One-electron oxidation of ds(5'-GGG-3') and ds(5'-G(8OG)G-3') and the nature of hole distribution: a density functional theory (DFT) study.

Of particular interest in radiation-induced charge transfer processes in DNA is the extent of hole localization immediately after ionization and subsequent relaxation. To address this, we considered double stranded oligomers containing guanine (G) and 8-oxoguanine (8OG), i.e., ds(5'-GGG-3') and ds(5'-G8OGG-3') in B-DNA conformation. Using DFT, we calculated a variety of properties, viz., vertical and adiabatic ionization potentials, spin density distributions in oxidized stacks, solvent and solute reorganization energies and one-electron oxidation potential (E0) in the aqueous phase. Calculations for the vertical state of the -GGG- cation radical showed that the spin was found mainly (67%) on the middle G. However, upon relaxation to the adiabatic -GGG- cation radical, the spin localized (96%) on the 5'-G, as observed in experiments. Hole localizations on the middle G and 3'-G were higher in energy by 0.5 kcal mol-1 and 0.4 kcal mol-1, respectively, than that of 5'-G. In the -G8OGG- cation radical, the spin localized only on the 8OG in both vertical and adiabatic states. The calculated vertical ionization potentials of -GGG- and -G8OGG- stacks were found to be lower than that of the vertical ionization potential of a single G in DNA. The calculated E0 values of -GGG- and -G8OGG- stacks are 1.15 and 0.90 V, respectively, which owing to stacking effects are substantially lower than the corresponding experimental E0 values of their monomers (1.49 and 1.18 V, respectively). SOMO to HOMO level switching is observed in these oxidized stacks. Consequently, our calculations predict that local double oxidations in DNA will form triplet diradical states, which are especially significant for high LET radiations.

Comprehensive model for X-ray-induced damage in protein crystallography.

Acquisition of X-ray crystallographic data is always accompanied by structural degradation owing to the absorption of energy. The application of high-fluency X-ray sources to large biomolecules has increased the importance of finding ways to curtail the onset of X-ray-induced damage. A significant effort has been under way with the aim of identifying strategies for protecting protein structure. A comprehensive model is presented that has the potential to explain, both qualitatively and quantitatively, the structural changes induced in crystalline protein at ∼100 K. The first step is to consider the qualitative question: what are the radiation-induced intermediates and expected end products? The aim of this paper is to assist in optimizing these strategies through a fundamental understanding of radiation physics and chemistry, with additional insight provided by theoretical calculations performed on the many schemes presented.

Calculation of Standard Reduction Potentials of Amino Acid Radicals and the Effects of Water and Incorporation into Peptides.

Guanine (Guo) is generally accepted as the most easily oxidized DNA base when cells are subjected to ionizing radiation; calculations of the standard reduction potential of the guanyl radical, Eo(Guo•+/Guo) are within ∼0.1 V of experimental values in aqueous solution extrapolated to standard conditions. While a number of experimental studies have shown some amino acid radicals have redox properties at pH 7 which suggest or confirm a capacity for radical "repair" by electron transfer from the amino acid to Guo•+ (or its deprotonated conjugate), the redox properties of the radicals of other amino acids, including methionine, lysine and cystine, are less well characterized. In addition, the effects of incorporation of the amino acids into peptides, or the effects of water of hydration on calculated potentials, have not been extensively studied. In this work, calculations of standard reduction potentials of radicals from model amino acids as they appear in histone proteins are performed. To predict redox properties at pH 7, acid dissociation constants (pKas) of both radical and ground state amino acids are required. In some instances these are not experimentally determined and calculated pKas have been derived for some common amino acids and compared with experimental values.

Calculations of the Energetics of Oxidation of Aqueous Nucleosides and the Effects of Prototropic Equilibria.

