Abstract
<title>Abstract</title> <p>Flavin-dependent photolyases and cryptochromes harness blue light for photoreduction, enabling repair of UV-damaged DNA and signaling. We investigated the photoreduction dynamics and protonation pathways of the prokaryotic 6-4 photolyase from Caulobacter crescentus (Cc(6-4)) by ultrafast UV/Vis spectroscopy, X-ray crystallography and molecular dynamics. The Cc(6-4) absorption spectrum is dominated by three cofactors: FAD, an [4Fe-4S]²⁺ cluster, and the DLZ antenna. FAD photoreduction uses a bridge-mediated tunneling mechanism, with tyrosine bridging forward electron transfer to FAD from the medial tryptophan of the electron-transfer triad. Subsequent protonation for stabilizing FAD•− is mediated by E402 and a nearby water as shown by protonation blockage in the E402Q mutant. The [4Fe-4S] cluster shows sub-picosecond oxidation, acting as a second light-driven electron injector, but recombines within 1.5 ps for lack of an electron acceptor. Overall, prokaryotic (6-4) photolyases rely on a unique FAD arrangement for bridge-mediated forward electron transfer and a distinct transient protonation pathway.</p>