Electron Attachment to DNA: The Protective Role of Amino Acids

We have studied the effect of amino acids on electron attachment properties of DNA nucleobases, taking cytosine as a model system. The equation of motion coupled cluster theory with an extended basis set has been used to simulate the electron-attached state of the DNA model system. Four selected amino acids, Arginine, Alanine, Lysine, and Glycine which form a major component of histone proteins are considered to investigate their role in electron attachment to DNA nucleobase. The electron attachment to cytosine in all the cytosine-amino acid dimer complexes follows a doorway mechanism, where the electron gets transferred from initial dipole-bound doorway state to the final nucleobase-bound state through the mixing of electronic and nuclear degrees of freedom. In higher amino acid concentration, amino acid-bound state acts as the doorway state, where the initial electron density is localized on the amino acid, away from the nucleobase. This leads to the physical shielding of nucleobase from the incoming extra electron. At the same time, the presence of amino acids can increase the stability of nucleobase-bound anionic state, which can suppress the dissociative electron attachment induced sugar-phosphate bond breaking.


Introduction
Ionizing (X-rays, Gamma rays) and particulate radiations (electrons, protons, neutrons, beta, and alpha particles) have been known for their damaging effect on DNA for many years. 1 One of the major applications of these phenomena is in the field of cancer treatment, where ionizing radiations are used to kill tumor cells. A major part of the radiation damage to genetic material is caused by secondary processes. 2,3 The ionizing radiations, even with 1 MeV of deposited energy, can generate a large number (~ 5 * 10 4 ) of secondary electrons 4 along with an array of other secondary products such as free radicals and cations 5 which are potentially harmful to DNA. A significant amount of work has been done in recent years to understand the role of secondary electrons in radiation damage to genetic materials. 3,[6][7][8][9] They have been shown to play a major role in various DNA damage processes such as base release, lesions, strand breaks, etc. [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] The electrons generated by the action of ionizing radiation, collide inelastically with the solvent molecules around DNA, and rapidly loose their kinetic energy to form low energy elctrons ( LEE), pre-solvated and aqueous electrons in a step-wise manner. 9,25,26 The LEE and presolvated electrons have been considered as one of the major contributors in the radiationinduced DNA damage. 10 Starting from the first experiment of Sanche's group, 10 various experiments [11][12][13][14][15] and simulations [27][28][29][30][31][32] have been reported on electron attachment-induced radiation damage.
Numerous attempts [27][28][29]33 were made to explain the exact mechanism of initial electron attachment to the DNA. A majority of the existing theoretical reports suggest a resonancebased pathway. 10,[34][35][36][37][38] Here, the incoming electron initially attaches to a π* orbital of the nucleobase, forming a shape or feshbach resonance state. [39][40][41][42] The electron from the π*-type resonance state subsequently gets transferred to the sugar-phosphate σ* (C-O) or sugarnucleobase (C−N) σ* state, resulting into the C−O/C−N bond cleavage. [43][44][45][46][47][48][49][50] We have recently reported a doorway mechanism of electron attachment to DNA nucleobases. 24,25,51 The initial electron attachment to nucleobases in the gas phase leads to the formation of a dipole-bound state, which acts as a doorway for electron capture. This has also been described previously by Sommerfeld in their earlier work. [52][53][54] The dipole-bound state subsequently gets converted into a valence-bound state. The potential energy surface of the ground and the first excited state of the DNA base anion shows an avoided crossing, and the transfer of electron takes place through a non-adiabatic process. A similar doorway mechanism has been observed for the nucleobase anion in the condensed phase, where the water-bound state acts as the doorway state. 25,55,56 The formation of stable anion through the doorway mechanism will be competitive with the resonance-based dissociative electron attachment in DNA.
The presence of other molecular components in close proximity to DNA, especially the proteins, can play a crucial role in determining the relative rate of the two pathways as well as the amount of induced damage. Numerous experimental and theoretical investigations have been reported in the literature on the role of protein on the radiation damage pathway. Solomun et. al. 57 have made the first attempt to study the role of a protein in LEE (3 eV) induced DNA strand break using fluorescence spectroscopy. They have reported that the protein is an effective inhibitor of radiation-induced DNA strand break. Experimental studies of arginine and glycine with a short DNA chain have also shown that a high concentration of amino acids in the samples results into protective action against LEE irradiation. 58 Additionally, gas phase studies on uracil-alanine and uracil-glycine complexes have observed a barrier-free proton transfer between anionic nucleobase and amino acids to stabilize excess negative charge. 59,60 Kohanoff and co-workers 8,61 have also revealed that the presence of glycine environment acts as a protective shield against the electron attachment to thymine. However, they have restricted their attention only to the ground state of the anion. Experimental studies have shown that the excited states of the anion play an important role in the electron attachment process to the DNA 62 . The accurate calculation of the ground and excited state of a complete DNA strand is not computationally feasible. The model systems can be helpful in getting an understanding of the role of protein in electron attachment-induced damages. In this study, we have investigated the mechanism of electron attachment to cytosine in the microsolvated and bulk amino acid solution.