Recently the calculated standard reduction potentials of the radical-cations of N-methyl substituted DNA bases have been reported that agree fairly well with the experimental results. However, there are issues reflecting the fact that the experimental results usually relate to the couple E(o)(Nuc(•),H(+)/NucH(+)), whereas the calculated results are for the E(o)(Nuc(•+)/Nuc) couple. To calculate the midpoint reduction potential at pH 7 (Em7), it is important to have accurate acid dissociation constants (pKs) for both ground-state bases and their radicals, and the effects of uncertainty in some of these values (e.g., that of the adenosine radical) must be considered. Calculations of the pKs of the radicals of the nucleic acid bases (as nucleosides) have been performed to explore the effects the various pKs have on calculating the values of Em7 and to see what improvements can be made with the accuracy of the calculations.

Calculated pK(a)'s of the DNA base radical ions.

For the DNA bases the pK(a)'s of the neutral molecules have been measured, and a review article by Steenken provides a convenient summary of the experimental pK(a)'s of the DNA radical ions. The purpose of the present work is to attempt to calculate the pK(a)'s of the DNA radical ions and to compare these results with the experimental values. The agreement between the calculated and experimental pK(a)'s for the guanine, cytosine, and thymine cations is very good but the pK(a) calculation on the adenine cation is not close to the experimental value. It is tempting to suggest that the pK(a) of the adenine cation is not actually ≤1 but is more likely somewhere near the calculated value of 3.9. Problems were encountered in calculating the pK(a)'s of the one-electron reduced nucleobases because optimizations tend to produce nonplanar structures. The calculations presented show close agreement between the calculated and experimental pK(a)'s for the thymine, cytosine, and adenine anions. Although there are no experimental data for the pK(a) of the guanine reduction product, the calculated value of 17.6 seems to be too high, given that for thymine the protonation is also at oxygen and results in a rather low pK(a).

DNA damage by the direct effect of ionizing radiation: products produced by two sequential one-electron oxidations.

It has long been assumed that the population of radicals trapped in irradiated DNA (that is, the radicals escaping recombination) would quantitatively account for the lesions observed in DNA. Recent results indicate that this is not the case. The yield of DNA lesions exceed the yield of trappable radicals. To account for a portion of this shortfall, it is thought that some of the initially formed 2'-deoxyribose radicals undergo a second oxidation by nearby base cation radicals to form 2'-carbocations. The carbocations react to give strand breaks and free base release. Schemes are presented to account for the major oxidation products observed including 8-oxoGua, 8-oxoAde, 5-OHMeUra, and free base release. Theoretical calculations were performed to ascertain the likelihood of the second oxidation step in these reaction pathways actually occurring, and to account for base sequence dependence and various levels of hydration.

Calculated vertical ionization energies of the common α-amino acids in the gas phase and in solution.

The vertical ionization energies of the low-lying conformers of the α-amino acids found in proteins have been calculated. Geometry optimizations were first performed at the B3LYP/6-311G(d,p) level of theory, and then reoptimized at the MP2/6-311G(d,p) level of theory. Vertical ionization energies were then computed by three methods, electron propagator in the partial third-order (P3) approximation, Outer-Valence-Green's Functions, and by evaluating the difference in the total energy between the cation radical and the neutral amino acid in the geometry of the neutral species. When available, the results are compared to the experimental vertical ionization energies. The vertical ionization energies calculated using the MP2/P3 method gave the best overall agreement with the experimental results. Next, the ionization energies in solution are calculated for the zwitterionic forms of the α-amino acids by using IEFPCM methods. To obtain the vertical ionization energy in solution, it is necessary to use the nonequilibrium polarizable continuum model (NEPCM), the results of which are reported here for the α-amino acids.

One-electron oxidation of individual DNA bases and DNA base stacks.

In calculations performed with DFT there is a tendency of the purine cation to be delocalized over several bases in the stack. Attempts have been made to see if methods other than DFT can be used to calculate localized cations in stacks of purines, and to relate the calculated hyperfine couplings with known experimental results. To calculate reliable hyperfine couplings it is necessary to have an adequate description of spin polarization which means that electron correlation must be treated properly. UMP2 theory has been shown to be unreliable in estimating spin densities due to overestimates of the doubles correction. Therefore attempts have been made to use quadratic configuration interaction (UQCISD) methods to treat electron correlation. Calculations on the individual DNA bases are presented to show that with UQCISD methods it is possible to calculate hyperfine couplings in good agreement with the experimental results. However these UQCISD calculations are far more time-consuming than DFT calculations. Calculations are then extended to two stacked guanine bases. Preliminary calculations with UMP2 or UQCISD theory on two stacked guanines lead to a cation localized on a single guanine base.

