The animal-like cryptochrome of Chlamydomonas reinhardtii (CraCRY) is a recently discovered photoreceptor that controls the transcriptional profile and sexual life cycle of this alga by both blue and red light. CraCRY has the uncommon feature of efficient formation and longevity of the semi-reduced neutral form of its FAD cofactor upon blue light illumination. Tyrosine Y 373 plays hereby a crucial role by elongating as fourth member the electron transfer (ET) chain that comprises the tryptophan triad otherwise found in most other cryptochromes and DNA photolyases. Here, we report the full mechanism of light-induced FADH • formation in CraCRY using transient absorption spectroscopy from hundreds of femtoseconds to seconds. Electron transfer starts from ultrafast reduction of excited FAD to FAD •by the proximal tryptophan (0.4 ps) and is followed by delocalized migration of the produced WH •+ radical along the tryptophan triad (3.7 and 55 ps). Oxidation of Y 373 by coupled ET to WH •+ and deprotonation then proceeds in ~800 ps, without any significant kinetic isotope effect, nor a pH effect between pH 6.5 and 9.0. The FAD •-/Y 373 • pair is formed with high quantum yield (~60%); its intrinsic decay by recombination is slow (~50 ms), favoring reduction of Y 373 • by extrinsic agents and protonation of FAD •to form the long-lived, red-light absorbing FADH • species. Possible mechanisms of tyrosine oxidation by ultrafast proton-coupled ET in CraCRY, a process about 40 times faster than the archetypal tyrosine-Z oxidation in photosystem II, are discussed in detail.

Photolyases are flavoenzymes repairing UV-induced lesions in DNA, which may be activated by a photoreduction of their FAD cofactor. In most photolyases, this photoreduction proceeds by electron transfer along a chain of three tryptophan (Trp) residues, connecting the flavin to the protein surface. Much less studied, animal (6-4) photolyases (repairing pyrimidine-pyrimidone (6-4) photoproducts) are particularly interesting as they were recently shown to have a longer electron transfer chain, counting four Trp residues. Using femtosecond polarized transient absorption spectroscopy, we performed a detailed analysis of the photoactivation reaction in the (6-4) photolyase of Xenopus laevis with oxidized FAD. We showed that the excited flavin is very quickly reduced (∼0.5 ps) by a nearby tryptophan residue, yielding FAD˙ and WH˙ radicals. Subsequent kinetic steps in the picosecond regime were assigned to the migration of the positive charge along the Trp tetrad, in competition with charge recombination. We propose that the positive charge is actually delocalized over various Trp residues during most of the dynamics and that charge recombination essentially occurs through the proximal tryptophanyl radical. Oxidation of the fourth tryptophan is thought to be reached about as fast as that of the third one (∼40 ps), based on a comparison with a mutant protein lacking the distal Trp, implying ultrafast electron transfer between these two residues. This unusual mechanism sheds light on the rich diversity of electron transfer pathways found in various photolyases, and evolution-related cryptochromes alike.