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

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

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