Effect of 5-deazaflavin on energy transduction during catalysis by Escherichia coli DNA photolyase.

DNA photolyase from Escherichia coli contains 1,5-dihydroFAD (FADH2) plus 5,10-methenyltetrahydropteroylpolyglutamate. The action spectrum observed for apoenzyme reconstituted with 5-deazaFADH2 (EdFADH2) matched its absorption spectrum after correction for the presence of a small amount of inactive 5-deazaFADox. The quantum yield for dimer repair with EdFADH2 (phi EdFADH2 = 0.110) was 6-fold lower than that observed with apoenzyme reconstituted with FADH2. Excited-state redox potential calculations indicate that 5-deazaFADH2 singlet is a better one-electron donor (E = -3.5 V) than FADH2 singlet (E = -2.7 V). Other studies indicate that the quantum yield for electron transfer from reduced flavin singlet to pyrimidine dimer (0.88) is unaffected when FADH2 is replaced by 5-deazaFADH2. Enhanced back electron transfer from pyrimidine dimer radical to flavin radical may account for the decreased quantum yield observed with EdFADH2 since, in the ground state, 5-deazaFADH. is a better oxidant than FADH.. The action spectrum observed for apoenzyme reconstituted with 5-deazaFADH2 plus 5,10-CH(+)-H4folate (EPtedFADH2) matched the absorption spectrum determined for enzyme-bound 5-deazaFADH2, indicating that the pterin chromophore was inactive as a sensitizer. This differs from results obtained with native enzyme, where pterin acts as a sensitizer via efficient singlet-singlet energy transfer to FADH2. The quantum yield for dimer repair by 5-deazaFADH2 bound to EPtedFADH2 (phi EPtedFADH2 = 0.0318) was 28.9% of that observed for EdFADH2. Spectroscopic studies indicate that singlet-singlet energy transfer in EPtedFADH2 is very efficient but only occurs in the "wrong" direction, i.e., from excited 5-deazaFADH2 to pterin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chromophore function and interaction in Escherichia coli DNA photolyase: reconstitution of the apoenzyme with pterin and/or flavin derivatives

Native DNA photolyase, as isolated from Escherichia coli, contains a neutral flavin radical (FADH.) plus a pterin chromophore (5,10-methenyltetrahydropteroylpolyglutamate) and can be converted to its physiologically significant form by reduction of FADH. to fully reduced flavin (FADH2) with dithionite or by photoreduction. Either FADH2 or the pterin chromophore in dithionite-reduced native enzyme can function as a sensitizer in catalysis. Various enzyme forms (EFADox, EFADH., EFADH2, EPteFADox, EPteFADH., EPteFADH2, EPte) containing stoichiometric amounts of FAD in either of its three oxidation states and/or 5,10-methenyltetrahydrofolate (Pte) have been prepared in reconstitution experiments. Studies with EFADox and EPte showed that these preparations retained the ability to bind the missing chromophore. The results suggest that there could be considerable flexibility in the biological assembly of holoenzyme since the order of binding of the enzyme's chromophores is apparently unimportant, the binding of FAD is unaffected by its redox state, and enzyme preparations containing only one chromophore are reasonably stable. The same catalytic properties are observed with dithionite-reduced native enzyme or EFADH2. These preparations do not exhibit a lag in catalytic assays whereas lags are observed with preparations containing FADox or FADH. in the presence or absence of pterin. Photochemical studies show that these lags can be attributed to enzyme activation under assay conditions in a reaction involving photoreduction of enzyme-bound FADox or FADH. to FADH2. EPte is catalytically inactive, but catalytic activity is restored upon reconstitution of EPte with FADox. The results show that pterin is not required for dimer repair when FADH2 acts as the sensitizer but that FADH2 is required when dimer repair is initiated by excitation of the pterin chromophore. The relative intensity of pterin fluorescence in EPte, EPteFADH., EPteFADox, or EPteFADH2 has been used to estimate the efficiency of pterin singlet quenching by FADH. (93%), FADox (90%), or FADH2 (58%). Energy transfer from the excited pterin to flavin is energetically feasible and may account for the observed quenching of pterin fluorescence and also explain why photoreduction of FADox or FADH. is accelerated by the pterin chromophore. An irreversible photobleaching of the pterin chromophore is accelerated by FADH2 in a reaction that is accompanied by a transient oxidation of FADH2 to FADH.. Both pterin bleaching and FADH2 oxidation are inhibited by substrate.     

