Flavin-dependent enzymes, such as photolyases and cryptochromes, harness blue light to drive photoreduction for the repair of UV-damaged DNA and signaling. Here, we investigate the photoreduction dynamics and protonation pathways of the prokaryotic 6-4 photolyase from Caulobacter crescentus (Cc(6-4)) using ultrafast UV/Vis spectroscopy, X-ray crystallography and molecular dynamics. The Cc(6-4) absorption spectrum is dominated by its three cofactors: FAD, an 4Fe-4S2+ cluster, and DLZ. FAD photoreduction proceeds via a super-exchange mechanism, with tyrosine serving as primary electron bridge, facilitating forward electron transfer from a tryptophan, the second residue in the highly conserved electron transfer triad. Protonation, crucial for stabilizing the reduced FAD•−, is mediated by residue E402 and a nearby water molecule. Mutagenesis of this residue to glutamine blocks protonation, highlighting its critical role. Finally, the photochemical activity of the 4Fe-4S cluster is reflected by sub-picosecond oxidation thus making the cluster to a second light-driven electron injector besides the fully reduced flavin cofactor. Given the absence of an ultimate electron acceptor, this cluster undergoes fast recombination within 1.5 ps. Overall, prokaryotic (6-4) photolyases depend for forward, but not backward electron transfer on a unique arrangement of the proximal aromatic residue allowing super-exchange, while protonation of the FAD•− state requires a transient protonation pathway.
Trunk et al. (Tue,) studied this question.