Charge separation and charge delocalization identified in long-living states of photoexcited DNA

Base stacking in DNA is related to long-living excited states whose molecular nature is still under debate. To elucidate the molecular background we study well-defined oligonucleotides with natural bases, which allow selective UV excitation of one single base in the strand. IR probing in the picosecond regime enables us to dissect the contribution of different single bases to the excited state. All investigated oligonucleotides show long-living states on the 100-ps time scale, which are not observable in a mixture of single bases. The fraction of these states is well correlated with the stacking probabilities and reaches values up to 0.4. The long-living states show characteristic absorbance bands that can be assigned to charge-transfer states by comparing them to marker bands of radical cation and anion spectra. The charge separation is directed by the redox potential of the involved bases and thus controlled by the sequence. The spatial dimension of this charge separation was investigated in longer oligonucleotides, where bridging sequences separate the excited base from a sensor base with a characteristic marker band. After excitation we observe a bleach of all involved bases. The contribution of the sensor base is observable even if the bridge is composed of several bases. This result can be explained by a charge delocalization along a well-stacked domain in the strand. The presence of charged radicals in DNA strands after light absorption may cause reactions—oxidative or reductive damage—currently not considered in DNA photochemistry.DNA photophysics is crucial for the understanding of light-induced damage of the genetic code (1). The excited state of single DNA bases is known to decay extremely fast on the subpicosecond time scale, predominantly via internal conversion (2, 3). This ultrafast decay is assumed to suppress destructive decay channels, thereby protecting the DNA from photodamage and avoiding disintegration of the genetic information. In contrast to this ultrafast deactivation of single nucleobases, the biological relevant DNA strands show further long-living states (4, 5). Several explanations for these long-living states and the size of their spatial extent have been discussed in the literature (5–9). Delocalized excitons (9); excitons that decay to charge-separated states or neutral excimer states (10, 11); exciplexes located on two neighboring bases (5, 8, 12, 13); or even excited single bases, where steric interactions in the DNA strand impedes the ultrafast decay (14), have been proposed. Further computations suggest a decay of an initially populated delocalized exciton to localized neutral or charged excimer states (15–17). However, to our knowledge, a final understanding of the nature of these long-living states has not been reached. Related experiments were performed in the last decade to investigate charge transport processes in DNA, motivated by DNA electronics and oxidative damage (18, 19). Charge transport was initiated by photoexcitation of modified DNA bases or chromophores and followed by transient absorption (20–23). The transport mechanism was described by charge-hopping, superexchange, or transfer of charge along delocalized domains in DNA (18).Until now, most experimental investigations of the long-living state were performed with transient absorption spectroscopy in the UV-visible (UV/Vis) regime (5, 9, 12) or with time-resolved fluorescence (10, 24, 25). Due to the broad, featureless, and overlapping absorption bands of the different DNA bases in this spectral region, it is difficult to investigate the molecular origin of the long-living states using these methods. A further drawback is the unselective and simultaneous excitation of several bases used in most experiments. To circumvent these problems, we used for the present study well-defined oligonucleotides, which enable selective excitation of one single base. Observation of the long-living excited states was performed via time-resolved IR spectroscopy, which can profit from the many “fingerprint” vibrational bands (26, 27). IR spectroscopy is able to distinguish between different DNA bases and their molecular states. It can also reveal changes in the electronic structure and identify charge-separated states.In this study we used single-stranded DNA, in which π stacking between neighboring bases leads to structured domains, similar to the structure in a double helix (28). This interaction is known to be crucial for the long-living states (5). The investigation of single-stranded DNA enables us to construct special sequences, where only one base can be selectively excited. We used the natural bases 2′-deoxyuridine (U), 2′-deoxyadenosine (A), 5-methyl-2′-deoxycytidine (mC), and 2′-deoxyguanosine (G). The nucleobase U occurs naturally in RNA and is similar to the DNA base thymine but shows a blue-shifted absorbance spectrum. mC occurs with a frequency of 4–5% in mammalian DNA (29) and plays an important role as an epigenetic marker (30). The UV/Vis absorbance of mC and G are red-shifted in comparison with A and U, which allows selective excitation at 295 nm in oligonucleotides consisting of mC, A, and U (Fig. 1 A and B) or G and A. This selectivity can only be obtained in single-stranded DNA because G and its complementary base mC have overlapping absorbance bands in the UV range (Fig. S1). Selectivity in probing is based on the significant differences in the IR-absorption spectra of these bases, which display distinct marker bands for each base (Fig. 1 A and E).Open in a separate windowFig. 1.Selective excitation of mC in mCUA and probing of characteristic A, U, and mC marker bands in the IR. (A, B, and E) Picosecond UV light pulses at 295 nm allow selective excitation of mC (shown in bold) in mixed DNA sequences consisting of mC, U, and A. (B) Absorbance spectra of 2′-deoxyadenosine monophosphate (A), 2′-deoxy-5-methylcytidine (mC), and uridine monophosphate (U). (C) Time-resolved absorption difference (color-coded) plotted vs. wavenumber and delay time for mCUA and (D) for a mixture of the corresponding monomers. (E) Probing the individual contribution of each base is possible in the IR at 1,625 cm⁻¹ (A), 1,655 cm⁻¹ (U), and 1,667 cm⁻¹ (mC) (marked by dashed lines). (F) Transients at 1,667 cm⁻¹ for mCUA and the mixture of monomers.With the combination of selective excitation and selective probing we are able to elucidate the nature of the long-living states in DNA strands. Investigation of dinucleotides clearly shows that light absorption in DNA leads to charge separation between stacked neighboring bases, which recombine on the 100-ps time scale. In longer oligonucleotides we observe simultaneous bleach of several bases, which points to a delocalization of the charges along the strand. Our results show that charge transfer in DNA is a natural process, induced by UV-light absorption of DNA.     