One-electron oxidation of 2'-deoxyadenosine-5'-phosphate: comparisons of theoretical calculations with experimental values.

A recent paper by Hou et al. (Hou, R.; Gu, J.; Xie, Y.; Yi, X.; Schaefer, H. F. J. Phys. Chem. B 2005, 109, 22053) on 2'-deoxyadenosine-5'-phosphate (5'-dAMP) reports calculations on one-electron oxidation of the 5'-dAMP anion. The paper presents a very interesting observation that, for the radical produced by electron removal, the unpaired spin density resides on both the phosphate and the adenine base moieties. There are also indications that this radical has a weakened C5'-O5' bond, and it is said that this may be the origin of a single-strand break in DNA. New calculations have been performed to show that the spin density on the phosphate is dependent on the charge on the phosphate. The use of the B3LYP method with the 6-31G(d) basis set yields results very similar to those obtained with the much larger B3LYP/DZP++ basis set in computing the structures of one electron oxidized 5'-dAMP. New calculations on the isotropic hyperfine couplings in 5'-dAMP are presented to show under just what conditions one might expect to see small amounts of unpaired spin density on the phosphates. Results show that this may occur in gas-phase studies of nucleotides but, most likely, not in DNA.

Ionization energy thresholds of microhydrated adenine and its tautomers.

In the present work the vertical and adiabatic ionization energy thresholds (IET) of adenine, and its amino and imino tautomers complexed with 1-3 water molecules are presented. The vertical and adiabatic IETs have been calculated at the B3LYP and P3 levels of theory, using the standard 6-31++G(d,p) basis set. The results show that there is hardly any effect of microhydration on the vertical DeltaIET of adenine, which is at odds with the experimental values determined by Kim et al. (J. Phys. Chem. 1996, 100, 7933). In an attempt to assign the experimental DeltaIET values, calculations have been performed on the microhydrated amino and imino tautomers of adenine. Vertical DeltaIET calculations and adiabatic DeltaIET calculations on adenine N7H tautomers complexed with water are in better agreement with the experimental results than are calculations involving the canonical (N9H) form of adenine.

Ionization energies of the nucleotides.

The vertical ionization energies of the four nucleotides have been computed. Geometries have been chosen to mimic orientations as they appear in B-DNA. The negative charge on the phosphate was neutralized by protonation, and also by the inclusion of counterions. Calculations have been performed with electron propagator methods (P3), Møller-Plesset second-order perturbation theory, and density functional theory to determine the nature of the orbitals associated with the highest lying ionization energies. Calculations at the MP2/6-311G(d,p)//P3/6-311G(d,p) level of theory yield vertical ionization energies for 5'-dTMP 9.05 eV, for 5'-dCMP 8.40 eV, for 5'-dAMP 8.16 eV and for 5'-dGMP 7.96 eV. In all cases the highest occupied molecular orbital resides on the base moieties.

Theoretical elucidation of conflicting experimental data on vertical ionization potentials of microhydrated thymine.

In a recent article we reported calculations of the ionization energy thresholds (IET) of microhydrated thymine (Close; et al. J. Phys. Chem. A, 2006, 110, 7485). Calculations showed a distinct effect of microhydration on the IET's of thymine. The first water molecule was seen to decrease the IET by about 0.1 eV, and the second and third water molecules caused a further decrease of less than 0.1 eV each. These changes in IET calculated for the canonical form of thymine with 1-3 waters of hydration are smaller than the experimental values determined by Kim et al. (J. Phys. Chem. C 1996, 100, 7933). In the present study it has been shown that there is considerable reorientation of the water molecules in microhydrated thymine upon ionization. This leads to the expectation that the experimental ionization energies may therefore represent an adiabatic process. The results presented here show that the changes in experimental ionization energies determined by Kim et al. for microhydrated thymine are in good agreement with the calculated adiabatic ionization energies.

