Primary Photochemistry of the Dark- and Light-Adapted States of the YtvA Protein from <i>Bacillus subtilis</i>

The primary (100 fs to 10 ns) and secondary (10 ns to 100 μs) photodynamics in the type II light–oxygen–voltage (LOV) domain from the blue light YtvA photoreceptor extracted from <i>Bacillus subtilis</i> were explored with transient absorption spectroscopy. The photodynamics of full-length YtvA were characterized after femtosecond 400 nm excitation of both the dark-adapted D₄₄₇ state and the light-adapted S₃₉₀ state. The S₃₉₀ state relaxes on a 43 min time scale at room temperature back into D₄₄₇, which is weakly accelerated by the introduction of imidazole. This is ascribed to an obstructed cavity in YtvA that hinders access to the embedded FMN chromophore and is more open in type I LOV domains. The primary photochemistry of dark-adapted YtvA is qualitatively similar to that of the type I LOV domains, including AsLOV2 from <i>Avena sativa</i>, but exhibits an appreciably higher (60% greater) terminal triplet yield, estimated near the maximal Φ_ISC value of ≈78%; the other 22% decays via non-triplet-generating fluorescence. The subsequent secondary dynamics are inhomogeneous, with three triplet populations co-evolving: the faster-decaying ^IT* population (38% occupancy) with a 200 ns decay time is nonproductive in generating the S₃₉₀ adduct state, a slower ^IIT* population (57% occupancy) exhibits a high yield (Φ_adduct ≈ 100%) in generating S₃₉₀ and a third (5%) ^IIIT*population persists (>100 μs) with unresolved photoactivity. The ultrafast photoswitching dynamics of the S₃₉₀ state appreciably differ from those previously resolved for the type I AcLOV2 domain from <i>Adiantum capillus-veneris</i> [Kennis, J. T., et al. (2004) <i>J. Am. Chem. Soc. 126</i>, 4512], with a low-yield dissociation (Φ_dis ≈ 2.5%) reaction, which is due to an ultrafast recombination reaction, following photodissociation, and is absent in AcLOV2, which results in the increased photoswitching activity of the latter domain

Subpicosecond Excited-State Proton Transfer Preceding Isomerization During the Photorecovery of Photoactive Yellow Protein

The ultrafast excited-state dynamics underlying the receptor state photorecovery is resolved in the M100A mutant of the photoactive yellow protein (PYP) from Halorhodospira halophila. The M100A PYP mutant, with its distinctly slower photocycle than wt PYP, allows isolation of the pB signaling state for study of the photodynamics of the protonated chromophore <i>cis-p</i>-coumaric acid. Transient absorption signals indicate a subpicosecond excited-state proton-transfer reaction in the pB state that results in chromophore deprotonation prior to the cis−trans isomerization required in the photorecovery dynamics of the pG state. Two terminal photoproducts are observed, a blue-absorbing species presumed to be deprotonated <i>trans-p</i>-coumaric acid and an ultraviolet-absorbing protonated photoproduct. These two photoproducts are hypothesized to originate from an equilibrium of open and closed folded forms of the signaling state, I₂ and I₂′