Spectroscopic and Photochemical Characterization of the Red‐Light Sensitive Photosensory Module of Cph2 from Synechocystis PCC 6803

Abstract

Cyanobacterial phytochromes are a diverse family of light receptors controlling various biological functions including phototaxis. In addition to canonical bona fide phytochromes of the well characterized Cph1/plant-like clade, cyanobacteria also harbor phytochromes that absorb green, violet or blue light. The Synechocystis PCC 6803 Cph2 photoreceptor, a phototaxis inhibitor, is unconventional in bearing two distinct chromophore-binding GAF domains. Whereas the C-terminal GAF domain is most likely involved in blue-light perception, the first two domains correspond to a Cph1-like photosensory module lacking the PAS domain. Biochemical and spectroscopic studies show that this region switches between red (P(r) ) and far-red (P(fr) ) absorbing states. Unlike Cph1, the P(fr) state of Cph2 decays rapidly in darkness. Mutations close to the PCB chromophore further destabilize the P(fr) state without drastically affecting the spectroscopic features such as the quantum efficiency of P(r) →P(fr) conversion, fluorescence, or the Resonance-Raman signature of the chromophore. Overall, the PAS-less photosensory module of Cph2 resembles Cph1 including its mode of isomerisation, but the P(fr) state is unstable.

Phototransformation of the Red Light Sensor Cyanobacterial Phytochrome 2 from Synechocystis Species Depends on Its Tongue Motifs

“…Difference spectra are calculated with A Pr Ϫ A Photoequilibrium and shown in blue. Deconvoluted, pure P fr (or red light-adapted) spectra are presented in green (9). At the photoequilibrium, a portion of 59% P fr -like state for S385A, 75% P alt for W389A, and 59% P fr state for W389F are obtained in comparison with SynCph2(1-2) (60% (9)).…”

“…(1-2) and mutants were produced and purified as described (9); the measurements with the PCB-assembled proteins were performed in Tris buffer (50 mM Tris, 300 mM NaCl, 5 mM EDTA, pH 8.0). For flash photolysis, the protein sample was diluted to an absorbance of 0.5 at the P r maximum using a cuvette of a 1-cm path length.…”

“…SynCph2 covalently attaches phycocyanobilin (PCB) via a thioether linkage that is partly solvent-exposed due to the missing PAS domain (8). Similar to plant phytochrome B, but unlike SynCph1 and PhyA, the P fr state of this photosensory module undergoes dark reversion to its P r state (9). Besides the UV-visible spectra, resonance Raman spectra of SynCph2(1-2), which are highly sensitive to structural changes at the chromophore-binding site, resemble closely the spectral features of SynCph1 in both states, P r and P fr .…”

“…The common PAS-GAF-PHY sensor module (PAS (Period/ARNT/Singleminded), GAF (cGMP phosphodiesterase/adenylyl cyclase/ FhlA), and PHY (phytochrome)), which defines canonical group I phytochromes (10), is here altered to a GAF-GAF bidomain (group II phytochromes) as the N-terminal photosensory module. This SynCph2(1-2) module exhibits P r /P fr photochromicity known from group I phytochromes (9), where the PHY domain has been structurally recognized as a GAF domain as well (11). SynCph2 covalently attaches phycocyanobilin (PCB) via a thioether linkage that is partly solvent-exposed due to the missing PAS domain (8).…”

“…First of all, SynCph2 is a bimodule photoreceptor (ϳ145 kDa) with a complex domain architecture, GAF1-GAF2-GGDEF1*-EAL-GAF3-GGDEF2 (8,9). The common PAS-GAF-PHY sensor module (PAS (Period/ARNT/Singleminded), GAF (cGMP phosphodiesterase/adenylyl cyclase/ FhlA), and PHY (phytochrome)), which defines canonical group I phytochromes (10), is here altered to a GAF-GAF bidomain (group II phytochromes) as the N-terminal photosensory module.…”