Determination of redox potentials for the Watson-Crick base pairs, DNA nucleosides, and relevant nucleoside analogues.

Redox potentials for the DNA nucleobases and nucleosides, various relevant nucleoside analogues, Watson-Crick base pairs, and seven organic dyes are presented based on DFT/B3LYP/6-31++G(d,p) and B3YLP/6-311+G(2df,p)//B3LYP/6-31+G* levels of calculations. The values are determined from an experimentally calibrated set of equations that correlate the vertical ionization (electron affinity) energy of 20 organic molecules with their experimental reversible oxidation (reduction) potential. Our results are in good agreement with those estimated experimentally for the DNA nucleosides in acetonitrile solutions (Seidel et al. J. Phys. Chem. 1996, 100, 5541). We have found that nucleosides with anti conformation exhibit lower oxidation potentials than the corresponding syn conformers. The lowering in the oxidation potential is due to the formation of an intramolecular hydrogen bonding interaction between the 5'-OH group of the sugar and the N3 of the purine bases or C2=O of the pyrimidine bases in the syn conformation. Pairing of adenine or guanine with its complementary pyrimidine base decreases its oxidation potential by 0.15 or 0.28 V, respectively. The calculated energy difference between the oxidation potential for the G.C base pair and that of the guanine base is in good agreement with the experimental value estimated recently (0.34 V: Caruso, T.; et al. J. Am. Chem. Soc. 2005, 127, 15040). The complete and consistent set of reversible redox values determined in this work for the DNA constituents is expected to be of considerable value to those studying charge and electronic energy transfer in DNA.

Reductive damage in directly ionized DNA: saturation of the C5=C6 bond of cytosine in d(CGCG)(2) crystals.

Electron paramagnetic resonance (EPR) was used to study an oligodeoxynucleotide duplex of d(CGCG)(2) that is known to crystallize in Z-form. After X irradiation at 4 K, EPR data were collected on single crystals and polycrystalline samples as a function of annealing temperature and dose. A radical produced by the net gain of a hydrogen atom at C6 and a proton at N3, Cyt(C6+H, N3+H(+))(+*), is identified. This radical had not been positively identified in polymeric DNA previously. The Cyt(C6+H, N3+H(+))(+*) makes up about 4% of the total radical population at 4 K, increasing to about 10-15% after the DNA is annealed to 240 K. There appears to be neither an increase nor a decrease in the absolute concentration of Cyt(C6+H, N3+H(+))(+*) upon annealing from 4 K to 240 K. Additionally, the presence of another radical, one due to the net gain of hydrogen at C5 of cytosine, the Cyt(C5+H)(*), is implicated. Together, these two radicals appear to account for 60-80% of the reduced species in DNA that has been irradiated at 4 K and annealed to 240 K.

Calculating hyperfine couplings in large ionic crystals containing hundreds of QM atoms: subsystem DFT is the key.

We present an application of the linear scaling frozen density embedding (FDE) formulation of subsystem DFT to the calculation of isotropic hyperfine coupling constants (hfcc's) of atoms belonging to a guanine radical cation embedded in a guanine hydrochloride monohydrate crystal. The model systems range from an isolated guanine to a 15,000 atom QM/MM cluster where the QM region is comprised of 36 protonated guanine cations, 36 chlorine anions, and 42 water molecules. Our calculations show that the embedding effects of the surrounding crystal cannot be reproduced by small model systems nor by a pure QM/MM procedure. Instead, a large QM region is needed to fully capture the complicated nature of the embedding effects in this system. The unprecedented system size for a relativistic all-electron isotropic hfcc calculation can be approached in this work because the local nature of the electronic structure of the organic crystals considered is fully captured by the FDE approach.

Electron transfer in amino acid.nucleic acid base complexes: EPR, ENDOR, and DFT study of X-irradiated N-formylglycine.cytosine complex crystals.