Action mechanism of Escherichia coli DNA photolyase. II. Role of the chromophores in catalysis

DNA photolyase repairs pyrimidine dimers in DNA in a reaction that requires visible light. Photolyase from Escherichia coli is normally isolated as a blue protein and contains 2 chromophores: a blue FAD radical plus a second chromophore that exhibits an absorption maximum at 360 nm when free in solution. Oxidation of the FAD radical is accompanied by a reversible loss of activity which is proportional to the fraction of the enzyme flavin converted to FADox. Quantitative reduction of the radical to fully reduced FAD causes a 3-fold increase in activity. The results show that a reduced flavin is required for activity and suggest that flavin may act as an electron donor in catalysis. Comparison of the absorption spectrum calculated for the protein-bound second chromophore (lambda max = 390 nm) with fluorescence data and with the relative action spectrum for dimer repair indicates that the second chromophore is the fluorophore in photolyase and that it does act as a sensitizer in catalysis. On the other hand, enzyme preparations containing diminished amounts of the second chromophore do not exhibit correspondingly lower activity. This suggests that reduced flavin may also act as a sensitizer in catalysis. The blue color of the enzyme is lost upon reduction of the FAD radical. The fully reduced E. coli enzyme exhibits absorption and fluorescence properties very similar to yeast photolyase. This indicates that the two enzymes probably contain similar chromophores but are isolated in different forms with respect to the redox state of the flavin.     

Reconstitution of Escherichia coli DNA photolyase with various folate derivatives

DNA photolyase from Escherichia coli contains both flavin and pterin. However, the isolated enzyme is depleted with respect to the pterin chromophore (0.5 mol of pterin/mol of flavin). The extinction coefficient of the pterin chromophore at 360 nm is underestimated by a method used in earlier studies which assumes stoichiometric amounts of pterin and flavin. The extinction coefficient of the pterin chromophore, determined on the basis of its (p-aminobenzoyl)polyglutamate content (epsilon 360 = 25.7 x 10(3) M-1 cm-1), is in good agreement with that expected for a 5,10-methenyltetrahydrofolate derivative. Also consistent with this structure, the pterin chromophore could be reversibly hydrolyzed to yield a 10-formyltetrahydrofolate derivative or reduced to yield a 5-methyltetrahydrofolate derivative. The isolated enzyme could be reconstituted with various folate derivatives to yield enzyme that contained equimolar amounts of pterin and flavin. Similar results were obtained in reconstitution studies with the natural pterin chromophore, with 5,10-methenyltetrahydrofolate, and with 10-formyltetrahydrofolate. The results show that the polyglutamate moiety, previously identified in the natural chromophore, is not critical for binding. Reconstitution with the natural pterin chromophore did not affect catalytic activity. The latter is consistent with our previous studies which show that, although the pterin chromophore acts as a sensitizer in native enzyme, it is not essential for dimer repair which can occur at the same rate under saturating light with flavin (1,5-dihydro-FAD) as the only chromophore.     

Characterization of Arabidopsis photolyase enzymes and analysis of their role in protection from ultraviolet-B radiation

DNA photolyases are enzymes which mediate the light-dependent repair (photoreactivation) of UV-induced damage products in DNA by direct reversal of base damage rather than via excision repair pathways. Arabidopsis thaliana contains two photolyases specific for photoreactivation of either cyclobutane pyrimidine dimers (CPDs) or pyrimidine (6-4)pyrimidones (6-4PPs), the two major UV-B-induced photoproducts in DNA. Reduced FADH and a reduced pterin were identified as cofactors of the native Arabidopsis CPD photolyase protein. This is the first report of the chromophore composition of any native class II CPD photolyase protein to our knowledge. CPD photolyase protein levels vary between tissues and with leaf age and are highest in flowers and leaves of 3-5-week-old Arabidopsis plants. White light or UV-B irradiation induces CPD photolyase expression in Arabidopsis tissues. This contrasts with the 6-4PP photolyase protein which is constitutively expressed and not regulated by either white or UV-B light. Arabidopsis CPD and 6-4PP photolyase enzymes can remove UV-B-induced photoproducts from DNA in planta even when plants are grown under enhanced levels of UV-B irradiation and at elevated temperatures although the rate of removal of CPDs is slower at high growth temperatures. These studies indicate that Arabidopsis possesses the photorepair capacity to respond effectively to increased UV-B-induced DNA damage under conditions predicted to be representative of increases in UV-B irradiation levels at the Earth's surface and global warming in the twenty-first century.     

