Flavin coenzymes are universally found in biological redox reactions. DNA photolyases with their flavin chromophore (FAD) utilize blue light for DNA repair and photoreduction. The latter process involves two single-electron transfers to FAD with an intermittent protonation step to prime the enzyme active for DNA repair. Here we use time-resolved serial femtosecond X-ray crystallography to describe how light-driven electron transfers trigger subsequent nanosecond-to-microsecond entanglement between FAD and its Asn/Arg-Asp redox sensor triad. We found that this key feature within the photolyase-cryptochrome family regulates FAD re-hybridization and protonation. After first electron transfer, the FAD •isoalloxazine ring twists strongly when the arginine closes in to stabilize the negative charge. Subsequent breakage of the arginine-aspartate salt bridge promotes proton transfer from arginine to FAD •-. Our molecular movies demonstrate how the protein environment of redox cofactors organizes multiple electron/proton transfer events in an ordered fashion, which could be applicable to other redox systems such as photosynthesis.

(6-4) photolyases [(6-4)PLs] are flavoproteins that use blue light to repair the ultraviolet-induced pyrimidine(6-4)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 Trp in particular, is discussed.

Photolyases (PLs) are flavoproteins able to repair cross-links formed between adjacent pyrimidine bases in DNA in a light-dependent manner via an electron transfer. The catalytically active redox state of the flavin chromophore for the DNA repair is a fully reduced form of flavin adenine dinucleotide (FADH–). PLs and their relative, cryptochromes (CRYs), share a physicochemical process attributable to the light-dependent reduction of the chromophore via an ultrafast successive electron transfer through exclusively conserved three tryptophan side chains. In some (6–4) PLs and animal CRYs, an additional tryptophan participates in this photoactivation process. In a search for the intrinsic difference between the Trp triad and tetrad, a water molecule proximal to the second and third Trp was found in the reported crystal structure of Arabidopsis thaliana (6–4) PL. Here, we investigated the involvement of the water molecule in photoactivation. Molecular dynamics simulations indicated that the water molecule is stably captured in the binding site, while mutation of S412 increased water displacement from the binding site. Photochemical analysis of recombinant proteins revealed that the S412A mutation significantly decelerated the FAD photoreduction as compared to the wild type. The hydrogen-bonding network including the water molecule would play a key role in the stabilization of the FAD–Trp radical pair.

Photolyases are flavoenzymes responsible for light-driven repair of carcinogenic crosslinks formed in DNA by UV exposure. They possess two non-covalently bound chromophores: flavin adenine dinucleotide (FAD) as a catalytic center and an auxiliary antenna chromophore that harvests photons and transfers solar energy to the catalytic center. Although the energy transfer reaction has been characterized by time-resolved spectroscopy, it is strikingly important to understand how well natural biological systems organize the chromophores for the efficient energy transfer. Here, we comprehensively characterized the binding of 8-hydroxy-7,8-didemethyl-5-deazariboflavin (8-HDF) to Xenopus (6–4) photolyase. In silico simulations indicated that a hydrophobic amino acid residue located at the entrance of the binding site dominates translocation of a loop upon binding of 8-HDF, and a mutation of this residue caused dysfunction of the efficient energy transfer in the DNA repair reaction. Mutational analyses of the protein combined with modification of the chromophore suggested that Coulombic interactions between positively charged residues in the protein and the phenoxide moiety in 8-HDF play a key role in accommodation of 8-HDF in the proper direction. This study provides a clear evidence that Xenopus (6–4) photolyase can utilize 8-HDF as the light-harvesting chromophore. The obtained new insights into binding of the natural antenna molecule will be helpful for the development of artificial light-harvesting chromophores and future characterization of the energy transfer in (6–4) photolyase by spectroscopic studies.

Abstract(6–4) Photolyases ((6–4) PLs) are ubiquitous photoenzymes that use the energy of sunlight to catalyze the repair of carcinogenic UV-induced DNA lesions, pyrimidine(6–4)pyrimidone photoproducts. To repair DNA, (6–4) PLs must first undergo so-called photoactivation, in which their excited flavin adenine dinucleotide (FAD) cofactor is reduced in one or two steps to catalytically active FADH− via a chain of three or four conserved tryptophan residues, transiently forming FAD•−/FADH− ⋯ TrpH•+ pairs separated by distances of 15 to 20 Å. Photolyases and related photoreceptors cryptochromes use a plethora of tricks to prevent charge recombination of photoinduced donor–acceptor pairs, such as chain branching and elongation, rapid deprotonation of TrpH•+ or protonation of FAD•−. Here, we address Arabidopsis thaliana (6–4) PL (At64) photoactivation by combining molecular biology, in vivo survival assays, static and time-resolved spectroscopy and computational methods. We conclude that At64 photoactivation is astonishingly efficient compared to related proteins—due to two factors: exceptionally low losses of photoinduced radical pairs through ultrafast recombination and prevention of solvent access to the terminal Trp3H•+, which significantly extends its lifetime. We propose that a highly conserved histidine residue adjacent to the 3rd Trp plays a key role in Trp3H•+ stabilization.