between the MD results and the two available X-ray structures of the (6-4)PHR/T(6-4)C complex consisted of the active site interactions corresponding to the following key heavy atom distances; D1: NHis365…N4`(6-4)pp; D2: NHis365…O2`FADH–; D3: NHis365…NHis369; and D4: NHis369…OTyr423. As shown in Fig. 3, all the interactions were stable throughout the simulation. More importantly, the average distances were comparable to those in the X-ray structures (Table 1), suggesting that His365 and His369 have the same protonation states in the T(6-4)T and T(6-4)C complexes. Furthermore, general active 11



site interactions during the MD simulation in the present study showed stable binding of the substrate using a binding mode similar to that obtained via the MD simulation with T(6-4)T.29 These similarities also implied the transferability of the repair mechanism between the two substrates, as previously concluded based on the X-ray structures.27,30 Our simulation also showed strong hydrogen-bond interaction between HIP365 and N4` of the substrate, suggesting the formation of NH3 as previously proposed.27

Figure 3. Molecular representation of the investigated active site interaction (right) and key distance fluctuation between pairs of heavy atoms throughout the MD (left).

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Table 1. Key heavy atom distances (average ± standard deviation in angstrom, Å) during the MD compared to those of the X-ray structures (PDB ID: 2WB2 and 2WQ7). MD

2WB227

2WQ730

D1

2.9 ± 0.2

3.3

3.0

D2

2.8 ± 0.1

2.8

2.9

D3

3.1 ± 0.2

2.9

2.7

D4

3.0 ± 0.2

2.6

2.6

Differences in electron transfer pathways. The representative structure of the MD was optimized using QM/MM to determine the structure of a pre-reactive complex (PRC) prior to electron transfer (ET) (see the Methods). As seen in Fig. 4, active site interactions in T(6-4)C are similar to T(6-4)T, wherein His365 is strongly hydrogen-bonded to the N4` atom of the substrate with a hydrogen bond distance of 1.94 Å but does not form any direct/indirect hydrogen-bonding interaction with the N3` atom. Furthermore, N4`-H is also hydrogen-bonded to a water molecule in the active site. Note that a water also was observed in the X-ray structures of the (6-4)PHR/T(6-4)T and (6-4)PHR /T(6-4)C complexes.11,27,30 In the case of T(6-4)T, the corresponding water, which orients the O4`-H of the substrate towards HIP365 to form a hydrogen bond in the active site, was suggested to be important for catalysis.28 In the T(6-4)C 13



calculation, Lys246, was found to adapt an orientation similar to that in T(6-4)T, forming hydrogen bonds with Gln299, FADH–, adenine, and a phosphate group. The 5`base of the substrate maintains hydrogen-bonding interactions with FADH–, adenine, and Gln299, a pattern similar to that seen with the T(6-4)T structure.

Figure 4. Molecular representation of the QM/MM optimized active site before ET (PRC:

14


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pre-reactive complex), highlighting the main hydrogen-bonding interactions (broken lines) and their distances (in Å).

Starting from the PRC structure, the formation of the reactive complex (RC) upon ET was investigated. Unlike the case for the T(6-4)T structure,28 it was found that the 5` base radical is unstable and the RC only occurs as the 3` base radical. This difference between the T(6-4)T and T(6-4)C models was due to the lower electronegativity of NH2 compared to OH, which reduces the stability of anionic radical formation on the 5`base. This difference was explored further using isolated models of T(6-4)C and T(6-4)T dimers at the um06-2x/6-311G+(2df,p) level of theory in a continuum model (integral equation formalism polarizable continuum model)43 with a dielectric constant of 20 to simulate the polar protein environment (Fig. 5). The PRC structure of T(6-4)T showed that both 3` and 5` orbitals have positive electron affinity; however, in the case of the PRC of T(6-4)C, the 5` orbital has negative electron affinity and exists as the third-lowest excited state. Upon ET, the 5`RC structure of T(6-4)T showed that the 5` orbital is now the highest occupied molecular orbital (HOMO) and the 3` orbital becomes the lowest unoccupied molecular orbital (LUMO). Starting from the 5`RC of T(6-4)T, we substituted OH with NH2 and removed the extra methyl group, then optimized the structure at the 3`base,

15



keeping the C4=O4 and C2=O2 bonds fixed to enforce the formation of the 5`RC radical state. However, the HOMO of T(6-4)C represents a 3`RC radical (Fig. 5). The same procedure also was repeated by fixing the C4=O4 and C4–N3 bonds as oxyanion and single bond, respectively, but the radical only forms the 3`RC. These results clearly showed that the substitution of OH with NH2 changes where the electron goes in the RC.

