What accounts for the different functions in photolyases and cryptochromes: a computational study of proton transfers to FAD.

Photolyases (PL) and cryptochromes (CRY) are light-sensitive flavoproteins, respectively, involved in DNA repair and signal transduction. Their activation is triggered by an electron transfer process, which partially or fully reduces the photo-activated FAD cofactor. The full reduction additionally requires a proton transfer to the isoalloxazine ring. In plant CRY, an efficient proton transfer takes place within several μs, enabled by a conserved aspartate working as a proton donor, whereas in E. coli PL a proton transfer occurs in the 4 s timescale without any obvious proton donor, indicating the presence of a long-range proton transfer pathway. Unexpectedly, the insertion of an aspartate as a proton donor in a suitable position for proton transfer in E. coli PL does not initiate a transfer process similar to plant CRY, but even prevents the formation of a protonated FAD. In the present work, thanks to a combination of classical molecular dynamics and state-of-the-art DFTB3/MM simulations, we identify a proton transfer pathway from bulk to FAD in E. coli PL associated with a free energy profile in agreement with the experimental kinetics data. The free energy profiles of the proton transfer between aspartate and FAD show an inversion of the driving force between plant CRY and E. coli PL mutants. In the latter, the proton transfer from the aspartate is faster than in plant CRY but also thermodynamically disfavoured, in agreement with the experimental data. Our results further illustrate the fine tuning of the electrostatic FAD environment and the adaptability of the FAD pocket to ensure the divergent functions of the members of the PL-CRY family.