Theoretical study of excitation energy transfer in DNA photolyase

Photolyase (PL) is a DNA repair enzyme which splits UV light-induced thymine dimers on DNA by an electron transfer reaction occurring between the photoactivated FADH(-) cofactor and the DNA dimer in the DNA/PL complex. The crystal structure of the DNA/photolyase complex from Anacystis nidulans has been solved. Here, using the experimental crystal structure, we re-examine the details of the repair electron transfer reaction and address the question of energy transfer from the antenna HDF to the redox active FADH(-) cofactor. The photoactivation of FADH(-) immediately preceding the electron transfer is a key step in the repair mechanism that is largely left unexamined theoretically. An important butterfly thermal motion of flavin is identified in ab initio calculations; we propose its role in the back electron transfer from DNA to photolyase. Molecular dynamics simulation of the whole protein/DNA complex is carried out to obtain relevant cofactor conformations for ZINDO/S spectroscopic absorption and fluorescence calculations. We find that significant thermal broadening of the spectral lines, due to protein dynamics, as well as the alignment of the donor HDF and the acceptor FADH(-) transition dipole moments both contribute to the efficiency of energy transfer. The geometric factor of F?rster's dipolar coupling is calculated to be 1.82, a large increase from the experimentally estimated 0.67. Using F?rster's mechanism, we find that the energy transfer occurs with remarkable efficiency, comparable with the experimentally determined value of 98%.     

Resonance Raman spectroscopy of the neutral radical Trp306 in DNA photolyase

The resonance Raman spectrum of the tryptophan neutral radical in a protein, Escherichia coli photolyase, is reported for the first time. The data compare very well to a solution study and computational predictions, and tentative assignments are made for the observed vibrations. This important new result demonstrates the potential of time-resolved resonance Raman spectroscopy as a powerful tool to investigate these radicals in protein electron-transfer processes and in enzymatic reactions in real time.     

Polarized transient absorption to resolve electron transfer between tryptophans in DNA photolyase

Transient absorption spectroscopy is a powerful tool for studying biological electron-transfer chains, provided that their members give rise to distinct changes of their absorption spectra. There are, however, chains that contain identical molecules, so that electron transfer between them does not change net absorption. An example is the chain flavin adenine dinucleotide (FAD)-W382-W359-W306 in DNA photolyase from E. coli. Upon absorption of a photon, the excited state of FADH* (neutral FAD radical) abstracts an electron from the tryptophan residue W382 in approximately 30 ps (monitored by transient absorption). The cation radical W382*+ is presumably reduced by W359 and W359*+ by W306. The latter two reactions could not be monitored directly so far because the absorption changes of the partners compensate in each step. To overcome this difficulty, we used linearly polarized flashes for excitation of FADH*, thus inducing a preferential axis in the a priori unoriented sample (photoselection). Because W359 and W306 are very differently oriented within the protein, detection with polarized light should allow us to distinguish them. To demonstrate this, W306 was mutated to redox-inert phenylalanine. We show that the resulting anisotropy spectrum of the initial absorption changes (measured at 10 ns time resolution) is in line with W359 being oxidized. The corresponding spectrum in wildtype photolyase is clearly different and identifies W306 as the oxidized species. These findings set an upper limit of 10 ns for electron transfer from W306 to W359*+ in wildtype DNA photolyase, consistent with previous, more indirect evidence [Aubert, C.; Vos, M. H.; Mathis, P.; Eker, A. P. M.; Brettel, K. Nature 2000, 405, 586-590].     

Computational studies of DNA photolyase

The electron transfer catalyzed (ETC) repair of the DNA photolesion cyclobutane pyrimidine dimer (CPD) is mediated by the enzyme DNA photolyase. Due to its importance as part of the cancer prevention mechanism in many organisms, but also due to its unique mechanism, this DNA photoreactivation is a topic of intense study. The progress in the application of computational methods to three aspects of the ETC repair of CPD is reviewed: (i) electronic structure calculations of the cycloreversion of the CPD radical cation and radical anion, (ii) MD simulations of the DNA photolyase and its complex to photodamaged DNA, and (iii) the structure and dynamics of photodamaged DNA. The contributions of this work to the overall understanding of the reaction and its relationship to the available experimental work are highlighted.     