Single crystals of the 1:1 complex of the nucleic acid base cytosine and the dipeptide N-formylglycine (C.NFG) have been irradiated at 10 and 273 K to doses of about 70 kGy and studied at temperatures between 10 and 293 K using 24 GHz (K-band) and 9.5 GHz (X-band) electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR), and ENDOR-induced EPR (EIE) spectroscopy. In this complex, the cytosine base is hydrogen bonded at positions N3 and N4 to the carboxylic group of the dipeptide, and the N3 position of cytosine has become protonated by the carboxylic group. At 10 K, two major radicals were characterized and identified. One of these (R1) is ascribed to the decarboxylated N-formylglycine one-electron oxidized species. The other (R2) is the N3-protonated cytosine one-electron reduced species. A third minority species (R3) appears to be a different conformation or protonation state of the one-electron reduced cytosine radical. Upon warming, the R2 and R3 radicals decay at about 100 K, and at 295 K, the only cytosine-centered radicals present are the C5 and C6 H-addition radicals (R5, R6). The R1 radical decays at about 150 K, and a glycine backbone radical (R4) grows in slowly. Thus, in the complex, a complete separation of initial oxidation and reduction events occurs, with oxidation localized at the dipeptide moiety, whereas reduction occurs at the nucleic acid base moiety. DFT calculations indicate that this separation is driven by large differences in electron affinities and ionization potentials between the two constituents of the complex. Once the initial oxidation and reduction products are trapped, no further electron transfer between the two constituents of the complex takes place.

Influence of microhydration on the ionization energy thresholds of thymine: comparisons of theoretical calculations with experimental values.

In the present study the ionization energy thresholds (IETs) of thymine, and thymine keto-enol tautomers, have been calculated (with the B3LYP, and P3 levels of theory using the standard 6-31++G(d,p) basis set) with 1-3 water molecules placed in the first hydration shell. Calculations show there is a distinct effect of microhydration on the IET of thymine. The first water molecule is seen to decrease the IET by about 0.1 eV, whereas the second and third water molecules cause a further decrease of less than 0.1 eV each. The changes in IET calculated here for thymine with 1-3 waters of hydration are smaller than the experimental values determined by Kim et al. (J. Phys. Chem. 1996, 100, 7933). Therefore calculations have been performed on the microhydrated keto-enol tautomers of thymine. The calculated results on the keto-enol tautomers are seen to be in better agreement with the experimental results. However, the keto-enol thymine tautomers are considerably higher in energy than the canonical form of thymine, and there is presently no good evidence that these thymine tautomers are actually present in a supersonic jet-cooled experiment.

The influence of microhydration on the ionization energy thresholds of uracil and thymine.

In the present study the ionization energy thresholds (IET's) of uracil and thymine have been calculated (with the B3LYP, PMP2, and P3 levels of theory using the standard 6-31++G(d,p) basis set) with one to three water molecules placed in the first hydration shell. Then (B3LYP) polarizable continuum model (PCM) calculations were performed with one to three waters of the hydration shell included. Calculations show there is a distinct effect of microhydration on uracil and thymine. For uracil, one added water results in a decrease in the IET of about 0.15 eV. The second and third water molecules cause a further decrease by about 0.07 eV each. For thymine, the first water molecule is seen to decrease the IET by about 0.1 eV, while the second and third water molecules cause a further decrease of less than 0.1 eV each. The changes in IET calculated here for thymine with one to three waters of hydration are smaller than the experimental values determined by Kim et al. (Kim, S. K.; Lee, W.; Herschbach, D. R. J. Phys. Chem. 1996, 100, 7933). Preliminary results presented here indicate that the experimental results may involve keto-enol tautomers of thymine. The results of placing the microhydrated structures of uracil and thymine in a PCM cavity was seen to make very little difference in the IET when compared to the IET of ordinary uracil or thymine in a PCM cavity. The implications are that accurate calculations of the IET's of uracil and thymine can be obtained by simply considering long-range solvation effects.