Femtosecond Photodynamics of the Red/Green Cyanobacteriochrome NpR6012g4 from <i>Nostoc punctiforme</i>. 2. Reverse Dynamics

Phytochromes are red/far-red photosensory proteins that utilize photoisomerization of a linear tetrapyrrole (bilin) chromophore to photoconvert reversibly between red- and far-red-absorbing forms (P_r and P_fr, respectively). Cyanobacteriochromes (CBCRs) are related photosensory proteins with more diverse spectral sensitivity. The mechanisms that underlie this spectral diversity have not yet been fully elucidated. One of the main CBCR subfamilies photoconverts between a red-absorbing 15<i>Z</i> ground state, like the familiar P_r state of phytochromes, and a green-absorbing photoproduct (^15<i>E</i>P_g). We have previously used the red/green CBCR NpR6012g4 from the cyanobacterium <i>Nostoc punctiforme</i> to examine ultrafast photodynamics of the forward photoreaction. Here, we examine the reverse reaction. Using excitation-interleaved transient absorption spectroscopy with broadband detection and multicomponent global analysis, we observed multiphasic excited-state dynamics. Interleaved excitation allowed us to identify wavelength-dependent shifts in the ground-state bleach that equilibrated on a 200 ps time scale, indicating ground-state heterogeneity. Compared to the previously studied forward reaction, the reverse reaction has much faster excited-state decay time constants and significantly higher photoproduct yield. This work thus demonstrates striking differences between the forward and reverse reactions of NpR6012g4 and provides clear evidence of ground-state heterogeneity in the phytochrome superfamily

Femtosecond Photodynamics of the Red/Green Cyanobacteriochrome NpR6012g4 from <i>Nostoc punctiforme</i>. 1. Forward Dynamics

Phytochromes are well-known red/far-red photosensory proteins that utilize the photoisomerization of a linear tetrapyrrole (bilin) chromophore to detect the ratio of red to far-red light. Cyanobacteriochromes (CBCRs) are related photosensory proteins with a bilin-binding GAF domain, but much more diverse spectral sensitivity, with five recognized subfamilies of CBCRs described to date. The mechanisms that underlie this spectral diversity have not yet been fully elucidated. One of the main CBCR subfamilies photoconverts between a red-absorbing ground state, like the familiar P_r state of phytochromes, and a green-absorbing photoproduct (P_g). Here, we examine the ultrafast forward photodynamics of the red/green CBCR NpR6012g4 from the <i>NpR6012</i> locus of the nitrogen-fixing cyanobacterium <i>Nostoc punctiforme</i>. Using transient absorption spectroscopy with broadband detection and multicomponent global analysis, we observed multiphasic excited-state dynamics that induces the forward reaction (red-absorbing to green-absorbing), which we interpret as arising from ground-state heterogeneity. Excited-state decays with lifetimes of 55 and 345 ps generate the primary photoproduct (Lumi-R), and the fastest decay (5 ps) did not produce Lumi-R. Although the photoinduced kinetics of Npr6012g4 is comparable with that of the Cph1 phytochrome isolated from <i>Synechocystis</i> cyanobacteria, NpR6012g4 exhibits a ≥2–3-fold higher photochemical quantum yield. Understanding the structural basis of this enhanced quantum yield may prove to be useful in increasing the photochemical efficiency of other bilin-based photosensors

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

Unraveling the Primary Isomerization Dynamics in Cyanobacterial Phytochrome Cph1 with Multipulse Manipulations

The ultrafast mechanisms underlying the initial photoisomerization (P_r → Lumi-R) in the forward reaction of the cyanobacterial photoreceptor Cph1 were explored with multipulse pump–dump–probe transient spectroscopy. A recently postulated multipopulation model was used to fit the transient pump–dump–probe and dump-induced depletion signals. We observed dump-induced depletion of the Lumi-R photoproduct, demonstrating that photoisomerization occurs via evolution on both the excited- and ground-state electronic surfaces. Excited-state equilibrium was not observed, as shown via the absence of a dump-induced excited-state “Le Châtelier redistribution” of excited-state populations. The importance of incorporating the inhomogeneous dynamics of Cph1 in interpreting measured transient data is discussed

Chemical Inhomogeneity in the Ultrafast Dynamics of the DXCF Cyanobacteriochrome Tlr0924

Cyanobacteriochromes (CBCRs) are diverse biliprotein photosensors distantly related to the red/far-red photoreceptors of the phytochrome family. There are several subfamilies of CBCRs, displaying varied spectral responses spanning the entire visible region. Tlr0924 belongs to the DXCF subfamily that utilizes the Cys residue in a conserved Asp-Xaa-Cys-Phe (DXCF) motif to form a second covalent linkage to the chromophore, resulting in a blue-absorbing dark state. Photoconversion leads to elimination of this linkage, resulting in a green-absorbing photoproduct. Tlr0924 initially incorporates phycocyanobilin (PCB) as a chromophore, exhibiting a blue/orange photocycle, but slowly isomerizes PCB to phycoviolobilin (PVB) to yield a blue/green photocycle. Ultrafast transient absorption spectroscopy was used to study both forward and reverse reaction photodynamics of the recombinant GAF domain of Tlr0924. Primary photoproducts were identified, as were subsequent intermediates at 1 ms. PCB and PVB population photodynamics were decomposed using global target analysis. PCB and PVB populations exhibit similar and parallel photocycles in Tlr0924, but the PVB population exhibits faster excited-state decay in both reaction directions. On the basis of longer time analysis, we show that the photochemical coordinate (15,16-isomerization) and second-linkage coordinate (elimination or bond formation at C10) are separate processes in both directions