Toward an artificial oxidative DNA photolyase

The design, synthesis, structure, and binding affinity of two dioptic receptors for the selective molecular recognition of the cis,syn cyclobutane pyrimidine dimer are reported. The design is based on two 2,6-di(acetamino)pyridine recognition units that are covalently linked via triple bonds to an anthraquinone functional spacer unit. The convergent synthesis uses a modified Sonogashira reaction involving a zinc transmetalation as the key step. The crystal structure of one of the receptors reveals a supramolecular 1D polymer with strong interactions mediated by shape self-complementarity, pi-stacking, and hydrogen bonding between adjacent molecules. Hydrogen bonding between adjacent strands enforces a parallel orientation, which leads to a noncentrosymmetric crystal structure of the highly polar compound. The receptor has an association constant of K(a) = 1.0 x 10(3) M(-1) with the cis,syn pyrimidine dimer, whereas binding of the trans,syn isomer is approximately 1 order of magnitude weaker.     

Femtosecond dynamics of flavin cofactor in DNA photolyase: radical reduction, local solvation, and charge recombination

We report here our femtosecond studies of the photoreduction dynamics of the neutral radical flavin (FADH) cofactor in E. coli photolyase, a process converting the inactive form to the biologically active one, a fully reduced deprotonated flavin FADH(-). The observed temporal absorption evolution revealed two initial electron-transfer reactions, occurring in 11 and 42 ps with the neighboring aromatic residues of W382 and F366, respectively. The new transient absorption, observed at 550 nm previously in photolyase, was found from the excited-state neutral radical and is probably caused by strong interactions with the adenine moiety through the flavin U-shaped configuration and the highly polar/charged surrounding residues. The solvation dynamics from the locally ordered water molecules in the active site was observed to occur in approximately 2 ps. These ultrafast ordered-water motions are critical to stabilizing the photoreduction product FADH(-) instantaneously to prevent fast charge recombination. The back electron-transfer reaction was found to occur in approximately 3 ns. This slow process, consistent with ultrafast stabilization of the catalytic cofactor, favors photoreduction in photolyase.     

Energetics of electron transfer reactions in polar solvents

The free energy change of an electron transfer reaction in a polar solvent is rigorously analyzed within the framework of the dielectric continuum model. An appropriate expression for the electrostatic energy between the two product ions separated by R is derived. The present result does not support a recent claim by Suppan that, if R is close to the contact distance, the electrostatic energy should be much larger in magnitude than estimated from the usual expression −e²/ε_sR.     

Energetics of cholesterol transfer between lipid bilayers

It is believed that natural biological membranes contain domains of lipid ordered phase enriched in cholesterol and sphingomyelin. Although the existence of these domains, called lipid rafts, is still not firmly established for natural membranes, direct microscopic observations and phase diagrams obtained from the study of three-component mixtures containing saturated phospholipids, unsaturated phospholipids, and cholesterol demonstrate the existence of lipid rafts in synthetic membranes. The presence of the domains or rafts in these membranes is often ascribed to the preferential interactions between cholesterol and saturated phospholipids, for example, between cholesterol and sphingomyelin. In this work, we calculate, using molecular dynamics computer simulation technique, the free energy of cholesterol transfer from the bilayer containing unsaturated phosphatidylcholine lipid molecules to the bilayer containing sphingomyelin molecules and find that the affinity of cholesterol to sphingomyelin is higher. Our calculations of the free-energy components, energy and entropy, show that cholesterol transfer is exothermic and promoted by the favorable change in the lipid-lipid interactions near cholesterol and not by the favorable energy of cholesterol-sphingomyelin interaction, as assumed previously.     

Substrate binding modulates the reduction potential of DNA photolyase

The reduction potential of the (FADH-/FADH*) couple in DNA photolyase was measured, and the value was found to be significantly higher than the values estimated in the literature. In the absence of substrate, the enzyme has a reduction potential of 16 +/- 6 mV vs NHE. In the presence of excess substrate the reduction potential increases to 81 +/- 8 mV vs NHE. The increase in reduction potential has physiological relevance since it gives the catalytic state greater resistance to oxidation. This is the first measurement of a reduction potential for this class of DNA-repair enzymes and the larger family of blue-light photoreceptors.     

