Origin of the transient electron paramagnetic resonance signals in DNA photolyase

DNA photolyase repairs pyrimidine dimer lesions in DNA through light-induce d electron donation to the dimer, During isolation of the enzyme, the flavi n cofactor necessary for catalytic activity becomes one-electron-oxidized t o a semiquinone radical. In the absence of external reducing agents, the fl avin can be cycled through the semiquinone radical to the fully reduced sta te with light-induced electron transfer from a nearby tryptophan residue. T his cycle provides a convenient means of studying the process of electron t ransfer within the protein by using transient EPR. By studying the excitati on wavelength dependence of the time-resolved EPR signals we observe, we sh ow that the spin-polarized EPR signal reported earlier from this laboratory as being initiated by semiquinone photochemistry actually originates from the fully oxidized form of the flavin cofactor. Exciting the semiquinone fo rm of the flavin produces two transient EPR signals: a fast signal that is limited by the time response of the instrument and a slower signal with a l ifetime of approximately 6 ms. The fast component appears to correlate with a dismutation reaction occurring with the flavin, The longer lifetime proc ess occurs on a time scale that agrees with transient absorption data publi shed earlier; the magnetic field dependence of the amplitude of this kineti c component is consistent with redox chemistry that involves electron trans fer between flavin and tryptophan, We also report a new procedure for the r apid isolation of DNA photolyase.