Figure 5. Molecular representation of the T(6-4)T and T(6-4)C small models and their corresponding orbitals before ET (PRC) and after ET (RC). Red and green orbital energies reflect positive and negative values, respectively.

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Role of Lys246. The previous theoretical study suggested that Lys246 plays an important role in catalysis.28 Therefore, we optimized an alternative reactive complex (RC`), in which Lys246 is hydrogen-bonded to the O4 of the 5` base with a small rotation of the χ4 dihedral angle from -50.9° in RC to -85.5° in RC`. This step occurs via a transition state (TSLys) with a low free energy barrier of 1.7 kcal mol-1. Note that this TS was obtained by scanning the NLys246…O4 distance. Since the energy of RC` is more stable than that of RC (by -3.3 kcal mol-1), RC` was chosen as the starting reactive complex for the following mechanism investigation. Despite the conformational change of Lys, the structure of RC` is similar to those of RC and PRC, thereby maintaining all of the aforementioned interactions (Figs. 4 and 6).

Figure 6. Molecular representation of the QM/MM optimized structures for the proton transfer

17



reaction from His365 to NH2.

Scheme 2 shows that ET leads to the elongation of the C4`-N3` bond from 1.31 to 1.38 Å, providing adaptation of the electron addition to the 3`base. Although the His365…NH2 hydrogen bond is maintained, the distance is elongated from 1.94 to 2.12 Å. The structure of RC upon back electron transfer (post-BET) to FADH– was further optimized. Similar to the T(6-4)T study, the energy difference between RC and post-BET is large (-1.2 eV), which explains the low efficiency in (6-4)PHR. However, this difference is smaller than the previously calculated value, -1.69 eV in T(6-4)T28, which suggests some differences in the electron dynamics in the repair cycle. Unfortunately, PHR electron dynamics in the T(6-4)C structure have not been well defined experimentally. Therefore, we calculated a ratio of back and forward electron transfer (BET/FET) catalytic rates using Marcus theory calculations as in the previous study.28 Assuming that the electronic factor is invariant, the BET/FET ratio is obtained by the following equation:

where (λ1) and (λ2) are the reorganization energy for FET and BET, respectively. ∆Ε1 and ∆Ε2 are the energy differences between PRC and RC and that between RC and post-BET, respectively.

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The obtained BET/FET ratios in RC and RC` were 93% and 23%, respectively. These values also suggest slower BET in the RC of T(6-4)C compared to that in the case of T(6-4)T (99%)28 and also highlights the role of Lys rotation, which reduces BET and allows for catalysis. The BET difference between RC and RC` originates primarily from the lower reorganization energy in the case of RC`, which yields a value of 0.04 eV (Table S3).

Scheme 2. Schematic representation of the electron transfer reaction forming the Reactive Complex (RC). Structural differences in the active site with selected distances (in Å) are highlighted in red text. H

H

N

N

H

H N

O

N O

H H

CH3

O

O

H

H H

1.94

2.12

H

N

ET

N

N

H

CH

N

O

N

CH

1.31 N

H

N

H

O

1.38 N

3'

CH3

H

5'

N

N

3'

PRC O

5'

RC O

Mechanism of the first proton transfer. Based on the mechanism suggested by Glas et al.27 as well as by our recent study on T(6-4)T,28 we investigated the possibility of proton transfer from 19



the protonated His365 to the substrate NH2 group as the first step in the repair process. As shown in Figs. 6 and 7, this step occurs via TS1 with a small free energy barrier of 0.4 kcal mol-1, which results in the formation of the first intermediate (I1). I1 is slightly less stable compared to RC`, with an energy difference of 0.1 kcal mol-1. The structure of TS1 showed that the proton is almost transferred to NH2, with a short proton…NH2 distance of 1.12 Å. In I1, the newly-formed NH3 maintains a strong hydrogen bond (1.56 Å) with the neutral His365 residue. Although the same protonation reaction directly from PRC also was tested, the proton moved back to His365 during optimization. Likewise, this step was tested directly from RC (prior to the Lys rotation), but the calculated energy barrier was higher than the protonation from RC`. Therefore, we judged that the most plausible pathway to I1 is that shown in Figs. 6 and 7.