The electronic structure of the flavin cofactor in DNA photolyase.

Density functional theory is used to calculate the electronic structure of the neutral flavin radical, FADH(*), formed in the light-induced electron-transfer reaction of DNA repair in cis,syn-cyclobutane pyrimidine dimer photolyases. Using the hybrid B3LYP functional together with the double-zeta basis set EPR-II, (1)H, (13)C, (15)N, and (17)O isotropic and anisotropic hyperfine couplings are calculated and explained by reference to the electron densities of the highest occupied molecular orbital and of the unpaired spin distribution on the radical. Comparison of calculated and experimental hyperfine couplings obtained from EPR and ENDOR/TRIPLE resonance leads to a refined structure for the FAD cofactor in Escherichia coli DNA photolyase. Hydrogen bonding at N3H, O4, and N5H results in significant changes in the unpaired spin density distribution and hyperfine coupling constants. The calculated electronic structure of FADH(*) provides evidence for a superexchange-mediated electron transfer between the cyclobutane pyrimidine dimer lesion and the 7,8-dimethyl isoalloxazine moiety of the flavin cofactor via the adenine moiety.     

Energetics of direct and water-mediated proton-coupled electron transfer

Proton-coupled electron transfer (PCET) is an elementary chemical reaction crucial for biological oxidoreduction. We perform quantum chemical calculations to study the direct and water-mediated PCET between two stacked tyrosines, TyrO(?) + TyrOH → TyrOH + TyrO(?), to mimic a key step in the catalytic reaction of class Ia ribonucleotide reductase (RNR). The energy surfaces of electronic ground and excited states are separated by a large gap of ~20 kcal mol(-1), indicative of an electronically adiabatic transfer mechanism. In response to chemical substitutions of the proton donor, the energy of the transition state for direct PCET shifts by exactly half of the change in energetic driving force, resulting in a linear free energy relation with a Br?nsted slope of ?. In contrast, for water-mediated PCET, we observe integer Br?nsted slopes of 1 and 0 for proton acceptor and donor modifications, respectively. Our calculations suggest that the π-stacking of the tyrosine dimer in RNR results in strong electronic coupling and adiabatic PCET. Water participation in the PCET can be identified perturbatively in a Br?nsted analysis.     

Role of the middle residue in the triple tryptophan electron transfer chain of DNA photolyase: ultrafast spectroscopy of a Trp-->Phe mutant

Photoreduction of the semi-reduced flavin adenine dinucleotide cofactor FADH* in DNA photolyase from Escherichia coli into FADH- involves three tryptophan (W) residues that form a closely spaced electron-transfer chain FADH*-W382-W359-W306. To investigate this process, we have constructed a mutant photolyase in which W359 is replaced by phenylalanine (F). Monitoring its photoproducts by femtosecond spectroscopy, the excited-state FADH* was found to decay in approximately 30 ps, similar as in wild type (WT) photolyase. In contrast to WT, however, in W359F mutant photolyase the ground-state FADH* fully recovered virtually concomitantly with the decay of its excited state and, despite the presence of the primary electron donor W382, no measurable flavin reduction was observed at any time. Thus, W359F photolyase appears to behave like many other flavoproteins, where flavin excited states are quenched by very short-lived oxidation of aromatic residues. Our analysis indicates that both charge recombination of the primary charge separation state FADH-W382*+ and (in WT) electron transfer from W359 to W382*+ occur with time constants <4 ps, considerably faster than the initial W382-->FADH* electron-transfer step. Our results provide a first experimental indication that electron transfer between aromatic residues can take place on the time scale of approximately 10(-12) s.     

Hydride transfer versus hydrogen radical transfer in thymidylate synthase

The nature of a H-transfer in the thymidylate synthase catalyzed reaction was investigated by comparison of the wild-type enzyme with the W80M mutant. The nature of the H-transfer was not affected, as indicated by intrinsic isotope effects and their temperature dependence. These findings support a single-step hydride transfer instead of a two-step radical transfer.     

