Photoreduction of inactive DNA photolyase: Correlated triplet-doublet radical pair polarization of tryptophan in the enzyme

UV radiation has many harmful effects on DNA, the most significant of which is the formation of pyrimidine dimers between adjacent thymines that occur in the nucleotide sequence. All organisms have mechanisms to repair these dimers. One of the most interesting is that carried out by DNA photolyase, which repairs pyrimidine dimers through a visible light-initiated photoreaction.;During isolation of the enzyme, one electron oxidation of the flavin produces the semiquinone form of the chromophore. The enzyme can be photoreactivated to its active form by electron transfer from an amino acid side chain to the flavin semiquinone. The electron transfer reaction generates a protein bound radical that can be reduced by an exogenous electron donor to form the active enzyme.;Time resolved electron paramagnetic resonance was used to detect the transient radical and to study the photoreactivating reaction. In agreement with previous optical studies, we have identified the donor to the flavin semiquinone as tryptophan-306 by using specific deuteration, ;We have also found that the transient tryptophan radical is formed initially in a spin polarized state. Spin polarization results from a non-Boltzman distribution of the electron spin energy levels. The transient tryptophan radical in photolyase has certain transitions in emission, which is hallmark of a spin polarized radical. Spin polarization results from interactions between two radicals in a radical pair. The polarization in photolyase is distinct from other radical pairs because the transitions alternate between emission and absorption, and arises from a doublet-triplet radical pair. This pattern has been observed for doublet-doublet radical pairs and is explained by the presence of an exchange interaction between the radicals during the observation time by EPR. We propose that spin polarization in photolyase reflects the same phenomenon but the interaction arises from a triplet-doublet radical pair.