flavin-adenine-dinucleotide and factor-420

Cryptochromes are ubiquitous flavin-binding light sensors closely related to DNA-repairing photolyases. The animal-like cryptochrome

Topics: Chlamydomonas; Chlamydomonas reinhardtii; Color; Cryptochromes; Deoxyribodipyrimidine Photo-Lyase; Dinitrocresols; Flavin-Adenine Dinucleotide; Flavins; Light; Riboflavin; Spectrophotometry, Ultraviolet; Spectroscopy, Fourier Transform Infrared

Photoantenna is essential to energy transduction in photoinduced biological machinery. A photoenzyme, photolyase, has a light-harvesting pigment of methenyltetrahydrofolate (MTHF) that transfers its excitation energy to the catalytic flavin cofactor FADH¯ to enhance DNA-repair efficiency. Here we report our systematic characterization and direct determination of the ultrafast dynamics of resonance energy transfer from excited MTHF to three flavin redox states in E. coli photolyase by capturing the intermediates formed through the energy transfer and thus excluding the electron-transfer quenching pathway. We observed 170 ps for excitation energy transferring to the fully reduced hydroquinone FADH¯, 20 ps to the fully oxidized FAD, and 18 ps to the neutral semiquinone FADH(•), and the corresponding orientation factors (κ(2)) were determined to be 2.84, 1.53 and 1.26, respectively, perfectly matching with our calculated theoretical values. Thus, under physiological conditions and over the course of evolution, photolyase has adopted the optimized orientation of its photopigment to efficiently convert solar energy for repair of damaged DNA.

Topics: Deoxyribodipyrimidine Photo-Lyase; Energy Transfer; Escherichia coli; Escherichia coli Proteins; Flavin-Adenine Dinucleotide; Folic Acid; Oxidation-Reduction; Riboflavin; Spectrum Analysis

DNA photolyase is a unique flavoenzyme that repairs UV-induced DNA lesions using the energy of visible light. Anacystis nidulans photolyase contains a light-harvesting chromophore, 8-hydroxy-5-deazaflavin (8-HDF), and flavin adenine dinucleotide (FAD) which, in contrast to the 8-HDF chromophore, is indispensable for catalytic activity. This work reports the crystallization and structure at 1.8 A resolution of DNA photolyase devoid of its 8-HDF chromophore (apophotolyase). The overall three-dimensional structure is similar to that of the holoenzyme, indicating that the presence of 8-HDF is not essential for the correct folding of the enzyme. Structural changes include an additional phosphate group, a different conformation for Arg11 and slight rearrangements of Met47, Asp101 and Asp382, which replace part of the 8-HDF molecule in the chromophore-binding pocket. The apophotolyase can be efficiently reconstituted with synthetic 8-hydroxy-5-deazariboflavin, despite the orientation of Arg11 and the presence of the phosphate group in the 8-HDF pocket. Red light or X-rays reduced the FAD chromophore in apophotolyase crystals, as observed by single-crystal spectrophotometry. The structural effects of FAD reduction were determined by comparison of three data sets that were successively collected at 100 K, while the degree of reduction was monitored online by changes in the light absorption of the crystals. X-ray-induced conformational changes were confined to the active site of the protein. They include sub-ångström movements of the O(2) and N(5) atoms of the flavin group as well as the O(delta) atoms of the surrounding amino acids Asp380 and Asn386.

Topics: Apoproteins; Binding Sites; Crystallization; Crystallography, X-Ray; Cyanobacteria; Deoxyribodipyrimidine Photo-Lyase; Flavin-Adenine Dinucleotide; Models, Molecular; Oxidation-Reduction; Photochemistry; Protein Structure, Tertiary; Riboflavin; Spectrum Analysis

Topics: Amino Acid Sequence; Apoenzymes; Aspergillus nidulans; Binding Sites; Catalysis; Chromatography; Chromatography, Affinity; Chromatography, Gel; Coenzymes; Deoxyribodipyrimidine Photo-Lyase; Deuterium; DNA Repair; Durapatite; Electron Spin Resonance Spectroscopy; Escherichia coli; Flavin-Adenine Dinucleotide; Pyrimidine Dimers; Radioisotope Dilution Technique; Riboflavin; Spectrophotometry; Spectrophotometry, Ultraviolet; Tetrahydrofolates; Tryptophan

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.

