Action mechanism of Escherichia coli DNA photolyase. II. Role of the chromophores in catalysis.
DNA photolyase repairs pyrimidine dimers in DNA in a reaction that requires visible light. Photolyase from Escherichia coli is normally isolated as a blue protein and contains 2 chromophores: a blue FAD radical plus a second chromophore that exhibits an absorption maximum at 360 nm when free in solution. Oxidation of the FAD radical is accompanied by a reversible loss of activity which is proportional to the fraction of the enzyme flavin converted to FADox. Quantitative reduction of the radical to fully reduced FAD causes a 3-fold increase in activity. The results show that a reduced flavin is required for activity and suggest that flavin may act as an electron donor in catalysis. Comparison of the absorption spectrum calculated for the protein-bound second chromophore (lambda max = 390 nm) with fluorescence data and with the relative action spectrum for dimer repair indicates that the second chromophore is the fluorophore in photolyase and that it does act as a sensitizer in catalysis. On the other hand, enzyme preparations containing diminished amounts of the second chromophore do not exhibit correspondingly lower activity. This suggests that reduced flavin may also act as a sensitizer in catalysis. The blue color of the enzyme is lost upon reduction of the FAD radical. The fully reduced E. coli enzyme exhibits absorption and fluorescence properties very similar to yeast photolyase. This indicates that the two enzymes probably contain similar chromophores but are isolated in different forms with respect to the redox state of the flavin.
D002384:Catalysis; D004247:DNA / Q000378:metabolism*; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000378:metabolism*; D004227:Dithionite / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D008190:Lyases / Q000378:metabolism*; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D011740:Pyrimidine Dimers / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D011740:Pyrimidine Dimers; D005182:Flavin-Adenine Dinucleotide; D004227:Dithionite; D004247:DNA; D008190:Lyases; D004255:Deoxyribodipyrimidine Photo-Lyase