The folate cofactor of Escherichia coli DNA photolyase acts catalytically

Escherichia coli DNA photolyase catalyzes the light-driven (300-500 nm) repair of pyrimidine dimers formed between adjacent pyrimidine bases in DNA exposed to UV light (200-300 nm). The light-driven repair process is facilitated by two enzyme-bound cofactors, FADH2 and 5,10-methenyltetrahydrofolate. The function of the folate has been characterized in greater detail in this series of experiments. Investigations of the relative binding affinities of photolyase for the monoglutamate and polyglutamate forms of 5,10-methenyltetrahydrofolate show that the enzyme has a greater affinity for the naturally occurring polyglutamate forms of the folate and that the exogenously added monoglutamate derivative is less tightly associated with the protein. Multiple turnover experiments reveal that the folate remains bound to photolyase even after 10 turnovers of the enzyme. Examination of the rates of repair by photolyase containing stoichiometric folate in the presence or absence of free folate under multiple turnover conditions and at micromolar concentrations of enzyme also demonstrates that the folate acts catalytically. The stimulation of turnover by exogenous folate seen at low concentrations of photolyase is shown to be due to the lower affinity of photolyase for the monoglutamate derivative used in reconstitution procedures. These results demonstrate that the folate of E. coli DNA photolyase is a bona fide cofactor and does not decompose or dissociate during multiple turnovers of the enzyme.     

Active DNA photolyase encoded by a baculovirus from the insect Chrysodeixis chalcites

The genome of Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV) contains two open reading frames, Cc-phr1 and Cc-phr2, which encode putative class II CPD-DNA photolyases. CPD-photolyases repair UV-induced pyrimidine cyclobutane dimers using visible light as an energy source. Expression of Cc-phr2 provided photolyase deficient Escherichia coli cells with photoreactivating activity indicating that Cc-phr2 encodes an active photolyase. In contrast, Cc-phr1 did not rescue the photolyase deficiency. Cc-phr2 was overexpressed in E. coli and the resulting photolyase was purified till apparent homogeneity. Spectral measurements indicated the presence of FAD, but a second chromophore appeared to be absent. Recombinant Cc-phr2 photolyase was found to bind specifically F0 (8-hydroxy-7,8-didemethyl-5-deazariboflavin), which is an antenna chromophore present in various photolyases.. After reconstitution, FAD and F0 were present in approximately equimolar amounts. In reconstituted photolyase the F0 chromophore is functionally active as judged from the increase in the in vitro repair activity. This study demonstrates for the first time that a functional photolyase is encoded by an insect virus, which may have implications for the design of a new generation of baculoviruses with improved performance in insect pest control.     

Active site of Escherichia coli DNA photolyase: Asn378 is crucial both for stabilizing the neutral flavin radical cofactor and for DNA repair

Escherichia coli DNA photolyase repairs cyclobutane pyrimidine dimer (CPD) in UV-damaged DNA through a photoinduced electron transfer mechanism. The catalytic activity of the enzyme requires fully reduced FAD (FADH (-)). After purification in vitro, the cofactor FADH (-) in photolyase is oxidized into the neutral radical form FADH (*) under aerobic conditions and the enzyme loses its repair function. We have constructed a mutant photolyase in which asparagine 378 (N378) is replaced with serine (S). In comparison with wild-type photolyase, we found N378S mutant photolyase containing oxidized FAD (FAD ox) but not FADH (*) after routine purification procedures, but evidence shows that the mutant protein contains FADH (-) in vivo as the wild type. Although N378S mutant photolyase is photoreducable and capable of binding CPD in DNA, the activity assays indicate the mutant protein is catalytically inert. We conclude that the Asn378 residue of E. coli photolyase is crucial both for stabilizing the neutral flavin radical cofactor and for catalysis.     

