Involvement of electron transfer in the photoreaction of zebrafish Cryptochrome-DASH

Photoreaction of a blue-light photoreceptor Cryptochrome-DASH (Cry-DASH), a new member of the Cryptochrome family, from zebrafish was studied by UV-visible absorption spectroscopy in aqueous solutions at 293 K. Zebrafish Cry-DASH binds two chromophores, a flavin adenine dinucleotide (FAD) and a N5,N10-methenyl-5,6,7,8-tetrahydrofolate (MTHF) noncovalently. The bound FAD exists in the oxidized form (FAD(ox)) in the dark. Blue light converts FAD(ox) to the neutral radical form (FADH*). Formed FADH* is transformed to the fully reduced form FADH(2) (or FADH(-)) by successive light irradiation, or reverts to FAD(ox). FADH(2) (or FADH(-)) reverts to FADH* or possibly to FAD(ox) directly. The effect of dithiothreitol suggests a possible electron transfer between FAD in zebrafish Cry-DASH and reductants in the external medium. This is the first report on the photoreaction pathway and kinetics of a vertebrate Cry-DASH family protein.     

Effect of the cyclobutane cytidine dimer on the properties of Escherichia coli DNA photolyase

Cyclobutane pyrimidine dimer (CPD) photolyases are structure specific DNA-repair enzymes that specialize in the repair of CPDs, the major photoproducts that are formed upon irradiation of DNA with ultraviolet light. The purified enzyme binds a flavin adenine dinucleotide (FAD), which is in the neutral radical semiquinone (FADH(*)) form. The CPDs are repaired by a light-driven, electron transfer from the anionic hydroquinone (FADH(-)) singlet excited state to the CPD, which is followed by reductive cleavage of the cyclobutane ring and subsequent monomerization of the pyrimidine bases. CPDs formed between two adjacent thymidine bases (T< >T) are repaired with greater efficiency than those formed between two adjacent cytidine bases (C< >C). In this paper, we investigate the changes in Escherichia coli photolyase that are induced upon binding to DNA containing C< >C lesions using resonance Raman, UV-vis absorption, and transient absorption spectroscopies, spectroelectrochemistry, and computational chemistry. The binding of photolyase to a C< >C lesion modifies the energy levels of FADH(*), the rate of charge recombination between FADH(-) and Trp(306)(*), and protein-FADH(*) interactions differently than binding to a T< >T lesion. However, the reduction potential of the FADH(-)/FADH(*) couple is modified in the same way with both substrates. Our calculations show that the permanent electric dipole moment of C< >C is stronger (12.1 D) and oriented differently than that of T< >T (8.7 D). The possible role of the electric dipole moment of the CPD in modifying the physicochemical properties of photolyase as well as in affecting CPD repair will be discussed.     

PHOTOACTIVATION OF NITRATE REDUCTASE FROM NEUROSPORA CRASSA

Abstract— The photoactivation of nitrate reductase from Neurospora crassa was studied in partially purified extracts. The inactive enzyme [inactivated by reduction in the presence of potassium cyanide] could be reactivated by chemical oxidation with ferricyanide or by irradiation with blue light. The enzyme contains a short electron transfer chain consisting of flavin adenine dinucleotide, cytochrome b ₅₅₇ and molybdenum which normally transfers electrons from reduced pyridine nucleotide to nitrate. This overall activity, which was negligible in the inactive enzyme, was restored to approximately 70% of the ferricyanide control by irradiation. However, nitrate reduction using reduced methylviologen as reducing power, which was also negligible in the inactive enzyme, was photoactivated to 100%. The diaphorase activity of the enzyme mediated by the flavin adenine dinucleotide, which was fully active in the inactivated enzyme, was inhibited approximately 30% by the irradiation treatment. The action spectrum for photoactivation showed that a flavin was the photoreceptor chromophore. Photoactivation occurs only in the presence of oxygen.     

