Abstract
<jats:title>Abstract</jats:title> <jats:p> Light-oxygen-voltage (LOV) domains are blue-light photoreceptors of plants, algae and fungi, and among the most widely used tools in optogenetics. They switch on by forming a covalent thioether bond between a conserved cysteine and their flavin chromophore, in a reaction that needs a proton to cross from the cysteine to the flavin through a pocket containing essentially no water. Its mechanism has been debated for two decades <jats:sup>1</jats:sup> , and because the chemistry is over within a microsecond its elementary steps have stayed hidden. Here we combine 10 time-resolved serial femtosecond crystallography snapshots and infrared spectroscopy with QM/MM calculations to resolve the entire sequence of events at 1.4 Å resolution: from excited-state distortion of the flavin ring (10–100 ps), through hydration of a surface channel (10 ns) and a single ordered water reaching the active site as the reactive cysteine shifts between its conformations (100–500 ns), to the thioether bond itself, caught half-formed at 1 µs (half the molecules reacted, half still poised) and complete at 10–100 µs. That water bridges the cysteine and the flavin and shuttles the proton, lowering the barrier from ∼35 to ∼15 kcal/mol and accelerating the reaction by roughly fourteen orders of magnitude (without it, the half-life would be ∼237,000 years), then departs before the bond forms. Proteins can therefore hydrate a dehydrated active site transiently and on demand to overcome otherwise prohibitive reaction barriers, a catalytic strategy that reaches well beyond photoreceptors. </jats:p>