title: Characteristic structure and environment in FAD cofactor of (6-4) photolyase along function revealed by resonance Raman spectroscopy.
abstract: A pyrimidine-pyrimidone (6-4) photoproduct and a cyclobutane pyrimidine dimer (CPD) are major DNA lesions induced by ultraviolet irradiation, and (6-4) photolyase, an enzyme with flavin adenine dinucleotide (FAD) as a cofactor, repairs the former specifically by light illumination. We investigated resonance Raman spectra of (6-4) photolyase from Arabidopsis thaliana having neutral semiquinoid and oxidized forms of FAD, which were selectively intensity enhanced by excitations at 568.2 and 488.0 nm, respectively. DFT calculations were carried out for the first time on the neutral semiquinone. The marker band of a neutral semiquinone at 1606 cm(-1) in H(2)O, whose frequency is the lowest among various flavoenzymes, apparently splits into two comparable bands at 1594 and 1608 cm(-1) in D(2)O, and similarly, that at 1522 cm(-1) in H(2)O does into three bands at 1456, 1508, and 1536 cm(-1) in D(2)O. This D(2)O effect was recognized only after being oxidized once and photoreduced to form a semiquinone again, but not by simple H/D exchange of solvent. Some Raman bands of the oxidized form were observed at significantly low frequencies (1621, 1576 cm(-1)) and with band splittings (1508/1493, 1346/1320 cm(-1)). These Raman spectral characteristics indicate strong H-bonding interactions (at N5-H, N1), a fairly hydrophobic environment, and an electron-lacking feature in benzene ring of the FAD cofactor, which seems to specifically control the reactivity of (6-4) photolyase.
authors.affiliation: Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Myodaiji, Okazaki, Aichi 444-8787, Japan.
authors.forename: Jiang
authors.initials: J
authors.lastname: Li
authors.forename: Takeshi
authors.initials: T
authors.lastname: Uchida
authors.forename: Takehiro
authors.initials: T
authors.lastname: Ohta
authors.forename: Takeshi
authors.initials: T
authors.lastname: Todo
authors.forename: Teizo
authors.initials: T
authors.lastname: Kitagawa
mesh_terms: D000595:Amino Acid Sequence; D001554:Benzene / Q000737:chemistry; D003067:Coenzymes / Q000737:chemistry*; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry*; D005415:Flavins / Q000737:chemistry; D006860:Hydrogen Bonding; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D011740:Pyrimidine Dimers / Q000737:chemistry*; D011789:Quantum Theory; D016415:Sequence Alignment; D013059:Spectrum Analysis, Raman
chemical_list: D003067:Coenzymes; D005415:Flavins; D011740:Pyrimidine Dimers; C001922:lumiflavin; D005182:Flavin-Adenine Dinucleotide; C098727:pyrimidine(6-4)pyrimidone photolyase; D004255:Deoxyribodipyrimidine Photo-Lyase; D001554:Benzene