Free energies of the Gln tautomerization and rotation mechanism of dark-state recovery in blue light-using flavin proteins

Blue light-using flavin (BLUF) proteins are light-sensors that regulate responsive movement, gene expression and enzyme activity in diverse organisms. Their signaling times range from seconds to minutes, indicating a uniquely flexible dark-state recovery mechanism. Unlike other light-sensors, the flavin chromophore is non-covalently bonded to the protein. Hence, the switching occurs via a change in the protein-flavin hydrogen-bond network, involving conserved residues transferring protons, tautomerizing, rotating, and approaching or leaving the chromophore pocket; triggering secondary structure displacements. The specific deactivation steps and residue roles have remained controversial. The detailed process is difficult to probe experimentally, and although simulations can track it, the computational effort is daunting. We combine forefront techniques to simulate, for the first time, explicit dynamics of the deactivation. A hybrid quantum mechanics/molecular mechanics scheme focuses the computational resolution in the flavins vicinity, while our path-based methods sample the mechanism of dark-state recovery with high efficiency. Our protocol delivers free-energy profiles for the deactivation of two BLUF proteins, BlrB and AppA; corroborating a proposed mechanism based on the rotation and tautomerization of a conserved Gln. We find that the conformation of a Trp and a Met near the flavin is crucial to modulate the rate-determining barrier, which differs significantly between the BlrB and AppA proteins. Our work evidences how specific variations of the deactivation mechanism control vast differences in signaling times.