The efficiency of photolyase and indole complexes to repair DNA containing dimers of pyrimidine: a theoretical analysis of the electron transfer reactions

We analyze the effects of competing reactions to the efficiency of enzymatic splitting of pyrimidine dimers formed in DNA by the incidence of ultraviolet radiation. This is accomplished with the aid of a formula that expresses the efficiency of the repair in terms of parameters that regulate the reaction rates for primary and for back long-range electron transfers taking place in the process. Comparison of experimental data with estimations on account of this formula supports early conjectures in the literature that attribute the relative high performance of the enzymatic complexes of photolyase to its ability to suppress the back reaction.     

Energetics of ligand binding to the DNA minor groove

In the present work the decomposition of the total Gibbs free energy of ligand-DNA binding onto various physical terms was accomplished for the group of nine DNA minor groove binders (MGB ligands) differing in both structure and charge state. The decomposition protocol includes the analysis of the most complete set of physical factors known to contribute to the complexation process, viz. the net change in the number of degrees of freedom (translational, rotational, vibrations of the chemical bonds and vibrations of the ligand as a whole within the binding site), the conformational entropy, van der Waals, electrostatic and hydrophobic interactions, the polyelectrolyte contribution and the net effect of changes in the number of hydrogen bonds. All of these processes are further decomposed into the interaction with the solvent and the interaction of the ligand with DNA. The principal outcome of the decomposition is the possibility of performing a comparative analysis of the energetic contribution of various physical terms and provide an answer to the question concerning what physical factors stabilize or destabilize the complexes of MGB ligands with DNA.     

原子转移自由基聚合研究进展

原子转移自由基聚合(Atom transfer radical polymerization,ATRP)是一种发展较快的可控/活性聚合技术,现已广泛应用于聚合物分子结构设计及众多功能高分子材料的合成.本文在综述了ATRP的反应机理的基础上,介绍了引发剂、催化剂、配体、单体等对ATRP的影响,同时综述了降低(或去除)金属盐含量的绿色、高效ATRP聚合体系,如引发剂持续再生活化ATRP,电子转移生成(再生)活化剂ATRP,铁催化体系,光催化体系等.近年来发展的无金属光诱导的有机催化ATRP聚合体系也做了综述.     

Substrate electric dipole moment exerts a pH-dependent effect on electron transfer in Escherichia coli photolyase

Transient absorption spectroscopy is used to demonstrate that the electric dipole moment of the substrate cyclobutane thymine dimer affects the charge recombination reaction between fully reduced flavin adenine dinucleotide (FADH-) and the neutral radical tryptophan 306 (Trp306*) in Escherichia coli DNA photolyase. At pH 7.4, the charge recombination is slowed by a factor of 1.75 in the presence of substrate, but not at pH 5.4. Photolyase does bind substrate at pH 5.4, and it seems that this pH effect originates from the conversion of FADH- to FADH2 at lower pH. The free-energy changes calculated from the electric field parameters and from the change in electron transfer rate are in good agreement and support the idea that the substrate electric dipole is responsible for the observed change in electron transfer rate. It is expected that the substrate electric field will also modify the physiologically important from excited 1FADH- to the substrate in the DNA repair reaction.     

Energetics and dynamics of proton transfer reactions along short water wires

Proton transfer (pT) reactions in biochemical processes are often mediated by chains of hydrogen-bonded water molecules. We use hybrid density functional calculations to study pT along quasi one-dimensional water arrays that connect an imidazolium-imidazole proton donor-acceptor pair. We characterize the structures of intermediates and transition states, the energetics, and the dynamics of the pT reactions, including vibrational contributions to kinetic isotope effects. In molecular dynamics simulations of pT transition paths, we find that for short water chains with four water molecules, the pT reactions are semi-concerted. The formation of a high-energy hydronium intermediate next to the proton-donating group is avoided by a simultaneous transfer of a proton from the donor to the first water molecule, and from the first water molecule into the water chain. Lowering the dielectric constant of the environment and increasing the water chain length both reduce the barrier for pT. We study the effect of the driving force on the energetics of the pT reaction by changing the proton affinity of the donor and acceptor groups through halogen and methyl substitutions. We find that the barrier of the pT reaction depends linearly on the proton affinity of the donor but is nearly independent of the proton affinity of the acceptor, corresponding to Br?nsted slopes of one and zero, respectively.