Origin of the multi-phasic quenching dynamics in the BLUF domains across the species.

Abstract

Blue light using flavin (BLUF) photoreceptors respond to light via one of nature's smallest photo-switching domains. Upon photo-activation, the flavin cofactor in the BLUF domain exhibits multi-phasic dynamics, quenched by a proton-coupled electron transfer reaction involving the conserved Tyr and Gln. The dynamic behavior varies drastically across different species, the origin of which remains controversial. Here, we incorporate site-specific fluorinated Trp into three BLUF proteins, i.e., AppA, OaPAC and SyPixD, and characterize the percentages for the W_out, W_inNH_in and W_inNH_out conformations using ¹⁹F nuclear magnetic resonance spectroscopy. Using femtosecond spectroscopy, we identify that one key W_inNH_in conformation can introduce a branching one-step proton transfer in AppA and a two-step proton transfer in OaPAC and SyPixD. Correlating the flavin quenching dynamics with the active-site structural heterogeneity, we conclude that the quenching rate is determined by the percentage of W_inNH_in, which encodes a Tyr-Gln configuration that is not conducive to proton transfer.