title: The active form of Escherichia coli DNA photolyase contains a fully reduced flavin and not a flavin radical, both in vivo and in vitro.
abstract: Escherichia coli DNA photolyase is a flavoprotein that when purified is blue in color and contains a stable neutral radical FAD (E-FADH). In the presence of a suitable electron donor (i.e., thiols, tyrosine, or NADH) the radical FAD adsorbs visible light and undergoes photoreduction to the fully reduced FAD (E-FADH2). The in vitro quantum yield of dimer repair for E-FADH is 0.07 while that of E-FADH2 approaches the in vivo value of 1. Electron paramagnetic resonance studies on whole cells indicate that the in vivo form of photolyase is E-FADH2 with enzyme containing radical FAD generated predominantly during the ammonium sulfate precipitation step of the purification. Activity measurements of E-FADH using long-wavelength photoreactivating light indicate that enzyme containing FAD in the radical form is not active in dimer repair. Dimer repair observed with E-FADH at shorter wavelengths is probably photoreduction of E-FADH followed by dimer repair by E-FADH2.
authors.affiliation: Department of Biochemistry, School of Medicine, University of North Carolina, Chapel Hill 27514.
authors.forename: G
authors.initials: G
authors.lastname: Payne
authors.forename: P F
authors.initials: PF
authors.lastname: Heelis
authors.forename: B R
authors.initials: BR
authors.lastname: Rohrs
authors.forename: A
authors.initials: A
authors.lastname: Sancar
mesh_terms: D004255:Deoxyribodipyrimidine Photo-Lyase / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D005609:Free Radicals; D007700:Kinetics; D008190:Lyases / Q000378:metabolism*; D010084:Oxidation-Reduction
chemical_list: D005609:Free Radicals; D005182:Flavin-Adenine Dinucleotide; D008190:Lyases; D004255:Deoxyribodipyrimidine Photo-Lyase