Evidence for concerted electron proton transfer in charge recombination between FADH- and 306Trp• in Escherichia coli photolyase

Proton-coupled electron-transfer (PCET) is a mechanism of great importance in protein electron transfer and enzyme catalysis, and the involvement of aromatic amino acids in this process is of much interest. The DNA repair enzyme photolyase provides a natural system that allows for the study of PCET using a neutral radical tryptophan (Trp(?)). In Escherichia coli photolyase, photoreduction of the flavin adenine dinucleotide (FAD) cofactor in its neutral radical semiquinone form (FADH(?)) results in the formation of FADH(-) and (306)Trp(?). Charge recombination between these two intermediates requires the uptake of a proton by (306)Trp(?). The rate constant of charge recombination has been measured as a function of temperature in the pH range from 5.5 to 10.0, and the data are analyzed with both classical Marcus and semi-classical Hopfield electron transfer theory. The reorganization energy associated with the charge recombination process shows a pH dependence ranging from 2.3 eV at pH ≤ 7 and 1.2 eV at pH(D) 10.0. These findings indicate that at least two mechanisms are involved in the charge recombination reaction. Global analysis of the data supports the hypothesis that PCET during charge recombination can follow two different mechanisms with an apparent switch around pH 6.5. At lower pH, concerted electron proton transfer (CEPT) is the favorable mechanism with a reorganization energy of 2.1-2.3 eV. At higher pH, a sequential mechanism becomes dominant with rate-limiting electron-transfer followed by proton uptake which has a reorganization energy of 1.0-1.3 eV. The observed 'inverse' deuterium isotope effect at pH < 8 can be explained by a solvent isotope effect that affects the free energy change of the reaction and masks the normal, mass-related kinetic isotope effect that is expected for a CEPT mechanism. To the best of our knowledge, this is the first time that a switch in PCET mechanism has been observed in a protein.     

Facile photoreduction of graphene oxide by an NAD(P)H model: Hantzsch 1,4-dihydropyridine

To make "clean" reduced GO sheets in high quality and in large scale, a natural reduced nicotinamine adenine dinucleotide NAD(P)H model, Hantzsch 1,4-dihydropyridine (HEH), is used as a mild organic photoreductant in this work. Benefiting from the intense absorption of HEH in the range of 300-420 nm, the graphene oxide (GO) can be readily reduced by HEH under UV light irradiation (λ > 320 nm) to afford single or few-layer reduced graphene oxide at room temperature. Studies on reduction extent reveal that both irradiation time and concentration ratio of HEH to GO are important for effective reduction of GO under UV light. The as-prepared photochemically reduced graphene oxide (PRGO) dispersion is stable without the need for any polymeric or surfactant stabilizers. Simply by extraction treatment, the "clean" PRGO sheets can be obtained in large quantities, and its conductivity approaches to 4680 S·m(-1) that is the highest value reported by photochemical approaches so far.     

Redox processes in mesoporous oxide membranes: layered TiO2 phytate and TiO2 flavin adenine dinucleotide films

Thin films of TiO2 (anatase) nanoparticles are assembled at an electrode surface via a layer-by-layer deposition process employing phytic acid, pyromellitic acid, or flavin adenine dinucleotide (FAD) as molecular binders. With all three types of binders, layers of typically 30 nm thickness are formed each deposition cycle. FAD as an electrochemically active component immobilized at the surface of the TiO2 particles is reduced to FADH2 and reoxidized in a chemically reversible two electron-two proton redox process. Two distinct voltammetric signals are observed for the immobilized FAD redox system associated with (i) hopping of electrons at the TiO2 surface (reversible) and (ii) conduction of electrons through the TiO2 assembly (irreversible). The conduction of electrons through the TiO2 assembly is possible by diffusion over considerable distances as well as through a "spacer" layer of TiO2 phytate. An order of magnitude (upper limit) estimate for the diffusion coefficient of electrons through TiO2 phytate, D(electron) approximately 10(-6) m(2) s(-1), is obtained from voltammetric data. Finally, it is demonstrated that the calcination of TiO2 assemblies causes dramatic changes in the electron transfer kinetics for the immobilized FAD/FADH2 redox system.     

