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

Noncanonical Photodynamics of the Orange/Green Cyanobacteriochrome Power Sensor NpF2164g7 from the PtxD Phototaxis Regulator of <i>Nostoc punctiforme</i>

Forward and reverse primary (<10 ns) and secondary (>10 ns) photodynamics of cyanobacteriochrome (CBCR) NpF2164g7 were characterized by global analysis of ultrafast broadband transient absorption measurements. NpF2164g7 is the most C-terminal bilin-binding GAF domain in the <i>Nostoc punctiforme</i> phototaxis sensor PtxD (locus Npun_F2164). Although a member of the canonical red/green CBCR subfamily phylogenetically, NpF2164g7 exhibits an orange-absorbing ^<i>15Z</i>P_o dark-adapted state instead of the typical red-absorbing ^<i>15Z</i>P_r dark-adapted state characteristic of this subfamily. The green-absorbing ^<i>15E</i>P_g photoproduct of NpF2164g7 is unstable, allowing this CBCR domain to function as a power sensor. Photoexcitation of the ^<i>15Z</i>P_o state triggers inhomogeneous excited-state dynamics with three spectrally and temporally distinguishable pathways to generate the light-adapted ^<i>15E</i>P_g state in high yield (estimated at 25–30%). Although observed in other CBCR domains, the inhomogeneity in NpF2164g7 extends far into secondary relaxation dynamics (10 ns −1 ms) through to formation of ^<i>15E</i>P_g. In the reverse direction, the primary dynamics after photoexcitation of ^<i>15E</i>P_g are qualitatively similar to those of other red/green CBCRs, but secondary dynamics involve a “pre-equilibrium” step before regenerating ^<i>15Z</i>P_o. The anomalous photodynamics of NpF2164g7 may reflect an evolutionary adaptation of CBCR sensors that function as broadband light intensity sensors

Primary and Secondary Photodynamics of the Violet/Orange Dual-Cysteine NpF2164g3 Cyanobacteriochrome Domain from <i>Nostoc punctiforme</i>

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors distantly related to phytochromes. Like phytochromes, CBCRs photointerconvert between two photostates that accompany photoisomerization of their bilin chromophores. While phytochromes typically exhibit red/far-red photocycles, CBCR photocycles are much more diverse, spanning the near-ultraviolet and the entire visible region. All CBCRs described to date have a conserved Cys residue covalently attached to the linear tetrapyrrole (bilin) chromophore; two CBCR subfamilies also exploit a second thioether linkage to the chromophore for detection of near-ultraviolet to blue light. Here, we present the photodynamic analysis of the insert-Cys CBCR NpF2164g3, a representative of the second class of two-cysteine CBCRs. Using broadband transient absorption pump–probe spectroscopy, we characterize the primary (100 fs to 10 ns) and secondary (10 ns to 1 ms) photodynamics in both directions, examining photodynamics over nine decades of time. Primary isomerization dynamics occur on a ∼10 ps time scale for both forward and reverse reactions. In contrast to previous studies on Tlr0924, a representative of the other class of two-cysteine CBCRs, formation and elimination of the second linkage are slower than the 1 ms experimental range probed here. These results extend our understanding of dual-cysteine CBCR photocycles in the phytochrome superfamily