Identification of a neutral flavin radical and characterization of a second chromophore in Escherichia coli DNA photolyase.
DNA photolyase from Escherichia coli is a blue protein exhibiting absorption maxima at 580, 475, and 384 nm. One of the two chromophores present in this enzyme has been identified as the blue neutral flavin adenine dinucleotide (FAD) radical on the basis, in part, of visible absorption and electron spin resonance (ESR) data. The enzyme-bound radical (epsilon 580 = 3.6 X 10(3) M-1 cm-1) is stable toward O2 or K3Fe(CN)6, is reversibly reduced by dithionite, and is converted to oxidized FAD upon aerobic denaturation. Disproportionation of the radical is observed upon anaerobic denaturation, consistent with an N-5 unsubstituted radical. The absorbance of the enzyme at lambda greater than 500 nm is due only to the FAD radical whereas the band at 384 nm reflects contributions from both the radical and a second chromophore. The latter is labile when protein free a neutral pH (lambda max = 360 nm, k = 5.5 X 10(-2) min -1 +/- O2), a reaction that is readily monitored by the loss of an intense absorption band at 360 nm following enzyme denaturation under conditions where radical oxidation is immediate. This decomposition is pH dependent and the chromophore is stable at acid pH. Native photolyase is fluorescent (emission lambda max = 470 nm, excitation lambda max = 398 nm). An unlikely fluorescent flavin radical can be excluded by the position of the emission maximum. The enzyme fluorescence is attributed to the second chromophore.
D000332:Aerobiosis; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000302:isolation & purification*; D004227:Dithionite / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis*; D005415:Flavins / Q000032:analysis*; D008190:Lyases / Q000302:isolation & purification*; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence
D005415:Flavins; D005182:Flavin-Adenine Dinucleotide; D004227:Dithionite; D008190:Lyases; D004255:Deoxyribodipyrimidine Photo-Lyase