The Photoconversion of Phytochrome Includes an Unproductive Shunt Reaction Pathway

Abstract

Phytochromes are modular bimodal photoswitches that control gene expression for morphogenetic processes in plants. These functions are triggered by photoinduced conversions between the inactive and active states of the photosensory module, denoted as Pr and Pfr, respectively. In the present time-resolved resonance Raman spectroscopic study of bacterial representatives of this photoreceptor family, we demonstrate that these phototransformations do not represent linear processes but include a branching reaction back to the initial state, prior to (de)activation of the output module. Thus, only a fraction of the photoreceptors undergoing the phototransformations can initiate the downstream signaling process, consistent with phytochrome's function as a sensor for more durable changes of light conditions.

Asymmetric activation mechanism of a homodimeric red light-regulated photoreceptor

“…At the same time, the fast reverting meta-R component is only slightly influenced by the conformational dynamics at the dimer interface. Most likely, the molecular mechanism of meta-R recovery follows a shunt-pathway similar to that described for Agrobacterium fabrum , Agp1 and Agp2 ( Buhrke et al, 2018 ). The different effects of the dimer interface on the recovery of the different biliverdin species are an additional indication for the structural asymmetry of light-activated Is PadC.…”

“…Interestingly, also the relative amplitudes contributing to individual phases at 710 and 750 nm appear to correlate for all constructs. While this observation might suggest that the recoveries do not involve photoproducts that significantly differ in their spectra from the P r or P fr absorbing species, it does not exclude the presence of closely related species such as for example meta-R states ( Borucki et al, 2005 ; Buhrke et al, 2018 ; Wagner et al, 2008 ). The observation of a non-isosbestic recovery ( Figure 1—figure supplement 2e ) further supports the involvement of at least one colored species in addition to P r and P fr .…”

“…Figure 1c ). This is either due to different biliverdin environments of the two protomers forming an Is PadC dimer or due to a steady-state equilibrium of P fr with preceding intermediates along the activation pathway ( Borucki et al, 2005 ; Buhrke et al, 2018 ; Dasgupta et al, 2009 ). By following the thermal reversion of 15 E biliverdin to its 15 Z isomer by means of quenching the protein after different dark adaptation times, mean lifetimes and amplitudes are observed that are comparable to those obtained for the native protein (inset of Figure 1e ), which indicates a direct correlation of the native spectral properties with the isomerization state of biliverdin.…”

Distinct chromophore–protein environments enable asymmetric activation of a bacteriophytochrome-activated diguanylate cyclase

“…Either the Meta-Rc or the Pfr-like state (or both) constitutes a stage of bifurcation. Previous timeresolved RR spectroscopic studies have demonstrated a thermal short-cut reaction back to the Pr from the Meta-state, in competition to its decay route to Pfr (22). Clearly, this proposed general reaction mechanism must currently be considered as a hypothesis that deserves further experiments for its verification or refinement.…”

“…The spectra are also highly similar to the prototypical histidine kinase Bph Agp1, confirming the typical 15Z configuration of BV in the Pr state in the truncated construct. Agp1 was chosen as a reference system for this study, because in addition to the large body of spectroscopic (16,17,22) and crystallographic data (23), the vibrational mode assign- Structural asymmetry of a phytochrome ment was recently validated by isotopic labeling experiments complemented by state-of-the-art QM/MM calculations (19). Following this assignment, we attribute the typical N-H inplane (i.p.)…”