Ultrafast Oxidation of a Tyrosine by Proton-Coupled Electron Transfer Promotes Light Activation of an Animal-like Cryptochrome

Abstract

The animal-like cryptochrome of <i>Chlamydomonas reinhardtii</i> (<i>Cr</i>aCRY) is a recently discovered photoreceptor that controls the transcriptional profile and sexual life cycle of this alga by both blue and red light. <i>Cr</i>aCRY has the uncommon feature of efficient formation and longevity of the semireduced neutral form of its FAD cofactor upon blue light illumination. Tyrosine Y₃₇₃ plays a crucial role by elongating , as fourth member, the electron transfer (ET) chain found in most other cryptochromes and DNA photolyases, which comprises a conserved tryptophan triad. Here, we report the full mechanism of light-induced FADH^• formation in <i>Cr</i>aCRY 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 (∼4 and ∼50 ps). Oxidation of Y₃₇₃ 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^•-/Y₃₇₃^• pair is formed with high quantum yield (∼60%); its intrinsic decay by recombination is slow (∼50 ms), favoring reduction of Y₃₇₃^• 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 <i>Cr</i>aCRY, a process about 40 times faster than the archetypal tyrosine-Z oxidation in photosystem II, are discussed in detail.