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

Abstract

The RcaE cyanobacteriochrome uses a linear tetrapyrrole chromophore to sense the ratio of green and red light to enable the Fremyella diplosiphon 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-Go was depleted, Lumi-Gr was unaffected, and Lumi-Gf was enhanced. This suggests that RcaE may be engineered to act either as an in vivo fluorescent probe (after single-pump excitation) or as an in vivo optogenetic sample (after pump and dump excitation).

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

“…The subsequent 100 ns -100 μs spectra shift slightly in peak wavelength and amplitude (Figure 2A; inset) before eventually evolving into exhibit comparable dynamics, the Lumi-G f population ( Figure 2D) 15, 16 exhibits a unique ~5 μs decay timescale, indicating it independently evolves as implied by previous interpretations of the inhomogeneous primary dynamics.…”

“…the multicomponent global analysis formalism that has been successfully applied to characterizing the photodynamics of other CBCR systems. 16,22,23,[26][27][28][35][36][37] The complete methodology of this approach is described elsewhere, 38 but the gist involves decomposing the transient data into a set of distinct populations with time-independent spectra and timedependent amplitudes. For the data discussed here, the constituent populations are assumed to evolve via activated barrier-crossing dynamics, which can be described by first-order kinetics:…”

“…Depending on the spectral properties of the incident light and the secondary dynamics (>10 ns) coupling these primary dynamics to the terminal 15,16,22,23,[25][26][27] the secondary dynamics (10 ns -10 ms) of CBCRs have attracted less attention, with only four so characterized: AnPixJg2 from Nostoc sp. strain PCC 7120, 29,30 Slr1393g3 from Synechocystis sp.…”

“…Since the 550-ps instrument response is appreciably slower than the excited-state 15Z P g * kinetics, 15,16 the measured spectra primarily track the subsequent ground-state intermediates. These spectra are the combination of a negative absorption from a ground-state bleach (GSB, 450-560 nm) due to loss of the 15Z P g ground-state population and positive absorption from new photoproduct bands (560-710 nm).…”

“…These spectra are the combination of a negative absorption from a ground-state bleach (GSB, 450-560 nm) due to loss of the 15Z P g ground-state population and positive absorption from new photoproduct bands (560-710 nm). The 2 ns spectrum (black curve) peaks at 593 nm with a long wavelength tail 15,16 and consists of the Lumi-G o (~595 nm), Lumi-G r (~630 nm), and Lumi-G f (~700 nm) primary photoproducts. This spectrum then evolves into a blue-shifted spectrum peaking at ~575 nm with a substantial loss of amplitude of the red portion of the spectrum (10 ns spectrum; red curve); this evolution is primarily ascribed to the decay of Lumi-G r .…”