DNA repair catalyzed by Escherichia coli DNA photolyase containing only reduced flavin: elimination of the enzyme's second chromophore by reduction with sodium borohydride.
DNA photolyase from Escherichia coli contains FAD plus a partially characterized, second chromophore. In vivo, the flavin is fully reduced (FADH2), but oxidation to a stable, blue radical (FADH.) occurs during enzyme isolation. The second chromophore is irreversibly reduced by reaction of the enzyme with sodium borohydride or by photoreduction in the presence of dithiothreitol. A similar reaction occurs with the protein-free chromophore and sodium cyanoborohydride. Reduction of the second chromophore is accompanied by a complete loss of the chromophore's visible absorption and fluorescence but does not significantly affect catalytic activity. The results show that the enzyme can repair dimers by a pathway involving only FADH2. Enzyme-bound FADH2 is fluorescent and exhibits emission (505 nm) and absorption (360 nm) maxima similar to that expected for a 1,5-dihydroflavin derivative. It is proposed that dimer cleavage via the second chromophore independent pathway involves electron donation from excited FADH2 to pyrimidine dimer. Pyrimidine dimer radicals are unstable and spontaneously monomerize. Unmodified second chromophore can also act as a sensitizer in a pathway that requires FADH2. This pathway may be similar to that proposed for the second chromophore independent reaction except that excited FADH2 would be produced via energy transfer from the excited second chromophore. The fluorescence observed for enzyme-bound, unmodified second chromophore is quenched by FADH. and increases 6-fold when the latter is reduced, but the absorption spectrum (lambda max = 390 nm epsilon 390 = 12.7 x 10(3) M-1 cm-1) is independent of the redox state of the flavin.(ABSTRACT TRUNCATED AT 250 WORDS)
Jorns
M S
MS
Department of Biological Chemistry, Hahnemann University School of Medicine, Philadelphia, Pennsylvania 19102.
Wang
B
B
Jordan
S P
SP
D001894:Borohydrides / Q000494:pharmacology*; D004260:DNA Repair; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D007700:Kinetics; D008190:Lyases / Q000378:metabolism*; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D001894:Borohydrides; D005182:Flavin-Adenine Dinucleotide; D008190:Lyases; D004255:Deoxyribodipyrimidine Photo-Lyase