Effect of laser and LED on enzymatic production of ceramide

An enzyme (Phospholipase C Type I from Clostridium perfringens) was exposed to 0-810Jcm(-2) of energy using laser light at wavelengths 808, 532, 1064 and 1342nm and two LED light sources at wavelengths 810 and 640nm. Enzyme responses were evaluated by measuring ceramide concentration using high performance thin-layer chromatography (HPTLC) at 0.5, 1, 2, 3, 4, 6, 17, 24h after irradiation. The duration of effect was evaluated from the experimental data. The results show that enzyme activity can be increased by using both laser and LED sources whose wavelength is located within a certain range. The effect depends on the energy and wavelength of the light. The increase in enzyme activity continued for about 4h after irradiation. This study shows that the duration of irradiation should be included as one of the main laser parameters when reporting on the effects of laser irradiation on enzymes. We also find that laser sources and LED sources have the same effect on enzyme activity if the wavelength and absorbed energy are equal.     

New insights into the ultrafast photophysics of oxidized and reduced FAD in solution

The ultrafast photophysics of oxidized and reduced flavin adenine dinucleotide (FAD) in aqueous solution was studied by broadband UV-vis femtosecond transient absorption spectroscopy. We observed that oxidized FAD (FAD(ox)) in solution readily aggregates at submillimolar concentration. Upon excitation of FAD(ox), three excited-state lifetimes were found and assigned to three different species: the closed (stacked) conformation of the monomer (～5.4 ps), the open (extended) conformation of the monomer (～2.8 ns), and the dimer (～27 ps). In the case of the stacked conformation of the monomer, we show that intramolecular electron transfer from the adenine to the isoalloxazine ring occurs with a time constant of 5.4 ps and is followed by charge recombination on a faster time scale, namely, 390 fs. We additionally demonstrate that deprotonated reduced flavin (FADH(-)) undergoes biphotonic ionization under high excitation fluence and dissociates into a hydrated electron and the neutral semiquinone radical FADH(?).     

DNA PHOTOREACTIVATING ENZYME FROM SILKWORM

An ammonium-sulfate-precipitable (33–70%) fraction in extracts from eggs of silkworm Bombyx mori contains photoreactivating enzyme that reactivates the transforming activity of UV inactivated Hemophilus influenzae DNA. The action spectrum for in vitro photoreactivation with the enzyme has a broad peak around 365–385 nm, with a shoulder extending to 460 nm. This relatively higher photoreactivation efficiency at wavelengths longer than 450 nm seems to be a unique feature of DNA photoreactivating enzyme of silkworm. Using gel filtration, a mol wt of 42,000 was estimated for the enzyme. Optimum and isoionic pH of the enzyme were 7.2 and 5.4, respectively. These properties of silkworm enzyme are within the range of variations in reported biochemical characteristics of photoreactivating enzymes from different species.     

G-tensors of the flavin adenine dinucleotide radicals in glucose oxidase: a comparative multifrequency electron paramagnetic resonance and electron-nuclear double resonance study

The flavin adenine dinucleotide (FAD) cofactor of Aspergillus niger glucose oxidase (GO) in its anionic (FAD*-) and neutral (FADH*) radical form was investigated by electron paramagnetic resonance (EPR) at high microwave frequencies (93.9 and 360 GHz) and correspondingly high magnetic fields and by pulsed electron-nuclear double resonance (ENDOR) spectroscopy at 9.7 GHz. Because of the high spectral resolution of the frozen-solution continuous-wave EPR spectrum recorded at 360 GHz, the anisotropy of the g-tensor of FAD*- could be fully resolved. By least-squares fittings of spectral simulations to experimental data, the principal values of g have been established with high precision: gX=2.00429(3), gY=2.00389(3), gZ=2.00216(3) (X, Y, and Z are the principal axes of g) yielding giso=2.00345(3). The gY-component of FAD*- from GO is moderately shifted upon deprotonation of FADH*, rendering the g-tensor of FAD*- slightly more axially symmetric as compared to that of FADH*. In contrast, significantly altered proton hyperfine couplings were observed by ENDOR upon transforming the neutral FADH* radical into the anionic FAD*- radical by pH titration of GO. That the g-principal values of both protonation forms remain largely identical demonstrates the robustness of g against local changes in the electron-spin density distribution of flavins. Thus, in flavins, the g-tensor reflects more global changes in the electronic structure and, therefore, appears to be ideally suited to identify chemically different flavin radicals.     