Computational Details
The choice of cytosine as a nucleobase in the model system goes to its highest vertical electron affinity among all the nucleobases. 63,64 In the micro-solvation study, we have restricted our attention to cytosine-amino acid dimer complexes. Geometries of all the complexes were optimized using the RI-MP2/def2-TZVP level of theory. The cartesian coordinates for all the optimized dimers at neutral and anionic geometries are provided in the supporting information.
The EA-EOM-DLPNO-CCSD 65 /aug-cc-pVXZ (X=D, T) level of theory has been used to calculate single-point electron affinities. Additional 5s5p4d diffuse functions 66 were added in an even-tempered way to the positive end of the dipole moment vector. The calculated electron affinity values have been extrapolated to the complete basis set (CBS) limit. The RIJK approximation in ORCA has been used for Hartree-Fock calculations, and the NORMALPNO setting has been used for the DLPNO calculations. [67][68][69][70][71][72][73][74] To understand the behaviour of cytosine in a bulk amino acid medium, the cytosine molecule was placed in a cubic box of 40Å length and solvated with 827 glycine molecules. The number of glycine molecules to be put inside the box was calculated from the equilibrium density of glycine at room temperature. Classical molecular dynamics (MD) simulations were performed using the NAMD 75 software package to generate the distribution of glycine molecules around the cytosine molecule. A CHARMM compatible forcefield, parameterized with SwissParam 76 has been used for both cytosine and glycine. These forcefield files can be downloaded from our website. 77 The system was minimized initially with a non-bonded interaction cut-off of 12 Å, and positional constraints were applied on cytosine. After minimization, the system was slowly heated to 300 K and then equilibrated for 10 ns at a constant temperature and pressure of 300 K and 1 atmosphere, respectively. Periodic boundary conditions and particle mesh Ewald technique (PME) have been employed in the equilibration run. Subsequently, a 30 ns production run was carried out at constant volume and constant temperature (300 K).
Equilibration and production run were performed with a time integration step of 2 fs. More details of which have been given in the supporting information ( Figure S1). All the QM/MM calculations have been performed using ORCA quantum chemistry package. 78

