title: Fourier-transform infrared study of the photoactivation process of Xenopus (6-4) photolyase.
abstract: Photolyases (PHRs) are blue light-activated DNA repair enzymes that maintain genetic integrity by reverting UV-induced photoproducts into normal bases. The flavin adenine dinucleotide (FAD) chromophore of PHRs has four different redox states: oxidized (FAD(ox)), anion radical (FAD(•-)), neutral radical (FADH(•)), and fully reduced (FADH(-)). We combined difference Fourier-transform infrared (FTIR) spectroscopy with UV-visible spectroscopy to study the detailed photoactivation process of Xenopus (6-4) PHR. Two photons produce the enzymatically active, fully reduced PHR from oxidized FAD: FAD(ox) is converted to semiquinone via light-induced one-electron and one-proton transfers and then to FADH(-) by light-induced one-electron transfer. We successfully trapped FAD(•-) at 200 K, where electron transfer occurs but proton transfer does not. UV-visible spectroscopy following 450 nm illumination of FAD(ox) at 277 K defined the FADH(•)/FADH(-) mixture and allowed calculation of difference FTIR spectra among the four redox states. The absence of a characteristic C=O stretching vibration indicated that the proton donor is not a protonated carboxylic acid. Structural changes in Trp and Tyr are suggested by UV-visible and FTIR analysis of FAD(•-) at 200 K. Spectral analysis of amide I vibrations revealed structural perturbation of the protein's β-sheet during initial electron transfer (FAD(•-) formation), a transient increase in α-helicity during proton transfer (FADH(•) formation), and reversion to the initial amide I signal following subsequent electron transfer (FADH(-) formation). Consequently, in (6-4) PHR, unlike cryptochrome-DASH, formation of enzymatically active FADH(-) did not perturb α-helicity. Protein structural changes in the photoactivation of (6-4) PHR are discussed on the basis of these FTIR observations.
authors: Department of Frontier Materials, Nagoya Institute of Technology, Nagoya 466-8555, Japan.
authors: Daichi
authors: D
authors: Yamada
authors: Yu
authors: Y
authors: Zhang
authors: Tatsuya
authors: T
authors: Iwata
authors: Kenichi
authors: K
authors: Hitomi
authors: Elizabeth D
authors: ED
authors: Getzoff
authors: Hideki
authors: H
authors: Kandori
mesh_terms: D000818:Animals; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism*; D008027:Light; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D014981:Xenopus / Q000378:metabolism*; D029867:Xenopus Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D029867:Xenopus Proteins; D005182:Flavin-Adenine Dinucleotide; D004255:Deoxyribodipyrimidine Photo-Lyase