Photochemistry and electron-transfer mechanism of transition metal oxalato complexes excited in the charge transfer band

The photoredox reaction of trisoxalato cobaltate (III) has been studied by means of ultrafast extended x-ray absorption fine structure and optical transient spectroscopy after excitation in the charge-transfer band with 267-nm femtosecond pulses. The Co-O transient bond length changes and the optical spectra and kinetics have been measured and compared with those of ferrioxalate. Data presented here strongly suggest that both of these metal oxalato complexes operate under similar photoredox reaction mechanisms where the primary reaction involves the dissociation of a metal-oxygen bond. These results also indicate that excitation in the charge-transfer band is not a sufficient condition for the intramolecular electron transfer to be the dominant photochemistry reaction mechanism.     

Axial interactions in the mixed-valent CuA active site and role of the axial methionine in electron transfer

Within Cu-containing electron transfer active sites, the role of the axial ligand in type 1 sites is well defined, yet its role in the binuclear mixed-valent Cu_A sites is less clear. Recently, the mutation of the axial Met to Leu in a Cu_A site engineered into azurin (Cu_A Az) was found to have a limited effect on E⁰ relative to this mutation in blue copper (BC). Detailed low-temperature absorption and magnetic circular dichroism, resonance Raman, and electron paramagnetic resonance studies on Cu_A Az (WT) and its M123X (X = Q, L, H) axial ligand variants indicated stronger axial ligation in M123L/H. Spectroscopically validated density functional theory calculations show that the smaller ΔE⁰ is attributed to H₂O coordination to the Cu center in the M123L mutant in Cu_A but not in the equivalent BC variant. The comparable stabilization energy of the oxidized over the reduced state in Cu_A and BC (Cu_A ∼ 180 mV; BC ∼ 250 mV) indicates that the S(Met) influences E⁰ similarly in both. Electron delocalization over two Cu centers in Cu_A was found to minimize the Jahn–Teller distortion induced by the axial Met ligand and lower the inner-sphere reorganization energy. The Cu–S(Met) bond in oxidized Cu_A is weak (5.2 kcal/mol) but energetically similar to that of BC, which demonstrates that the protein matrix also serves an entatic role in keeping the Met bound to the active site to tune down E⁰ while maintaining a low reorganization energy required for rapid electron transfer under physiological conditions.Long-range electron transfer (ET) is vital to a wide range of biological processes, including two key energy transduction pathways essential for life: H₂O oxidation in photosynthesis and O₂ reduction in respiration (1, 2). Nature has adapted a conserved cupredoxin fold motif (i.e., the Greek-key β barrel) to construct two evolutionarily linked, but structurally distinct Cu-containing ET proteins (3–5). These are the mononuclear type 1 (T1) or blue copper (BC) and binuclear purple Cu_A proteins. The first coordination sphere of the classic BC sites [e.g., plastocyanin (Pc) and azurin (Az)] consists of a trigonally distorted tetrahedral environment where Cu resides in an equatorial plane formed by one S(Cys) and two N(His) ligands and has an axial S(Met) ligand (Fig. 1A) (6, 7). The binuclear purple Cu_A site consists of two bridging S(Cys) ligands and two equatorial N(His) ligands as well as an axial polypeptide backbone carbonyl oxygen [O(Gln) on Cu_O] and an axial thioether sulfur [S(Met) on Cu_M] (Fig. 1B) (8–11). Both sites carry out rapid, efficient long-range ET with rates on the order of 10³–10⁵ s⁻¹ (12, 13).Open in a separate windowFig. 