Active site of Escherichia coli DNA photolyase: mutations at Trp277 alter the selectivity of the enzyme without affecting the quantum yield of photorepair

Escherichia coli DNA photolyase repairs pyrimidine dimers by a photoinduced electron-transfer reaction. The enzyme binds to UV-damaged DNA independent of light (the dark reaction) and upon absorbing a 300-500-nm photon breaks the cyclobutane ring of the dimer (the light reaction) and thus restores the DNA. No structural information on the enzyme is available at present. However, comparison of the sequences of photolyases from five different organisms has identified highly conserved regions of homology. These regions are presumably involved in chromophore (flavin and folate) and substrate binding or catalysis. Trp277 (W277) in E. coli photolyase is conserved in all photolyases sequenced to date. We replaced this residue with Arg, Glu, Gln, His, and Phe by site-specific mutagenesis. Properties of the mutant proteins indicate that W277 is involved in binding to DNA but not in chromophore binding or catalysis. Of particular significance is the finding that compared to wild type W277R and W277E mutants have about 300- and 1000-fold lower affinity, respectively, for substrate but were indistinguishable from wild-type enzyme in their photochemical and photocatalytic properties.     

Photolyases from Saccharomyces cerevisiae and Escherichia coli recognize common binding determinants in DNA containing pyrimidine dimers.

DNA photolyases catalyze the light-dependent repair of pyrimidine dimers in DNA. The results of nucleotide sequence analysis and spectroscopic studies demonstrated that photolyases from Saccharomyces cerevisiae and Escherichia coli share 37% amino acid sequence homology and contain identical chromophores. Do the similarities between these two enzymes extend to their interactions with DNA containing pyrimidine dimers, or does the organization of DNA into nucleosomes in S. cerevisiae necessitate alternative or additional recognition determinants? To answer this question, we used chemical and enzymatic techniques to identify the contacts made on DNA by S. cerevisiae photolyase when it is bound to a pyrimidine dimer and compared these contacts with those made by E. coli photolyase and by a truncated derivative of the yeast enzyme when bound to the same substrate. We found evidence for a common set of interactions between the photolyases and specific phosphates in the backbones of both strands as well as for interactions with bases in both the major and minor grooves of dimer-containing DNA. Superimposed on this common pattern were significant differences in the contributions of specific contacts to the overall binding energy, in the interactions of the enzymes with groups on the complementary strand, and in the extent to which other DNA-binding proteins were excluded from the region around the dimer. These results provide strong evidence both for a conserved dimer-binding motif and for the evolution of new interactions that permit photolyases to also act as accessory proteins in nucleotide excision repair. The locations of the specific contacts made by the yeast enzyme indicate that the mechanism of nucleotide excision repair in this organism involves incision(s) at a distance from the pyrimidine dimer.     

Light-induced reactions of Escherichia coli DNA photolyase monitored by Fourier transform infrared spectroscopy

Cyclobutane-type pyrimidine dimers generated by ultraviolet irradiation of DNA can be cleaved by DNA photolyase. The enzyme-catalysed reaction is believed to be initiated by the light-induced transfer of an electron from the anionic FADH- chromophore of the enzyme to the pyrimidine dimer. In this contribution, first infrared experiments using a novel E109A mutant of Escherichia coli DNA photolyase, which is catalytically active but unable to bind the second cofactor methenyltetrahydrofolate, are described. A stable blue-coloured form of the enzyme carrying a neutral FADH radical cofactor can be interpreted as an intermediate analogue of the light-driven DNA repair reaction and can be reduced to the enzymatically active FADH- form by red-light irradiation. Difference Fourier transform infrared (FT-IR) spectroscopy was used to monitor vibronic bands of the blue radical form and of the fully reduced FADH- form of the enzyme. Preliminary band assignments are based on experiments with 15N-labelled enzyme and on experiments with D2O as solvent. Difference FT-IR measurements were also used to observe the formation of thymidine dimers by ultraviolet irradiation and their repair by light-driven photolyase catalysis. This study provides the basis for future time-resolved FT-IR studies which are aimed at an elucidation of a detailed molecular picture of the light-driven DNA repair process.     

