Identification of a neutral flavin radical and characterization of a second chromophore in Escherichia coli DNA photolyase

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Abstract
DNA photolyase from E. coli is a blue protein exhibiting absorption maxima at 580, 475 and 384 nm. One chromophore present in this enzyme was identified as the blue neutral FAD radical on the basis, in part, of visible absorption and ESR data. The enzyme-bound radical (.epsilon.580 = 3.6 .times. 103 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. > 500 nm is due only to the FAD radical, whereas the band at 384 nm reflects contributions from the radical and a 2nd chromophore. The latter is labile when protein free at neutral pH (.lambda.max =360 nm, k = 5.5 .times. 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 2nd chromophore.