Structural determinants underlying the adduct lifetime in the LOV proteins of <i>Pseudomonas putida</i>

The primary photochemistry is similar among the flavin-bound sensory domains of light–oxygen–voltage (LOV) photoreceptors, where upon blue-light illumination a covalent adduct formed on microseconds time scale between flavin chromophore and strictly conserved cysteine residue. In contrast, adduct-state decay kinetics vary from seconds to days or longer. molecular basis for this variation structurally LOV not fully understood. Here, we selected PpSB2-LOV, fast-cycling (τrec 3.5 min, 20 °C) short protein Pseudomonas putida that shares 67% sequence identity with slow-cycling 2467 homologous PpSB1-LOV. Based crystal structure PpSB2-LOV in dark state reported here, used comparative approach, which combined information dynamic (MD) simulations address mechanistic vastly different lifetimes two proteins. MD pointed toward dynamically distinct structural region, were subsequently targeted by site-directed mutagenesis introduced single- multisite substitutions exchanging them corresponding residues Collectively, data presented identify key amino acids Aβ-Bβ, Eα-Fα loops, Fα helix, such as E27 I66, play decisive role determining lifetime. Our results additionally suggest correlation solvent accessibility pocket add our understanding signaling will have important implications tuning behavior (on/off kinetics) LOV-based optogenetic tools.