Light-Regulated Synthesis of Cyclic-di-GMP by a Bidomain Construct of the Cyanobacteriochrome Tlr0924 (SesA) without Stable Dimerization

Phytochromes and cyanobacteriochromes (CBCRs) use double-bond photoisomerization of their linear tetrapyrrole (bilin) chromophores within cGMP-specific phosphodiesterases/adenylyl cyclases/FhlA (GAF) domain-containing photosensory modules to regulate activity of C-terminal output domains. CBCRs exhibit photocycles that are much more diverse than those of phytochromes and are often found in large modular proteins such as Tlr0924 (SesA), one of three blue light regulators of cell aggregation in the cyanobacterium <i>Thermosynechococcus elongatus</i>. Tlr0924 contains a single bilin-binding GAF domain adjacent to a C-terminal diguanylate cyclase (GGDEF) domain whose catalytic activity requires formation of a dimeric transition state presumably supported by a multidomain extension at its N-terminus. To probe the structural basis of light-mediated signal propagation from the photosensory input domain to a signaling output domain for a representative CBCR, these studies explore the properties of a bidomain GAF–GGDEF construct of Tlr0924 (Tlr0924Δ) that retains light-regulated diguanylate cyclase activity. Surprisingly, circular dichroism spectroscopy and size exclusion chromatography data do not support formation of stable dimers in either the blue-absorbing ^15<i>Z</i>P_b dark state or the green-absorbing ^15<i>E</i>P_g photoproduct state of Tlr0924Δ. Analysis of variants containing site-specific mutations reveals that proper signal transmission requires both chromophorylation of the GAF domain and individual residues within the amphipathic linker region between GAF and GGDEF domains. On the basis of these data, we propose a model in which bilin binding and light signals are propagated from the GAF domain via the linker to alter the equilibrium and interconversion dynamics between active and inactive conformations of the GGDEF domain to favor or disfavor formation of catalytically competent dimers

Protonation Heterogeneity Modulates the Ultrafast Photocycle Initiation Dynamics of Phytochrome Cph1

Phytochrome proteins utilize ultrafast photoisomerization of a linear tetrapyrrole chromophore to detect the ratio of red to far-red light. Femtosecond photodynamics in the PAS-GAF-PHY photosensory core of the Cph1 phytochrome from <i>Synechocystis</i> sp. PCC6803 (Cph1Δ) were resolved with a dual-excitation-wavelength-interleaved pump–probe (DEWI) approach with two excitation wavelengths (600 and 660 nm) at three pH values (6.5, 8.0, and 9.0). Observed spectral and kinetic heterogeneity in the excited-state dynamics were described with a self-consistent model comprised of three spectrally distinct populations with different protonation states (P_r-I, P_r-II, and P_r-III), each composed of multiple kinetically distinct subpopulations. Apparent partitioning among these populations is dictated by pH, temperature, and excitation wavelength. Our studies provide insight into photocycle initiation dynamics at physiological temperatures, implicate the low-pH/low-temperature P_r-I state as the photoactive state <i>in vitro</i>, and implicate an internal hydrogen-bonding network in regulating the photochemical quantum yield

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

Reactive Ground-State Pathways Are Not Ubiquitous in Red/Green Cyanobacteriochromes

Recent characterization of the red/green cyanobacteriochrome (CBCR) NpR6012g4 revealed a high quantum yield for its forward photoreaction [J. Am. Chem. Soc. 2012, 134, 130−133] that was ascribed to the activity of hidden, productive ground-state intermediates. The dynamics of the pathways involving these ground-state intermediates was resolved with femtosecond dispersed pump–dump–probe spectroscopy, the first such study reported for any CBCR. To address the ubiquity of such second-chance initiation dynamics (SCID) in CBCRs, we examined the closely related red/green CBCR NpF2164g6 from <i>Nostoc punctiforme</i>. Both NpF2164g6 and NpR6012g4 use phycocyanobilin as the chromophore precursor and exhibit similar excited-state dynamics. However, NpF2164g6 exhibits a lower quantum yield of 32% for the generation of the isomerized Lumi-R primary photoproduct, compared to 40% for NpR6012g4. This difference arises from significantly different ground-state dynamics between the two proteins, with the SCID mechanism deactivated in NpF2164g6. We present an integrated inhomogeneous target model that self-consistently fits the pump–probe and pump–dump–probe signals for both forward and reverse photoreactions in both proteins. This work demonstrates that reactive ground-state intermediates are not ubiquitous phenomena in CBCRs

Optically Guided Photoactivity: Coordinating Tautomerization, Photoisomerization, Inhomogeneity, and Reactive Intermediates within the RcaE Cyanobacteriochrome