Identification of a pterin derivative in Escherichia coli DNA photolyase

DNA photolyase from Escherichia coli contains reduced flavin adenine dinucleotide plus a second chromophore, partially characterized in previous studies. Both chromophores function as sensitizers in catalysis. The second chromophore has been identified as a 6-substituted pterin derivative. The compound is oxidized with permanganate to yield 6-carboxypterin or reduced with sodium cyanoborohydride to yield a 5,6,7,8-tetrahydropterin derivative. The second chromophore exhibits spectral properties (lambda max = 360, 255 nm, pH 2) similar to that observed for 7,8-dihydropterin cations. The compound does not exhibit a spectrally detectable pKa around 4 but is converted to a dication (lambda max = 346, 255 nm) in strong acid (pKa approximately 1). Similar ionization behavior is observed with 7,8-dihydropterin derivatives that are alkylated at N(5). The instability of the second chromophore in weakly alkaline solution is due to a fully reversible conversion to a labile bleached form. As compared with other pterin derivatives, the hydrolytic instability is unusual but is very similar to that observed for 5,6-dialkyl-7,8-dihydropterinium salts. It is proposed that the second chromophore is a 7,8-dihydropterin with substituents at positions 5 and 6. The discovery that a pterin derivative functions as a photosensitizer in DNA repair is apparently the first example of a photobiological function for pterins.     

Activity assay of His-tagged E. coli DNA photolyase by RP-HPLC and SE-HPLC

Escherichia coli DNA photolyase was expressed as C-terminal 6x histidine-fused protein. Purification of His-tagged E. coli DNA photolyase was developed using immobilized metal affinity chromatography with Chelating Sepharose Fast Flow. By one-step affinity chromatography, approximate 4.6 mg DNA photolyase was obtained from 400 ml E. coli culture. The purified His-tagged enzyme was combined with two chromophors, FADH and MTHF. Using the oligonucleotide containing cyclobutane pyrimidine dimer as substrate, both reversed-phase high-performance liquid chromatography and size-exclusion high-performance liquid chromatography were developed to measure the enzyme activity. The enzyme was found to be able to repair the cyclobutane pyrimidine dimer with the turnover rate of 2.4 dimers/photolyase molecule/min.     

Analysis of the role of intraprotein electron transfer in photoreactivation by DNA photolyase in vivo

Escherichia coli DNA photolyase contains FADH(-) as the catalytic cofactor. The cofactor becomes oxidized to the FADH(*) blue neutral radical during purification. The E-FADH(*) form of the enzyme is catalytically inert but can be converted to the active E-FADH(-) form by a photoreduction reaction that involves intraprotein electron transfer from Trp306. It is thought that the E-FADH(*) form is also transiently generated during pyrimidine dimer repair by photoinduced electron transfer, and it has been suggested that the FADH(*) that is generated after each round of catalysis must be photoreduced before the enzyme can engage in subsequent rounds of repair. In this study, we introduced the Trp306Phe mutation into the chromosomal gene and tested the non-photoreducible W306F mutant for photorepair in vivo. We find that both wild-type and W306F mutant photolyases carry out at least 25 rounds of photorepair at the same rate. We conclude that photoreduction by intraprotein electron transfer is not part of the photolyase photocycle under physiological conditions.     

Active site of DNA photolyase: tryptophan-306 is the intrinsic hydrogen atom donor essential for flavin radical photoreduction and DNA repair in vitro