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30.0

Relative Energies 17.1

20.0

(Kcal mol-1)

13.7

12.0 TS3 I3

9.8

10.0

0.0 0.0

RC

TS2

1.7 TSLYS

-3.3 -2.9 -3.2 TS1 I1 RC`

TS4 6.3 0.8

-2.8

TS5

I4 I2

-10.0

I5

-9.9

-20.0

-30.0

-37.4 -40.0

I5`

-45.4

-41.9 PC

TS6

-50.0

Figure 7. Free energy surface of the T(6-4)C repair mechanism in the NH3 formation pathway. Relative energies were obtained using the large-QM approach including 991 atoms in the QM layer.

NH2 transfer mechanism and pyrimidine restoration. According to the previously proposed mechanism, the next step in the repair process is expected to be C5-N4` bond breaking, which produces a free NH3 and induces NH2 transfer to the 3` base. This step was found to occur via TS2, wherein the C5-N4` bond is elongated to 1.74 from 1.48 Å in I1 (Fig. 8). The free energy barriers for TS2 are 13.0 and 13.1 kcal mol-1 from I1 and RC`, respectively (Fig. 7). This

21



transition state leads to the formation of I2, in which the C5-N4` bond is broken at a distance of 2.53 Å. The energy of I2 is slightly higher than that of I1, with a difference of 0.4 kcal mol-1. The structure of I2 (Fig. 8) shows that the NH3…C4` distance is shortened to 2.70 Å, which allows for the subsequent NH2 transfer. Additionally, in I2, His365 is strongly hydrogen-bonded to NH3, with a distance of 1.93 Å. Afterwards, NH3 nucleophilically attacks C4` via TS3, wherein the C4`…N4` distance is shortened to 1.91 Å. Similarly, the His365…NH3 distance is decreased to 1.87 Å. The barriers for TS3 were found to be 14.8 and 15.3 kcal mol-1 compared to I2 and RC` (Fig. 7), respectively. Additionally, in TS3, Lys246 is rotated and becomes hydrogen-bonded to N3` with a distance of 1.93 Å. This hydrogen bond is further shortened in the following intermediate (I3) to 1.71 Å, stabilizing the N3` upon NH2 transfer to the 3`base (see Fig. 8). As expected, the NH3 transfer in T(6-4)C occurs in a manner similar to the OH transfer in T(6-4)T.

22


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Figure 8. Molecular representation of the QM/MM-optimized transition states (TSs) and intermediates (Is) throughout the repair mechanism. Important reactive atoms are shown by tubes while the rest of the active site is shown as wires. Hydrogen bond distances are shown in Å.

23



The transfer of NH2 to the 3`base leads to a proton transfer in I3 from NH3 to His365. The energy of I3 is slightly less stable than TS3 by 1.7 kcal mol-1, due to the formation of a single bond between C4` and N3` (1.45 Å). We investigated the possibility of stabilizing N3` via proton abstraction from the protonated His365. This effect would occur via slight rotation of the His365 side chain as shown in TS4, in which the His365…NH2 and His365…N3` distances are 1.77 and 2.18 Å, respectively (Fig. 8). Once His365 rotates, the proton transfers during the optimization (and without a barrier) to N3`, which forms I4. The energies of TS4 and I4 are 17.1 and 0.8 kcal mol-1 (respectively) with respect to RC. The TS4 barrier is the rate-limiting step of the reaction, exhibiting an energy barrier of 20.4 kcal mol-1 from RC`, a value that is higher than that of the previously observed rate-limiting step in the T(6-4)T repair (13.4 kcal mol-1). This barrier difference, combined with lower driving force for BET in TC, suggests a difference in the electron dynamics between the T(6-4)T and T(6-4)C repairs: T(6-4)T has more driving force for BET and a lower rate-limiting barrier, while T(6-4)C has a lower driving energy for BET and a higher rate-limiting barrier, leading to overall similar repair efficiencies.23 As shown in Fig. 8, the structure of I4 shows that Lys246 is now rotated to the original position and His365 is hydrogen-bonded to N3`H, with a distance of 1.94 Å. Since the NH2 group is transferred to the 3`base, the following step is the dissociation of

