Dissecting the Ultrafast Stepwise Bidirectional Proton Relay in a Blue-Light Photoreceptor.

Proton relays through H-bond networks are essential in realizing the functionality of protein machines such as in photosynthesis and photoreceptors. It has been challenging to dissect the rates and energetics of individual proton-transfer steps during the proton relay. Here, we have designed a proton rocking blue light using a flavin (BLUF) domain with the flavin mononucleotide (FMN)-glutamic acid (E)-tryptophan (W) triad and have resolved the four individual proton-transfer steps kinetically using ultrafast spectroscopy. We have found that after the photo-induced charge separation forming FMN · - /E-COOH/WH · + , the proton first rapidly jumps from the bridging E-COOH to FMN - (τ fPT2 = 3.8 ps; KIE = 1.0), followed by a second proton transfer from WH · + to E-COO - (τ fPT1 = 336 ps; KIE = 2.6) which immediately rocks back to W · (τ rPT1 = 85 ps; KIE = 6.7), followed by a proton return from FMNH · to E-COO - (τ rPT2 = 34 ps; KIE = 3.3) with the final charge recombination between FMN · - and WH · + to close the reaction cycle. Our results revisited the Grotthuss mechanism on the ultrafast timescale using the BLUF domain as a paradigm protein.