Ultrafast transient mid IR to visible spectroscopy of fully reduced flavins

The light sensing apparatus of many organisms includes a flavoprotein. In any spectroscopic analysis of the photocycle of flavoproteins a detailed knowledge of the spectroscopy and excited state dynamics of potential intermediates is required. Here we correlate transient vibrational and electronic spectra of the two fully reduced forms of flavin adenine dinucleotide (FAD): FADH(-) and FADH(2). Ground and excited state frequencies of the characteristic carbonyl modes are observed and assigned with the aid of DFT calculations. Excited state decay and ground state recovery dynamics of the two states are reported. Excited state decay occurs on the picosecond timescale, in agreement with the low fluorescence yield, and is markedly non single exponential in FADH(-). Further, an unusual 'inverse' isotope effect is observed in the decay time of FADH(-), suggesting the involvement in the radiationless relaxation coordinate of an NH or hydrogen bond mode that strengthens in the excited electronic state. Ground state recovery also occurs on the picosecond time scale, consistent with radiationless decay by internal conversion, but is slower than the excited state decay.     

Role of adenine in thymine-dimer repair by reduced flavin-adenine dinucleotide

We present a study of excited-state behavior of reduced flavin cofactors using femtosecond optical transient absorption spectroscopy. The reduced flavin cofactors studied were in two protonation states: flavin-adenine dinucleotide (FADH2 and FADH-) and flavin-mononucleotide (FMNH2 and FMNH-). We find that FMNH- exhibits multiexponential decay dynamics due to the presence of two bent conformers of the isoalloxazine ring. FMNH2 exhibits an additional fast deactivation component that is assigned to an iminol tautomer. Reduced flavin cofactors also exhibit a long-lived component that is attributed to the semiquinone and the hydrated electron that are produced in photoinduced electron transfer to the solvent. The presence of adenine in FADH2 and FADH- further changes the excited-state dynamics due to intramolecular electron transfer from the isoalloxazine to the adenine moiety of cofactors. This electron transfer is more pronounced in FADH2 due to pi-stacking interactions between two moieties. We further studied cyclobutane thymine dimer (TT-dimer) repair via FADH- and FMNH- and found that the repair is much more efficient in the case of FADH-. These results suggest that the adenine moiety plays a significant role in the TT-dimer repair dynamics. Two possible explanations for the adenine mediation are presented: (i) a two-step electron transfer process, with the initial electron transfer occurring from flavin to adenine moiety of FADH-, followed by a second electron transfer from adenine to TT-dimer; (ii) the preconcentration of TT-dimer molecules around the flavin cofactor due to the hydrophobic nature of the adenine moiety.     

DESTRUCTION OF PHOTOREACTIVATING ENZYME BY 365 nm RADIATION*

Abstract— Following the observation that in vivo photoreactivation of 365-nm-induced pyrimidine dimers could not be observed chemically, a study was made of the inactivation of photoreactivating enzyme activity by this near-ultraviolet wavelength. It was observed that: (1) Dimers induced in extracted bacterial DNA by 365 nm radiation are completely photoreactivable and are monomerized as an exponential function of the photoreactivation time. (2) Photoreactivability of 254-nm-induced damage in Escherichia coli B/r Hcr is progressively destroyed in vivo as a function of the dose of 365 nm radiation. (3) The ability of the yeast photoreactivating enzyme to monomerize dimers induced at 365 nm in bacterial DNA is destroyed in vitro as a function of the dose of 365 nm radiation, and at a rate comparable to killing of E. coli. These results are consistent with biological measurements which indicate that photoreactivability of ultraviolet (near and far) lethal damage is reduced by exposure of the bacteria to 365 nm radiation.     

