A protonation-coupled feedback mechanism controls the signalling process in bathy phytochromes

Abstract

Phytochromes are bimodal photoswitches composed of a photosensor and an output module. Photoactivation of the sensor is initiated by a double bond isomerization of the tetrapyrrole chromophore and eventually leads to protein conformational changes. Recently determined structural models of phytochromes identify differences between the inactive and the signalling state but do not reveal the mechanism of photosensor activation or deactivation. Here, we report a vibrational spectroscopic study on bathy phytochromes that demonstrates that the formation of the photoactivated state and thus (de)activation of the output module is based on proton translocations in the chromophore pocket coupling chromophore and protein structural changes. These proton transfer steps, involving the tetrapyrrole and a nearby histidine, also enable thermal back-isomerization of the chromophore via keto-enol tautomerization to afford the initial dark state. Thus, the same proton re-arrangements inducing the (de)activation of the output module simultaneously initiate the reversal of this process, corresponding to a negative feedback mechanism.

Spectroscopic Investigation on the Primary Photoreaction of Bathy Phytochrome Agp2‐Pr of Agrobacterium fabrum: Isomerization in a pH‐dependent H‐bond Network

“…First we discuss the general slow‐down of the observed kinetics [Result (3)] considering the altered distribution of protons and H‐bonds upon an increase in pH from 6 to 9 …”

“…This peculiarity together with their spectral, structural and functional properties has further spurred several aspects in phytochrome research. This concerns in particular the functional role of structural heterogeneity, the functional role of the direction of the dark reversion as well as its mechanism, and the role of the Pr and the Pfr state for output module activation and deactivation …”

“…It was concluded that the BV protonation state in the Pr form is not caused by a specific amino acid residue, but is rather sensitive to subtle structural changes affecting the chromophore binding pocket with possible involvement of Asp 196, His 248, His 278 and Tyr 255. In an extended IR‐ and resonance‐Raman spectroscopic study it was shown that the deprotonation at ring B/C is paralleled by protonation of ring‐D oxygen, corresponding to an keto/enol tautomerization of the bilin chromophore, with the enol form prevailing at high pH. Furthermore, His 278 near ring D was suggested to be the titratable group, coupled to the intramolecular proton transfer.…”

The Crystal Structures of the N-terminal Photosensory Core Module of Agrobacterium Phytochrome Agp1 as Parallel and Anti-parallel Dimers

“…The phytochromes Agp1 and Agp2 from Agrobacterium have been extensively used as model systems for biochemical and spectroscopic studies related to chromophore protein interaction, light-induced conformational changes, or HK regulation, and it is desired to combine results from these studies with structural data (7,(32)(33)(34)(35)(36)(37). Crystallization of the PAS-GAF-PHY fragment of Agp1 (Agp1-PCM) with the wild-type sequence has been reported previously (38), but crystallographic structure refinement could not be carried out satisfactorily (7).…”

Structural communication between the chromophore‐binding pocket and the N‐terminal extension in plant phytochrome phyB

“…It has been shown previously that dark reversion kinetics of phyB is substantially accelerated upon removal of the NTE segment [7]. Also, the changes of the hydrogen bond interactions induced by NTE deletion might contribute to the acceleration of the dark reversion via favoring the transient formation of the enol form of the chromophore as the essential mechanistic step of thermal double bond isomerization [25]. This conformational heterogeneity has only been observed for cyanobacterial and prototypical bacteriophytochromes that, like plant phytochromes, thermally revert to the Pr state.…”