Light-Oxygen-Voltage (LOV) proteins show considerable variation in the lifetime of the adduct state. Here, we used a comparative approach selecting two homologous LOV proteins originating from Pseudomonas fluorescens – SBW25-LOV and Pf5-LOV. At 37°C, SBW25-LOV and Pf5-LOV exhibit adduct state lifetimes of 1470 min and 3.6 min, respectively. The cartoon representation on the left shows the light-dependent global structural changes in the dimeric short LOV proteins of Pseudomonadaceae, such as the rotation of the core domains relative to each other and the increased distances at the N- and C-terminal junctions. Display omitted •Short LOV (Light, Oxygen, Voltage) proteins from Pseudomonas fluorescens.•Crystal structures of SBW25-LOV (fully light-adapted), and Pf5-LOV (dark) state.•Light-induced rotation of core domains and splaying of N- and C-terminal helices.•Three non-conserved residues identified as crucial for tuning dark recovery kinetics.•Signal transduction mechanisms among Pseudomonadaceae LOV proteins is conserved. Light-Oxygen-Voltage (LOV) flavoproteins transduce a light signal into variable signaling outputs via a structural rearrangement in the sensory core domain, which is then relayed to fused effector domains via α-helical linker elements. Short LOV proteins from Pseudomonadaceae consist of a LOV sensory core and N- and C-terminal α-helices of variable length, providing a simple model system to study the molecular mechanism of allosteric activation. Here we report the crystal structures of two LOV proteins from Pseudomonas fluorescens - SBW25-LOV in the fully light-adapted state and Pf5-LOV in the dark-state. In a comparative analysis of the Pseudomonadaceae short LOVs, the structures demonstrate light-induced rotation of the core domains and splaying of the proximal A′α and Jα helices in the N and C-termini, highlighting evidence for a conserved signal transduction mechanism. Another distinguishing feature of the Pseudomonadaceae short LOV protein family is their highly variable dark recovery, ranging from seconds to days. Understanding this variability is crucial for tuning the signaling behavior of LOV-based optogenetic tools. At 37 °C, SBW25-LOV and Pf5-LOV exhibit adduct state lifetimes of 1470 min and 3.6 min, respectively. To investigate this remarkable difference in dark recovery rates, we targeted three residues lining the solvent channel entrance to the chromophore pocket where we introduced mutations by exchanging the non-conserved amino acids from SBW25-LOV into Pf5-LOV and vice versa. Dark recovery kinetics of the resulting mutants, as well as MD simulations and solvent cavity calculations on the crystal structures suggest a correlation between solvent accessibility and adduct lifetime.