1.The active sites of two previously published Cu ET proteins: (A) the monomeric T1 Cu Az from Pseudomonas aeruginosa (PDB ID code 4AZU) and (B) the binuclear purple Cu_A from T. thermophilus (PDB ID code 2CUA).Although BC proteins use a Cu⁺/Cu²⁺ redox couple, the binuclear Cu_A sites use a (Cu¹⁺–Cu¹⁺)/(Cu^1.5+–Cu^1.5+) redox cycle. The oxidized form of Cu_A is mixed-valent (MV), with a highly covalent Cu₂S₂ core that gives rise to its unique spectroscopic features. The unpaired electron is fully delocalized over the two Cu centers and exhibits a characteristic seven-line ^63,65Cu hyperfine splitting pattern in electron paramagnetic resonance (EPR) spectroscopy (14, 15). Maintaining valence delocalization even in the presence of a low symmetry protein environment has been attributed to the large electronic coupling (H_AB) resulting from a direct Cu–Cu σ bond and efficient superexchange facilitated by substantial Cu₂–S(Cys)₂ covalency. This strong electronic coupling between the two Cu’s leads to a Ψ → Ψ* (Cu–Cu σ → σ*) transition at ∼13,500 cm⁻¹ (16). Excitation into this transition using resonance Raman (RR) yields a large excited state distortion in the totally symmetric Cu₂S₂ core “accordion” mode (ν₁), a characteristic of Robin & Day class III MV delocalization (17–19). The two bridging S(Cys) ligands give rise to four in-plane S(p)-derived molecular orbitals (MOs) for S(Cys) → Ψ* charge transfer (CT) transitions. These have been assigned to absorption bands in the region of 20,000 cm⁻¹. Laser excitation into these CT transitions gives rise to RR enhancement of three additional Cu₂S₂ core vibrations (SI Appendix, Fig. S1A). The functional advantage of a valence delocalization in terms of rapid, long-range ET at low driving forces (∼45 mV) has been ascribed to lowering the reorganization energy (λ) by distributing structural rearrangements associated with redox over two Cu centers (20).In nature, the S(Met) ligand of BC is sometimes found to be replaced by other protein residues. These can either coordinate to Cu [e.g., O(Gln) in stellacyanin (St)] or leave the axial position vacant (e.g., Leu in the fungal laccases) (21, 22). In BC, it was found that variation of the axial ligand from O(Gln) to S(Met) to nothing can tune E⁰ over a 300 mV range (23). In nitrite reductase (NiR), the Cu²⁺–S(Met) bond strength could be experimentally determined and was found to be weak (4.6 kcal/mol) as its loss is compensated by an increased S(Cys) donor interaction with Cu. The low strength of this bond suggested an important role of the protein in keeping the S(Met) ligand bound at physiological temperature. The contribution of the protein in stabilizing the active site structure has been referred to as an entatic/rack state in bioinorganic chemistry (24, 25). For BC sites, the protein matrix provides the negative free energy required to overcome the entropically favored S(Met) bond loss. This plays an important role in ET function as S(Met) binding stabilizes the oxidized more than the reduced state of the Cu site and lowers E⁰ by ∼200 mV.In contrast to BC proteins, S(Met) is the only axial ligand found in naturally occurring Cu_A sites [cytochrome c oxidase (CcO), nitrous oxide reductase (N₂OR), nitric oxide reductase (NOR), terminal oxidase in Sulfolobus acidocaldarius (SoxH)] (26). Interestingly, in contrast to BC, the Met to Leu mutation in the Cu_A Az only led to a 16 mV increase in E⁰ (compared with an 86 mV increase for this mutant in BC Az) (27). This apparent difference in the extent of the axial ligand contribution to E⁰ relative to previous studies on BC has led us to further explore its contribution to function in Cu_A and whether or not it is entatic as in BC. We use a combination of spectroscopic methods [low-temperature (LT) absorption and magnetic circular dichroism (MCD), RR, and EPR] coupled to density functional theory (DFT) calculations to investigate the geometric and electronic structures of Cu_A Az and a series of its axial ligand variants (M123X; X = Q, L, H). The influence of the axial ligand on the E⁰ and λ are evaluated and compared with these properties in the well-understood BC site. Furthermore, the proposed involvement of Cu_A in ET pathways (28) as well as the entatic/rack nature of the Cu–S(Met) bond in Cu_A are evaluated and discussed.