Effect of micro solvation
To unravel the role of amino acids in the process of electron attachment to cytosine, four different amino acids (arginine, lysine, alanine and glycine) are chosen to form complexes with cytosine. Glycine is selected due to its nonpolar nature and the presence of a short side chain, which will keep the cost of the calculations down. Whereas arginine, alanine, and lysine are considered as they form a large part of histone proteins bonded to DNA in the biological environment. Table 1 presents the dipole moment and vertical electron affinity (VEA) corresponding to the dipole-bound state of all four complexes of cytosine and amino acid.
The addition of an electron to the neutral cytosine leads to the formation of a dipole-bound state where the excess electron is located away from the nuclear framework (See Figure 1).
The geometry distortion is minimal 51 in the dipole bound case which leads to almost identical values of VEA with Vertical Detachment Energy (VDE) and Adiabatic Electron Affinity (AEA) for the dipole bound state. All four amino acids, cytosine, and the considered cytosineamino acid complexes show the presence of a dipole-bound state.
The dipole moment and VEA values mentioned in Table 1  The valence-type anionic states of the cytosine-amino acid complexes are not stable at neutral geometry and are only bound in the anionic geometry. The VDE and AEA corresponding to the valence-bound state of all the complexes are also mentioned in Table 1. For the isolated amino acid, the excess electron is not bound even at the anionic geometry, which is reflected in negative AEA values of all the amino acids. The AEA value is negative for isolated cytosine also. However, the AEA value in the case of all the complexes is positive, depicting the formation of the stable valence-bound anionic state. The isolated cytosine has a larger (less negative) AEA value than the amino acids. Hence, one can assume that the excess electron in the valence-bound state of complexes should preferentially localize on the nucleobase. This is indeed observed in all four complexes (See Figure 1). The positive AEA values in the case of complexes can be related to the stabilizing effect of hydrogen bonding between the cytosine and the amino acids. Out of all the four complexes, the VDE is maximum in the case of cytosine-arginine complex, with a value of 1.57 eV. Higher stability of this complex can be attributed to a larger number of H-bonding interaction sites in arginine compared to the other three amino acids.
The amino acids can act as a protective agent for the nucleobase in two ways. 58 The initial dipole-bound states in cytosine-amino acid dimers act as a doorway for electron capture, and the electron subsequently gets transferred to the cytosine-bound state. To understand the mechanism of electron transfer from the dipole bound to the valence bound state of cytosine with and without the presence of amino acids, we have calculated the adiabatic potential energy curve (PEC) of ground and first excited state of the anion along a linear transit from dipole-bound to valence-bound geometry. Intermediate geometries along the linear transit can be derived using the following expression: Where RDB is the parameter (bond length, bond angle and dihedral angle) for the dipole-bound geometry, and RVB is the parameter corresponding to the geometry of the valence-bound state.
λ is the linear transition parameter, which varies from 0 to 1. The =0 leads to the dipolebound geometry, and =1 leads to the valence-bound geometry. The adiabatic potential energy curve in Figure 4 shows an avoided crossing between the ground and excited states of the anion, indicating a mixing of electronic and nuclear degrees of freedom. Such mixed states cannot be treated by standard Born-Oppenheimer approximation. 79 As a potential solution, one can switch to a diabatic representation where the electro-nuclear coupling is replaced by the electronic coupling between the two diabatic states. Two diabatic states were generated using morse potential fitting to the valence-bound and dipole-bound parts of the ground state adiabatic PEC. The PEC for all the four cytosine-amino acid complexes are presented in supporting information (See Figure S3). A two-state avoided crossing model potential 79 has been used to calculate the coupling between the two diabatic states as follows: where the diagonal elements V1 and V2 are morse potentials in the coordinate as per the following equation, keeping off-diagonal elements constant. The potential varies with as per the equation: One can observe from Table 2  where λ, W and ∆ 0 represent the reorganization energy, coupling constant, and free energy change between the two states (EVB -EDB), respectively.
The rate of electron transfer from dipole bound to valence-bound species is presented in Table   2 for isolated cytosine, monohydrated cytosine, and all the cytosine-amino acid complexes. It should be noted that the rate of transfer is increased to 5-6 folds in dimers in comparison to isolated cytosine. The cytosine-arginine complex shows the highest rate of electron transfer of 7.8410 11 sec -1 between the dipole and valence-bound state. The other three complexes show one-fold lower rate than that observed in the cytosine-arginine case. The rates in all four complexes are higher than that observed in the monohydrated cytosine 25 . The rapid formation of valence-bound anionic state leads to the scavenging of low-energy electrons by the nucleobase and suppresses the electron attachment induced bond breaking 24 .