DNA photolyases repair cyclobutadipyrimidines (Pyr()Pyr) in DNA by photoinduced electron transfer. The enzyme isolated from Escherichia coli contains methenyltetrahydrofolate (MTHF), which functions as photoantenna, and FADH2, which is the redox-active cofactor. During purification, FADH2 is oxidized to the blue neutral radical form, FADH., which has greatly diminished activity. Previous nanosecond flash photolysis studies [Heelis, P.F., Okamura, T., & Sancar, A. (1990) Biochemistry 29, 5694-5698] indicated that excitation of FADH. either directly by absorbing a photon or indirectly by electronic energy transfer from MTHF excited singlet state yielded an FADH. quartet which abstracted a hydrogen atom from a nearby tryptophan to generate the catalytically competent FADH2 from of the enzyme. Using site-directed mutagenesis, we replaced all 15 photolyase tryptophan residues by phenylalanine, individually, in order to identify the internal hydrogen atom donor responsible for photoreduction. We found that W306F mutation abolished photoreduction of FADH. without affecting the excited-state properties of FADH. or the substrate binding (KA approximately 10(9) M-1) of the enzyme. The specificity constant (kcat/km) was approximately 0 for the mutant enzyme in the absence of reducing agents in the reaction mixture, indicating that photoreduction of FADH. is an essential step for photorepair by photolyase in vitro. Chemical reduction of FADH. of the mutant enzyme restored the specificity constant to the wild-type level.     

Roles of FAD and 8-hydroxy-5-deazaflavin chromophores in photoreactivation by Anacystis nidulans DNA photolyase.

DNA photolyase from the cyanobacterium Anacystis nidulans contains two chromophores, flavin adenine dinucleotide (FADH2) and 8-hydroxy-5-deazaflavin (8-HDF) (Eker, A. P. M., Kooiman, P., Hessels, J. K. C., and Yasui, A. (1990) J. Biol. Chem. 265, 8009-8015). While evidence exists that the flavin chromophore (in FADH2 form) can catalyze photorepair directly and that the 8-HDF chromophore is the major photosensitizer in photoreactivation it was not known whether 8-HDF splits pyrimidine dimer directly or indirectly through energy transfer to FADH2 at the catalytic center. We constructed a plasmid which over-produces the A. nidulans photolyase in Escherichia coli and purified the enzyme from this organism. Apoenzyme was prepared and enzyme containing stoichiometric amounts of either or both chromophores was reconstituted. The substrate binding and catalytic activities of the apoenzyme (apoE), E-FADH2, E-8-HDF, E-FAD(ox)-8-HDF, and E-FADH2-8-HDF were investigated. We found that FAD is required for substrate binding and catalysis and that 8-HDF is not essential for binding DNA, and participates in catalysis only through energy transfer to FADH2. The quantum yields of energy transfer from 8-HDF to FADH2 and of electron transfer from FADH2 to thymine dimer are near unity.     

Energy transduction during catalysis by Escherichia coli DNA photolyase.

Native DNA photolyase from Escherichia coli contains 1,5-dihydroFAD (FADH2) plus 5,10-methenyltetrahydropteroylpolyglutamate. Quantum yield and action spectral data for thymine dimer repair were obtained by using a novel multiple turnover approach under aerobic conditions. This method assumes that catalysis proceeds via a (rapid-equilibrium) ordered mechanism with light as the second substrate, as verified in steady state kinetic studies. The action spectrum observed with native enzyme matched its absorption spectrum and an action spectrum simulated based on an energy transfer mechanism where dimer repair is initiated either by direct excitation of FADH2 or by pterin excitation followed by singlet-singlet energy transfer to FADH2. The quantum yield observed for dimer repair with native enzyme (phi Native = 0.722 +/- 0.0414) is similar to that observed with enzyme containing only FADH2 (phi EFADH2 = 0.655 +/- 0.0256), as expected owing to the high efficiency of energy transfer from the natural pterin to FADH2 [EET = 0.92]. The quantum yield observed for dimer repair decreased (2.1-fold) when the natural pterin was partially (68.8%) replaced with 5,10-CH(+)-H4folate (phi obs = 0.342 +/- 0.0149). This is consistent with the energy transfer mechanism (phi calc = 0.411 +/- 0.0118) since a 2-fold lower energy transfer efficiency is observed when the natural pterin is replaced with 5,10-CH(+)-H4folate (EET = 0.46) (Lipman & Jorns, 1992). The action spectrum observed for 5,10-CH(+)-H4folate-supplemented enzyme matched a simulated action spectrum which exhibited a small (5 nm) hypsochromic shift as compared with the absorption spectrum (lambda max = 385 nm).(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and characterization of a second chromophore of DNA photolyase from Thermus thermophilus HB27

