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

Abstract

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 Fremyella diplosiphon. 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 ((15Z)Pg → (15E)Pr), which we interpret as arising from ground-state inhomogeneity with different tautomers of the PCB chromophore. The reverse reaction ((15E)Pr → (15Z)Pg) 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.

Heterogeneous Photodynamics of the Pfr State in the Cyanobacterial Phytochrome Cph1

“…Other transient absorption studies of phytochromes and CBCRs have also reported multiphasic excited-state relaxation, typically interpreted as arising from coevolving subpopulations. 33 , 34 , 45 , 47 , 52 This is consistent with characterization of other photoreceptor systems such as PYP and phototropin, which also exhibit multiexponential kinetics attributed to inhomogeneity. 46 , 53 …”

“…The biphasic decay of ESA, the sequential analysis described above, and the simulation of P fr CD spectra 32 argue for the adoption of a parallel evolving target model for interpreting the narrowband transient data (Figure 8 A). By analogy to other phytochrome and CBCR systems, 21 , 22 , 33 , 34 , 45 we modeled this as arising because of an inhomogeneous ground state rather than bifurcation on a homogeneous population on a excited-state surface. In the absence of evidence to the contrary, we assumed that the two subpopulations possess identical ground-state and excited-state spectra.…”

“… 32 There is growing evidence that observed multiphasic excited-state dynamics in phytochromes and related PCB-incorporating GAF domains cyanobacteriochrome (CBCR) photosensors arises due to ground-state heterogeneity. 21 , 33 , 34 In recent temperature- and excitation wavelength-dependent studies of forward photoconversion of Cph1Δ, 21 we resolved five P r * excited-state decay pathways arising from photoactive ( Photo P r ) and blue-shifted fluorescent ( Fluor P r ) subpopulations (Figure 1 B, red and blue curves, respectively). Photo P r productively formed the primary Lumi-R F photoproduct on a 20 ps timescale, whereas Fluor P r did not yield Lumi-R F and exhibited longer excited-state lifetimes.…”

Tracking the secondary photodynamics of the green/red cyanobacteriochrome RcaE from Fremyella diplosiphon

“…The 15Z P g absorption spectrum (green curve) peaks in the green region with a non-Gaussian spectral lineshape that is attributed to two tautomers of the PCB chromophore with either the B-or C-ring deprotonated 39 that coexist in equilibrium.…”

“…The primary forward dynamics are strongly controlled by the inhomogeneity of the sample, ascribed in part to the two tautomers of the PCB chromophore. 15,16,39 Of the three primary Lumi photoproducts formed after excitation …”