We previously characterized a LOV protein PpSB2-LOV, present in the common soil bacterium Pseudomonas putida, that exhibits a plant phototropin LOV-like photochemistry [Krauss, U., Losi, A., Gartner, W., Jaeger, K. E., and Eggert, T. (2005) Phys. Chem. Chem. Phys. 7, 2804-2811]. Now, we have identified a second LOV homologue, PpSB1-LOV, found in the same organism with approximately 66% identical amino acids. Both proteins consist of a conserved LOV core flanked by short N- and C-terminal extensions but lack a fused effector domain. Although both proteins are highly similar in sequence, they display drastically different dark recovery kinetics. At 20 degrees C, PpSB2-LOV reverts with an average time constant of 137 s from the photoequilibrium to the dark state, whereas PpSB1-LOV exhibits an average dark recovery time constant of 1.48 x 10(5) s. Irrespective of the significant differences in their dark recovery behavior, both proteins showed nearly identical kinetics for the photochemically induced adduct formation. In order to elucidate the structural and mechanistic basis of these extremely different dark recovery time constants, we performed a mutational analysis. Six amino acids in a distance of up to 6 A from the flavin chromophore, which differ between the two proteins, were identified and interchanged by site-directed mutagenesis. The amino acid substitution R66I located near the FMN phosphate in LOV domains was identified in PpSB1-LOV to accelerate the dark recovery by 2 orders of magnitude. Vice versa, the corresponding substitution I66R slowed down the dark recovery in PpSB2-LOV by a factor of 10. Interestingly, the interchange of the C-terminal extensions between the two proteins also had a pronounced effect on the dark recovery time constants, thus highlighting a coupling of these protein regions to the chromophore binding pocket.