EXPERIMENTAL METHODS
5.4 Discussion
In this study, two main radicals in the L-lys·HCl·2H2O crystalirradiated at 66K were identified as
the carboxyl anion and the decarboxylation radical. For the carboxyl anion, we identified two neighboring protons transferred to the carboxyl group following the electron trapping: H1’→O1 and H2’→O2. Such proton transfer is common for carboxyl radical anions20-24. Miyagawa et al.27 proposed a proton-tunneling model to explain the proton addition to the alanine carboxyl anion as follows. Before irradiation, the energy surface for the proton in hydrogen bond of N-H…O (Fig. 5.7a) has the H in the deeper part of the potential well near the N, and quantum tunneling can take place only for excited vibrational states. After irradiation and formation of the carboxyl anion, additional negative charge is on the oxygen atom, which increases its proton affinity. As a result, the energy minimum on the side of O atom becomes deeper (Fig. 5.7b), and the proton can tunnel more easily through the energy barrier and bond to the O atom. Also, this tunneling model suggested that the tunneling rate (the proton transfer rate) is proportional to the hydrogen bond distance. That is, a shorter hydrogen bond may induce higher probability of the proton transfer.
Iwasaki et al. also applied the tunneling model to describe formation of OH radicals and of carboxyl radical in irradiated carboxylic acid28.A similar mechanism can explain the proton transfers in the lysine
carboxyl anion. For example, H2’…O2 is the shortest intermolecular hydrogen bond in the crystal, and thus H2’ will have the highest tunneling rate or transfer rate to form the O2-H2’ bond according to this model. In the two hydrogen bonds N2’-H1’…O1 and Ow1-H4’…O1, H1’ bonds to a side chain amino
model calculations, the energy for deprotonation of NH4+ is lower than that of H2O by ~900kJ/molIII.
Thus H1’ can more easily tunnel from the neighboring NH3+ to form the O1-H1’ bond than H4’ from the
water. The two proton transfers (to O1 and to O2) indicate that the proton affinity of the carboxyl group is sufficiently high to allowthe second proton transfer.
As described before, the identification of the decarboxylation radical was confirmed by the DFT calculation and WINSIM simulation, even though two coupling tensors could not be obtained from the experiments. Previous studies17 have found that the decarboxylation radical is a common intermediate from the oxidation product and originates from the carboxyl cation. In DNA/histone complexes, existing evidence indicates that electrons transfer from histone to DNA, thereby inducing a significant increase in the proportion of DNA anions, and leaving the oxidation products in the histone side. Thus, the detection and study of the mechanisms of decarboxylation radicals can be helpful for understanding the role of histone on the radiation damage mechanisms for DNA in vivo. In addition, the carboxyl oxygen atoms are general intermolecular hydrogen bond acceptors in amino acids. Thus, the direct result of decarboxylation is the breakage of the hydrogen bond. If the carboxyl oxygen of lysine in chromatin acts as hydrogen bond acceptors between DNA and histone, then it is reasonable to predict that decarboxylation in lysine (or in histone) can induce weakening of the DNA-histone association following irradiation. This effect was reported by Lloyd and Peacocke in 1965 29. However, this prediction needs to be verified by more
studies on the irradiation damage to DNA in vivo to show that the decarboxylation radical can be formed in chromatin and can induce the hydrogen bond breakage between histone and DNA.
III
In the modeling calculations, full optimization plus frequency calculation were performed on the molecules H2O and NH4+.
The initial coordinates of H2O came from cluster D in Fig. 5.2a and those of NH4+ came from cluster A in Fig.5.2a by
substituting an H for the methyl group. Then H4’ and H1’ (see Fig. 5.2a) were removed from H2O and from NH4+ respectively,
followed by full optimization and frequency calculations on the two structures. Single point energy calculations were performed
for each optimized geometry. The energies for deprotonation were given by DPE(H2O) = E(OH-)-E(H2O) and by DPE(NH4+) =
(a) (b)
Fig. 5.7 The sketch of energy curves for the proton in hydrogen bond N-H…O before the irradiation
damage (a) and after the irradiation and the carboxyl anion formed (b).
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CHAPTER 6.