24


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the C6–C4` bond. This reaction can occur easily via TS5 wherein this bond is elongated from 1.62 to 2.02 in I4. Figure 7 shows that the barriers for TS5 are 5.5 and 6.3 kcal mol-1 with reference to I4 and RC, respectively. The subsequent intermediate (I5) was found to be lower in energy compared to RC and RC` by -9.9 and -6.6 kcal mol-1, respectively, showing a thermodynamically-favorable reaction overall. The structure of I5 shows two detached pyrimidines with a C6…C4` distance of 3.43 Å (Fig. 8). Once I5 is formed, BET occurs, leading to I5`, which is -27.5 and -37.4 kcal mol-1 more stable than I5 and RC, respectively. These values show the favorability of BET upon C4`–C6 bond breaking. Although the structure of I5` possesses a repaired pyrimidine, N3` is still protonated. Therefore, we investigated a proton transfer from N3` to His365 via TS6, which would restore the natural protonation state of cytosine. The barrier of TS6 is lower than that of I5`, reflecting a barrier-less reaction for proton transfer. This final step leads to the formation of a product complex (PC) that is also slightly more stable than I5`, possessing an energy difference of -4.5 kcal mol-1. The overall mechanism shows a feasible reaction via a maximum rate-limiting step of 20.4 kcal mol-1 and a thermodynamically favorable PC with a stable energy of -41.9 kcal mol-1 with respect to RC. Instead of proton transfer to N3`, another mechanism was examined in which C4`–C6

25



bond breaking occurs directly from I3; however, the barrier for this step was slightly higher than TS4 by 1.6 kcal mol-1.

AZT mechanism is unfeasible. Although the abovementioned mechanism is energetically feasible, with a rate-limiting barrier of 20.4 kcal mol-1, we also considered the historically-proposed four-membered ring intermediate formation (AZT). Note that the AZT analog in T(6-4)T (oxetane) was previously shown to have an unfeasible barrier.18 As shown in Fig. 9, this pathway for T(6-4)C repair via AZT starts with a proton transfer from His365 to N3` via a low barrier of 0.2 kcal mol-1. This reaction leads to the formation of a stable intermediate (I1AZT) with an energy of -4.7 kcal mol-1. Finally, the four-membered ring intermediate occurs thorough TS2AZT, in which the C4`…N4` distance was scanned to form AZT. In TS2AZT, the C4`…N4` distance significantly decreases (to 1.52 Å) and the N4`…H distance slightly elongates (to 1.09 Å). As for the oxetane mechanism, the barriers for this reaction is unfeasible, exhibiting energy barriers of 60.0 and 55.3 kcal mol-1 with respect to I1AZT and RC, respectively. Therefore, these results exclude the possibility of AZT formation in the T(6-4)C repair mechanism. Similar to the oxetane mechanism, the barrier for this reaction is unfeasible with energy barrier of 60.0 and 55.3 kcal mol-1 with respect to I1AZT and RC, respectively. Note that similar high barrier of

26


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55 kcal mol-1 has been found for oxetane in T(6-4)T repair.18,25 Notably, these results exclude the possibility of AZT formation in the T(6-4)C repair mechanism.

Figure 9. Free energy surface of the T(6-4)C repair mechanism via the AZT formation pathway. Relative energies were obtained using the large-QM/MM approach, as applied for the NH3 pathway in earlier figures.