The RcaE cyanobacteriochrome uses a linear tetrapyrrole chromophore to sense the ratio of green and red light to enable the <i>Fremyella diplosiphon</i> cyanobacterium to control the expression of the photosynthetic infrastructure for efficient utilization of incident light. The femtosecond photodynamics of the embedded phycocyanobilin chromophore within RcaE were characterized with dispersed femtosecond pump–dump–probe spectroscopy, which resolved a complex interplay of excited-state proton transfer, photoisomerization, multilayered inhomogeneity, and reactive intermediates. These reactions were integrated within a central model that incorporated a rapid (200 fs) excited-state Le Châtelier redistribution between parallel evolving populations ascribed to different tautomers. Three photoproducts were resolved and originates from four independent subpopulations, each with different dump-induced behavior: Lumi-G_o was depleted, Lumi-G_r was unaffected, and Lumi-G_f was enhanced. This suggests that RcaE may be engineered to act either as an <i>in vivo</i> fluorescent probe (after single-pump excitation) or as an <i>in vivo</i> optogenetic sample (after pump and dump excitation)

Primary Photodynamics of the Green/Red-Absorbing Photoswitching Regulator of the Chromatic Adaptation E Domain from <i>Fremyella diplosiphon</i>

Phytochromes are red/far-red photosensory proteins that utilize the photoisomerization of a linear tetrapyrrole (bilin) chromophore to detect the red to far-red light ratio. Cyanobacteriochromes (CBCRs) are distantly related cyanobacterial photosensors with homologous bilin-binding GAF domains, but they exhibit greater spectral diversity. Different CBCR subfamilies have been described, with spectral sensitivity varying across the near-ultraviolet and throughout the visible spectrum, but all known CBCRs utilize photoisomerization of the bilin 15,16-double bond as the primary photochemical event. The first CBCR discovered was RcaE, responsible for tuning light harvesting to the incident color environment (complementary chromatic adaptation) in <i>Fremyella diplosiphon</i>. The green/red RcaE photocycle has recently been described in detail. We now extend this analysis by examining femtosecond photodynamics using ultrafast transient absorption techniques with broadband detection and multicomponent global analysis. Excited-state dynamics in both directions are significantly slower than those recently published for the red/green CBCR NpR6012g4. In the forward reaction, the primary Lumi-G photoproduct arises from the longer-lived excited-state populations, leading to a low photoproduct quantum yield. Using dual-excitation wavelength interleaved pump–probe spectroscopy, we observe multiphasic excited-state dynamics in the forward reaction (^15<i>Z</i>P_g → ^15<i>E</i>P_r), which we interpret as arising from ground-state inhomogeneity with different tautomers of the PCB chromophore. The reverse reaction (^15<i>E</i>P_r → ^15<i>Z</i>P_g) is characterized via pump–probe spectroscopy and also exhibits slow excited-state decay dynamics and a low photoproduct yield. These results provide the first description of excited-state dynamics for a green/red CBCR

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

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

Conservation and Diversity in the Primary Forward Photodynamics of Red/Green Cyanobacteriochromes

Phytochromes are red/far-red photosensory proteins that detect the ratio of red to far-red light. Crucial to light regulation of plant developmental biology, phytochromes are also found in fungi, bacteria, and eukaryotic algae. In addition to phytochromes, cyanobacteria also can contain distantly related cyanobacteriochromes (CBCRs) that, like phytochromes, utilize the photoisomerization of a linear tetrapyrrole (bilin) chromophore to convert between two photostates with distinct spectral properties. CBCRs exhibit a wide range of photostates spanning the visible and even near-ultraviolet spectrum. In both phytochromes and CBCRs, biosynthesis initially yields a holoprotein with bilin in the 15<i>Z</i> configuration, and the 15<i>E</i> photoproduct can often revert to the 15<i>Z</i> photostate in the absence of light (dark reversion). One CBCR subfamily, red/green CBCRs, typically exhibits red-absorbing dark states and green-absorbing photoproducts. Dark reversion is extremely variable in red/green CBCRs with known examples ranging from seconds to days. One red/green CBCR, NpR6012g4 from <i>Nostoc punctiforme,</i> is also known to exhibit forward photoconversion that has an unusually high quantum yield at ∼40% compared to 10–20% for phytochromes and CBCRs from other subfamilies. In the current study, we use time-resolved pump-probe absorption spectroscopy with broadband detection and multicomponent global analysis to characterize forward photoconversion of seven additional red/green CBCRs from <i>N. punctiforme</i> on an ultrafast time scale. Our results reveal that red/green CBCRs exhibit a conserved pathway for primary forward photoconversion but that considerable diversity exists in their excited-state lifetimes, photochemical quantum yields, and primary photoproduct stabilities