Cyclobutane pyrimidine dimer (CPD) photolyases use light to repair CPDs. For efficient light absorption, CPD photolyases use a second chromophore. We purified Thermus thermophilus CPD photolyase with its second chromophore. UV-visible absorption spectra, reverse-phase HPLC, and NMR analyses of the chromophores revealed that the second chromophore of the enzyme is flavin mononucleotide (FMN). To clarify the role of FMN in the CPD repair reaction, the enzyme without FMN (Enz-FMN(-) and that with a stoichiometric amount of FMN (Enz-FMN(+)) were both successfully obtained. The CPD repair activity of Enz-FMN(+) was higher than that of Enz-FMN(-), and the CPD repair activity ratio of Enz-FMN(+) and Enz-FMN(-) was dependent on the wavelength of light. These results suggest that FMN increases the light absorption efficiency of the enzyme. NMR analyses of Enz-FMN(+) and Enz-FMN(-) revealed that the binding mode of FMN is similar to that of 7,8-didemethyl-8-hydroxy-5-deazariboflavin in Anacystis nidulans CPD photolyase, and thus a direct electron transfer between FMN and CPD is not likely to occur. Based on these results, we concluded that FMN acts as a highly efficient light harvester that gathers light and transfers the energy to FAD.     

Role of enzyme-bound 5,10-methenyltetrahydropteroylpolyglutamate in catalysis by Escherichia coli DNA photolyase

DNA photolyase catalyzes the photoreversal of pyrimidine dimers. The enzymes from Escherichia coli and yeast contain a flavin chromophore and a folate cofactor, 5,10-methenyltetrahydropteroylpolyglutamate. E. coli DNA photolyase contains about 0.3 mol of folate/mol flavin, whereas the yeast photolyase contains the full complement of folate. E. coli DNA photolyase is reconstituted to a full complement of the folate by addition of 5,10-methenyltetrahydrofolate to cell lysates or purified enzyme samples. The reconstituted enzyme displays a higher photolytic cross section under limiting light. Treatment of photolyase with sodium borohydride or repeated camera flashing results in the disappearance of the absorption band at 384 nm and is correlated with the formation of modified products from the enzyme-bound 5,10-methenyltetrahydrofolate. Photolyase modified in this manner has a decreased photolytic cross section under limiting light. Borohydride reduction results in the formation of 5,10-methylenetetrahydrofolate and 5-methyltetrahydrofolate, both of which are released from the enzyme. Repeated camera flashing results in photodecomposition of the enzyme-bound 5,10-methenyltetrahydrofolate and release of the decomposition products. Finally, it is observed that photolyase binds 10-formyltetrahydrofolate and appears to cyclize it to form the 5,10-methenyltetrahydrofolate chromophore.     

DNA repair mechanism by photolyase: electron transfer path from the photolyase catalytic cofactor FADH(-) to DNA thymine dimer

Photolyase is an enzyme that catalyses photorepair of thymine dimers in UV damaged DNA by electron transfer reaction. The structure of the photolyase/DNA complex is unknown at present. Using crystal structure coordinates of the substrate-free enzyme from E. coli, we have recently built a computer molecular model of a thymine dimer docked to photolyase catalytic site and studied molecular dynamics of the system. In this paper, we present analysis of the electronic coupling and electron transfer pathway between the catalytic cofactor FADH(-) and the pyrimidine dimer by the method of interatomic tunneling currents. Electronic structure is treated in the extended Hückel approximation. The root mean square transfer matrix element is about 6 cm(-1), which is consistent with the experimentally determined rate of transfer. We find that electron transfer mechanism responsible for the repair utilizes an unusual folded conformation of FADH(-) in photolyases, in which the isoalloxazine ring of the flavin and the adenine are in close proximity, and the peculiar features of the docked orientation of the dimer. The tunneling currents show explicitly that despite of the close proximity between the donor and acceptor complexes, the electron transfer mechanism between the flavin and the thymine bases is not direct, but indirect, with the adenine acting as an intermediate. These calculations confirm the previously made conclusion based on an indirect evidence for such mechanism.