Conclusions In this study, we investigated the mechanism of T(6-4)C repair by (6-4)PHR using several computational

approaches,

including

MD

simulations,

QM/MM,

QM/QM/MM,

and

large-QM/MM. First, the transferability of the previously-proposed His365 and His369 27



protonation states were examined using MD simulations. The MD results showed the stability of active site interactions in comparison with the X-ray structures, suggesting the same protonation states as in the (6-4)PHR/T(6-4)T complex. Second, the forward and backward electron transfer steps were elucidated, showing the formation of a 3`RC wherein the BET/FET ratio was found to be 93%. This ratio emphasizes the dominance of the BET reaction in T(6-4)C repair, which is in agreement with the experimental results.15 Furthermore, the rotation of Lys246, forming RC`, was found to reduce the BET/FET ration to 23%, demonstrating that RC` is more stable than RC. Third, the repair mechanisms via the NH3 and AZT formations were elucidated. This comparison revealed that AZT formation was unfeasible due to the existence of a high-energy (60 kcal mol-1) barrier.

28


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Page 29 of 37

Scheme 3. Schematic representation of optimized stationary points of the T(6-4)C repair mechanism via NH3 formation. Important distances (in Å) are highlighted in red text. H

H

H

N

N

N

H

H

H N

O H

CH3

N

N O

O

H

2.12 N

H

H

H

CH3

N

H

O

H H

Proton transfer

N

O

O

H

H

N

H

C–N breaking

N

CH

O

N

H N

H H

H

CH

CH3 N

2.70

CH

O

N

O

1.38 N

5'

N

3'

N

5'

N

O H

H

N

N

N

N O

N

CH 3

H

H

N

O

CH3

H

N

H

H N

N

N

H N

CH 3

N

H

3'

H

1.72 H

N N

I2

N O

H

CH

5'

N

O

O

H C H

N

3'

I1

3'

RC'

C–N formation

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


O

C–C breaking

H

Proton transfer N

CH

O

N

CH

O

O

5'

N

I5

5'

N H

3'

N

I4

O

N

3'

5'

I3 O

We demonstrated that the T(6-4)C repair mechanism is similar to the H2O formation/activation in the T(6-4)T repair, where His365 transfers a proton (via a low-energy

29



barrier of 0.4 kcal mol-1) to NH2, which leads to I1 (Scheme 3). Afterwards the C4–N4 bond breaks, leading to the formation of a free NH3 group that nucleophilically attacks C4` on the 3` base. This attack further induces NH2 transfer followed by a proton transfer to His365. Finally, the C4`–C6 bond breaks via TS5 with barrier of 6.3 kcal mol-1, restoring the repaired pyrimidine. The whole reaction is exothermic, with a rate-limiting barrier of 20.4 kcal mol-1. Although the proposed mechanism of T(6-4)C repair is similar to that of T(6-4)T (described in a previous study),28 differences in electron dynamics were observed, especially in a higher-energy rate-limiting step and lower BET driving energy in the case of T(6-4)C. These differences compensate for the calculated lower-energy rate-limiting barrier and higher driving force in T(6-4)T, which explains similar quantum yields for both substrates when measured experimentally.41 Furthermore, the rotation of Lys, instead of the proton transfer step in T(6-4)T repair, was found to play an important role in decreasing BET. These results highlight the need for obtaining further experimental results on the T(6-4)C repair mechanism; specifically, electron dynamics and mutation of Lys are expected to be of use in confirming the proposed mechanism.

30


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Acknowledgements

We express our gratitude to Prof. Hideki Kandori and Dr. Daichi Yamada for helpful discussions and comments. This research was supported by MEXT/JSPS KAKENHI (No. JP25104002 and JP15H04357) to A.K., and by MEXT as "Priority Issue on Post-K Computer” (Building Innovative Drug Discovery Infrastructure through Functional Control of Biomolecular Systems) to A.K. The computations were performed in part using the supercomputers at the RCCS, The National Institute of Natural Science, and ISSP, The University of Tokyo. This research also used computational resources of the K computer provided by the RIKEN Advanced Institute for Computational Science through the HPCI System Research project (Project ID: hp150270, hp160207, hp170254, and hp180201).

Supporting Information Figure S1 and Table S1-S3.

Competing financial interests The authors declare no competing financial interests.

31



Corresponding author Correspondence to: Akio Kitao

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TOC

37


Cytosine

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