The light-induced electron transfer reaction of flavin cofactor photoactivation in Xenopus laevis (6 -4) photolyase has been studied by continuous-wave and time-resolved electron paramagnetic resonance spectroscopy. When the photoactivation is initiated from the fully oxidized form of the flavin, a neutral flavin radical is observed as a long-lived paramagnetic intermediate of two consecutive single-electron reductions under participation of redox-active amino acid residues. By time-resolved electron paramagnetic resonance, a spin-polarized transient radical-pair signal was detected that shows remarkable differences to the signals observed in the related cyclobutane pyrimidine dimer photolyase enzyme. In (6 -4) photolyase, a neutral tyrosine radical has been identified as the final electron donor, on the basis of the characteristic line width, hyperfine splitting pattern, and resonance magnetic field position of the tyrosine resonances of the transient radical pair. U ltraviolet light (Յ300 nm) damages cellular DNA by formation of cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts from adjacent pyrimidine bases on the same DNA strand (1). Such dimers may be restored to their monomeric form through the action of two photoactive (300ϽϽ500 nm) damage-specific DNA repair enzymes, named CPD photolyase (also called DNA photolyase) (2-6) and (6-4) photolyase (7,8). Both enzymes are found in various organisms and exhibit a 20-30% amino acid sequence identity (3, 9, 10). Two photoactive pigments are used in the DNA repair pathway. One is invariably a redox-active FAD (11,12), and the other so-called second chromophore, which acts as a light harvester, is a methenyltetrahydrofolate in most species (11,13,14) but 5-deazariboflavin in certain rare species that synthesize this compound (15,16). It has been proposed that the initial step in the DNA repair mechanism is a photoinduced single electron transfer (ET) from the FAD cofactor, which in the active enzyme is in its fully reduced form (12,17,18), FADH Ϫ , to the DNA lesion. This mechanism is supported by a previous electron paramagnetic resonance (EPR) study in which a photoinduced spin-polarized radical pair (RP) signal assigned to the flavin-CPD dimer complex was observed (19).If the enzyme is found in an inactive state with FAD either semireduced as neutral radical, FADH ⅐ , or fully oxidized, FAD ox (20), photolyases can undergo reversible ET reactions with the participation of amino acid residues to lower the redox state of the flavin cofactor to FADH Ϫ . This photoactivation process has recently attracted much experimental (20-26) and theoretical (27, 28) interest. In Escherichia coli CPD photolyase, cofactor photoactivation proceeds on a nanosecond time scale via a chain of tryptophan residues (W-382, -359, and -306) (29). W-306 is believed to be the final electron donor that is rereduced on a millisecond time scale either by back ET from the flavin or by exogenous reductants (30). In Anacystis nidulans CPD photolyase, however, a further ET step from a t...