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.     

PHOTOREACTIVATION OF THYMINE-STARVED ESCHERICHIA COLIBs-1

Abstract —Thymine starvation prior to 254 nm ultraviolet light (UV) exposures has been found to decrease the level of maximum photoreactivation in Escherichia coli B _s-1. The dark equilibrium level of photoreactivating enzyme-substrate complexes was determined from the levels of photoreactivation obtained with exposures to single flashes of high-intensity light. The kinetics indicate that photoreactivating enzyme concentration does not decrease as a result of thymine starvation. The UV sensitivities of normal and thymine-starved cells are found to be the same. Photoreactivation by sequential flashes shows a lesser number of total photorepairable lesions in starved cells. It is concluded that thymine starvation renders a portion of the dimers inaccessible to the photoreactivating enzyme, thus lowering the level of maximum photoreactivation.     

PHOTOREACTIVATION IN THE EXTREME HALOPHILIC ARCHAEBACTERIUM Halobacterium cutirubrum

Abstract— Photoreactivation in the extreme halophilic archaebacterium Halobacterium cutirubrum was studied both in vivo and in vitro. Cells irradiated with ultraviolet (UV)-fluences up to 350 J/m² could be completely photoreactivated, indicating very efficient repair of pyrimidine dimers in UV-irradiated DNA. Dark repair is apparently absent in Halobacterium since liquid holding under non-growth conditions did not influence the survival of UV-irradiated cells, while cells remained completely photoreactivable with no change in the kinetics of photoreactivation. Experiments with Halobacterium isolates of different carotenoid content indicated that carotenoids do not influence either UV-inactivation or photoreactivation. Small differences in the rates of UV-inactivation and photoreactivation could be assigned to the occurrence of gas vesicles. Flash experiments and the temperature dependence of photoreactivation indicated an enzymatical reaction. This was confirmed by in vitro experiments with partially purified photoreactivating enzyme. The in vivo action spectrum of photoreactivation showed a main band in the 400-470 nm region with a maximum at 440 nm. Comparison with action spectra of other microorganisms classified the Halobacterium enzyme as a 8-hydroxy-5-deazaflavin type photoreactivating enzyme.     

Interaction between thymine dimer and flavin-adenine dinucleotide: a DFT and direct ab initio molecular dynamics study

The interaction between the fully reduced flavin-adenine dinucleotide (FADH (-)) and thymine dimer (T) 2 has been investigated by means of density functional theory (DFT) calculations. The charges of FADH (-) and (T) 2 were calculated to be -0.9 and -0.1, respectively, at the ground state. By photoirradiation, an electron transfer occurred from FADH (-) to (T) 2 at the first excited state. Next, the reaction dynamics of electron capture of (T) 2 have been investigated by means of the direct ab initio molecular dynamics (MD) method (HF/3-21G(d) and B3LYP/6-31G(d) levels) in order to elucidate the mechanism of the repair process of thymine dimer caused by the photoenzyme. The thymine dimer has two C-C single bonds between thymine rings (C 5-C 5' and C 6-C 6' bonds) at the neutral state, which is expressed by (T) 2. After the electron capture of (T) 2, the C 5-C 5' bond was gradually elongated and then it was preferentially broken. The time scale of the C-C bond breaking and formation of the intermediate with a single bond (T) 2 (-) was estimated to be 100-150 fs. The present calculations confirmed that the repair reaction of thymine dimer takes place efficiently via an electron-transfer process from the FADH (-) enzyme.     

