Dynamics and mechanism of DNA repair by a bifunctional cryptochrome

Photolyase and cryptochrome belong to a group of structurally similar flavoproteins but with two distinct functions of DNA repair as a photoenzyme and signal transduction as a photoreceptor, respectively, under blue-light illumination. Here, we studied a recently discovered bifunctional Chlamydomonas reinhardtii cryptochrome (CraCRY) with focus on its repair of UV-induced pyrimidine-pyrimidone (6-4) photoproduct (6-4PP). We used femtosecond spectroscopy and site-directed mutagenesis to map out the critical elementary steps by following the dynamics of initial reactants, various intermediates, and final products. We observed initial direct ultrafast electron tunneling from the hydroquinone flavin cofactor to 6-4PP in 300 ps through an intervening adenine as a mediator, minimizing the electron bifurcation of a two-step hopping pathway bridged also by the adenine as an intermediate. The subsequent proton transfer (PT) from the neighboring histidine to anionic 6-4PP in 2 ns is critical and competes with the futile back electron transfer in 151 ps. Mutations of either of two histidines in the active site nearly abolish repair, indicating their essential role on repair reactivity and structural integrity. These results elucidate the electron-coupled PT mechanism and the repair photocycle of this bifunctional cryptochrome at the molecular level, further supporting that the hydroquinone flavin is the active state in vivo for dual functions or one of the two functions is excessive.