Effect of the local environment
Glycine, being the smallest of all the considered amino acids, is chosen to look into the effect of the local environment of glycine on the electron attachment to cytosine. Various conformers of the cytosine-glycine complex were generated using CREST 81 software and subsequently optimized using the XTB method 82 . The twelve lowest energy isomers were taken and further optimized at the RI-MP2/def2-TZVP level of theory. Figure 5 shows all the twelve cytosineglycine conformers generated using the CREST package, arranged in increasing order of their relative MP2 energy with respect to the most stable conformer a1. The twelve isomers of the cytosine-glycine complex can be categorized into four classes of position isomers based on their hydrogen bonding patterns. They are denoted by a, b, c, and d in Figure 5. Further calculations were performed for the most stable isomers in their respective category (a1, b1, c1 and d1). These are shown as encircled structures in Figure 5.
Dipole moment and VEA for dipole bound state, and VDE and AEA for valence bound state of these four conformers are mentioned in Table 3. The electron attachment to all the four conformers at neutral geometry leads to the formation of dipole bound anionic state (see Figure   6). Isomer b1 shows the highest dipole moment and, consequently, the highest VEA. The ground state at the anionic geometry is valence-bound in nature. One can see proton transfer from glycine to cytosine in isomers a1 and b1 in the anionic geometry. The AEA of b1 is also higher than other isomers. We have calculated the potential energy surface corresponding to the linear transit from dipole-bound to valence-bound geometry (see Figure S4) for all the four lowest energy conformers. The corresponding coupling constant and the rate of electron transfer is presented in Table 4.
Isomer c1 shows the highest coupling constant of 19.4 meV, with the rest of the three isomers showing almost half of the corresponding value. The rate of transfer from dipole-bound to valence-bound state is maximum for isomer b1, which is one order of magnitude higher than the rest of the three isomers and similar to that observed for cytosine in GC base-pair. 83 It shows that the electron attachment properties of cytosine in cytosine-glycine dimer depends upon the local interaction between cytosine and glycine. Therefore, the conclusions drawn about the electron attachment to cytosine in the presence of glycine from micro-solvation models may not be transferrable to the cytosine in a bulk glycine environment.

Effect of bulk glycine environment
Although a dimer model can be helpful in understanding the mechanism of the electron attachment process, to simulate the electron attachment process to DNA in a realistic environment, one needs to consider the effect of the bulk environment. A high amino acid concentration can increase the stability of cytosine anion with the formation of an extensive hydrogen-bond network.
To understand the effect of high amino acid concentration,  Therefore, the glycine-bound state acts as a doorway for the formation of a stable valence-type nucleobase-bound state. The transition from glycine-bound state to nucleobase-bond state was observed around 100-105 fs and is followed by glycine to cytosine proton transfer. This glycine to cytosine proton transfer occurs around 150 fs. To further understand the nature of the initial glycine-bound state, we have reperformed the EA-EOM-DLPNO-CCSD calculation at the snapshot from 0.5 fs by keeping the cytosine molecule as a ghost. The corresponding natural orbitals are presented in Figure S5. One can see that the VDE of the system with and without cytosine is almost identical and leads to nearly identical natural orbitals for the anionic state.
Therefore, this doorway state arises primarily due to the interaction of the electron with the glycine. Here, one can see a sharp peak around 1 Å in case of interaction observed in case a and around 3 Å for case b, which indicates that a short-range local structure of glycine is formed around the cytosine anion. This local structure of the glycine distribution leads to the preferential stabilization of the cytosine anion through proton transfer. This kind of protection can be referred to as a chemical mechanism of protection, where the glycine, by preferential stabilization of the valence-bound cytosine anion, increases its electron scavenging effect and reduces the availability of secondary electrons for resonance-induced strand breaking. One should note that, unlike the micro-solvated cytosine-amino acid system, where the dipole bound state has electron density situated towards cytosine, the bulk solvated system shows stabilization via physical shielding also. The initial electron density is localized on glycine and away from the nucleobase. It reduces the possibility of base-centered resonance states [84][85][86] which leads to DNA strand breaking. A similar trend has been observed in all the considered QM/MM dynamics trajectories generated from other snapshots. The radial distribution function depicted in Figure 9 has been generated from the 25 ns snapshot trajectory.

Conclusions
In this work, we have studied the effect of amino acids on the electron attachment behaviour          Figure 6: Natural orbitals corresponding to dipole and valence bound state of four isomers of Glycine + Cytosine.