Ultrafast dynamics and anionic active states of the flavin cofactor in cryptochrome and photolyase

We report here our systematic studies of the dynamics of four redox states of the flavin cofactor in both photolyases and insect type 1 cryptochromes. With femtosecond resolution, we observed ultrafast photoreduction of oxidized state flavin adenine dinucleotide (FAD) in subpicosecond and of neutral radical semiquinone (FADH(*)) in tens of picoseconds through intraprotein electron transfer mainly with a neighboring conserved tryptophan triad. Such ultrafast dynamics make these forms of flavin unlikely to be the functional states of the photolyase/cryptochrome family. In contrast, we find that upon excitation the anionic semiquinone (FAD(*-)) and hydroquinone (FADH(-)) have longer lifetimes that are compatible with high-efficiency intermolecular electron transfer reactions. In photolyases, the excited active state (FADH(-)*) has a long (nanosecond) lifetime optimal for DNA-repair function. In insect type 1 cryptochromes known to be blue-light photoreceptors the excited active form (FAD(*-)*) has complex deactivation dynamics on the time scale from a few to hundreds of picoseconds, which is believed to occur through conical intersection(s) with a flexible bending motion to modulate the functional channel. These unique properties of anionic flavins suggest a universal mechanism of electron transfer for the initial functional steps of the photolyase/cryptochrome blue-light photoreceptor family.     

Photochemical oxygen consumption, oxygen evolution and spectral changes during UVA irradiation of EMT6 spheroids.

Remarkable rates of oxygen consumption are observed via microelectrode measurements immediately upon the onset of 325 nm irradiation of multicell tumor spheroids. Consumption is irradiance dependent over the range 20-200 mW cm-2, and its magnitude is comparable to that observed previously in the same system using exogenous photosensitizers. Oscillations in the oxygen concentrations suggest that oxygen is also being evolved during irradiation. Oxygen evolution is likely the result of enzymatic dissociation of hydrogen peroxide, which is formed through UV-induced photochemistry. Irradiation of spheroids at 442 and at 514 nm produces a much more modest but detectable oxygen consumption. The dynamics of oxygen concentration changes are quite different at these wavelengths, suggesting a different photochemical mechanism. In these cases, initial oxygen depletion is followed immediately by a more gradual, monotonic increase in the oxygen concentration, consistent with irreversible photobleaching. No oscillations in the oxygen concentration are detectable. At 662 nm, no oxygen consumption was observed over the range of irradiances studied. Fluorescence spectra of cells prior to irradiation include contributions from anthranilic acid and reduced nicotinamide adenine dinucleotide (NADH). During 325 nm irradiation, anthranilic acid is rapidly and irreversibly bleached, while NADH emission undergoes only modest reduction.     

PHOTOREACTIVATING ENZYME FROM Streptomyces griseus—VI. ACTION SPECTRUM AND KINETICS OF PHOTOREACTIVATION

Abstract— A high resolution action spectrum for photoreactivation was determined using purified photoreactivating enzyme from Streptomyces griseus. Conversion of pyrimidine dimers in UV-irradiated DNA, the substrate for photoreactivating enzyme, was measured with a Haemophilus influenzae transformation assay. A high similarity was found between action spectrum (max. at 445 nm) and the long wavelength absorption band (max. at 443 nm)of photoreactivating enzyme. In addition to the400–470 nm region considerable photoreactivation was found with wavelengths between 280 and 320 nm. No evidence was obtained for the presence of nonenzymatic photoreactivation. Comparison of in vitro and in vivo action spectra revealed that the sharp peak at 313 nm found in vivo is probably the result of counteracting photoreactivation and inactivation effects. Comparison of the action spectrum with the absorption spectrum of 8-hydroxy-10-methyl-5-deazaisoalloxazine in an aprotic dipolar solvent (which serves as a model for the 8-hydroxy-5-deazaflavin chromophore in photoreactivating enzyme) indicates the possible presence of other chromophore(s) involved in the photorepair process. From kinetic measurements and flash experiments values were obtained for the rate constants of the photoreactivation reaction. The quantum yield of photoreactivation was estimated to be approximately 1.