Topics: Apoenzymes; Base Sequence; Chromatography, Ion Exchange; Cloning, Molecular; Codon; Coenzymes; Cyanobacteria; Deoxyribodipyrimidine Photo-Lyase; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Flavin-Adenine Dinucleotide; Light; Mutagenesis, Site-Directed; Plasmids; Recombinant Proteins; Restriction Mapping; Riboflavin; Spectrometry, Fluorescence; Spectrophotometry

Photoreactivating enzyme, which specifically monomerizes pyrimidine dimers in UV-irradiated DNA, was purified 21,000-fold from the cyanobacterium Anacystis nidulans to apparent homogeneity with 41% overall yield. The enzyme consists of a single protein chain with 53,000 molecular weight. Maximal activity was found at pH 6.2 and 0.1 M NaCl. Purified photoreactivating enzyme exhibits a marked absorption spectrum with a main band in the blue region (maximum 437 nm), a protein band (maximum 266 nm), and a low intensity band above 500 nm. The molar extinction coefficient of native enzyme was estimated 53,000 at 437 nm. The action spectrum for photoreactivation shows maximal activity at 440 nm and correlates closely with the 437-nm absorption band. The enzyme contains two different intrinsic chromophores in equimolar amounts, which were identified as 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO) and (reduced) FAD. The low intensity absorption band of native photoreactivating enzyme exhibits a shoulder at 498 and maxima at 588 and 634 nm. This band is attributed to a neutral FAD semiquinone radical which accounts for the major part of the FAD present in dark equilibrated enzyme. Preillumination at 585 nm bleaches the semiquinone spectrum due to formation of fully reduced FAD, but exposure to air in the dark restores the spectrum completely. On preillumination at 437 nm the disappearance of FAD semiquinone is more rapid, indicating that the photoreduction is sensitized by the 8-hydroxy-5-deazaflavin chromophore. The 8-hydroxy-5-deazaflavin and possibly also the reduced FAD chromophore appear to act as a primary photon acceptor in the photoreactivation process.

Topics: Amino Acid Sequence; Chromatography; Cyanobacteria; Deoxyribodipyrimidine Photo-Lyase; Flavin-Adenine Dinucleotide; Hydrogen-Ion Concentration; Light; Lyases; Molecular Sequence Data; Molecular Weight; Photochemistry; Protein Denaturation; Riboflavin; Spectrometry, Fluorescence; Spectrophotometry

Methanobacterium ruminantium was shown to possess a nicotinamide adenine dinucleotide phosphate (NADP)-linked factor 420 (F420)-dependent hydrogenase system. This system was also shown to be present in Methanobacterium strain MOH. The hydrogenase system of M. ruminantium also links directly to F420, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), methyl viologen, and Fe-3 plus. It has a pH optimum of about 8 and an apparent Km for F420 of about 5 x 10-6 M at pH 8 when NADP is the electron acceptor. The F420-NADP oxidoreductase activity is inactive toward nicotinamide adenine dinucleotide (nad) and no NADPH:NAD or FADH2(FMNH2):NAD transhydrogenase system was detected. Neither crude ferredoxin nor boiled crude extract of Clostridium pasteuranum could replace F420 in the NADP-linked hydrogenase reaction of M. ruminantium. Also, neitther F420 nor a curde "ferredoxin" fraction from M. ruminantium extracts could substitute for ferredoxin in the pyruvate-ferredoxin oxidoreductase reaction of C. pasteurianum.

Topics: Bacteria; Cell-Free System; Coenzymes; Electron Transport; Ferredoxins; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Hydrogen; Hydrogen-Ion Concentration; Indicators and Reagents; Iron; Manometry; NAD; NADP; Nucleotides; Oxidoreductases; Pyridines; Riboflavin; Spectrophotometry