Loss of Fourth Electron-Transferring Tryptophan in Animal (6-4) Photolyase Impairs DNA Repair Activity in Bacterial Cells

(6-4) photolyases [(6-4)PLs] are flavoproteins that use blue light to repair the ultraviolet-induced pyrimidine(64)pyrimidone photoproduct in DNA. Their flavin adenine dinucleotide (FAD) cofactor can be reduced to its repair-active FADH(-) form by a photoinduced electron transfer reaction. In animal (6-4)PLs, a chain of four Trp residues was suggested to be involved in a stepwise transfer of an oxidation hole from the flavin to the surface of the protein. Here, we investigated the effect of mutation of the fourth Trp on the DNA photorepair activity of Xenopus laevis (6-4)PL (Xl64) in bacterial cells. The photoreduction and photorepair properties of this mutant protein were independently characterized in vitro. Our results demonstrate that the mutation of the fourth Trp in Xl64 drastically impairs the DNA repair activity in cells and that this effect is due to the inhibition of the photoreduction process. We thereby show that the photoreductive formation of FADH-through the Trp tetrad is essential for the biological function of the animal (6-4)PL. The role of the Trp cascade, and of the fourth Tip in particular, is discussed.