Spectroscopic Investigation on the Primary Photoreaction of Bathy Phytochrome Agp2‐Pr of Agrobacterium fabrum: Isomerization in a pH‐dependent H‐bond Network
Abstract:Bathy phytochrome Agp2 from Agrobacterium fabrum exhibits an unusually low pKa =7.6 in the Pr state in contrast to a pKa >11 in the Pfr state, indicating a pH-dependent charge distribution and H-bond network in the Pr chromophore binding pocket around neutral pH. Here, we report on ultrafast UV/Vis absorption spectroscopy of the primary Pr photoisomerization of Agp2 at pH 6 and pH 9 and upon H2 O/D2 O buffer exchange. The triexponential Pr kinetics slows down at increased pH and pronounced pH-dependent kinetic… Show more
“…1 a). Despite the differences between the bilin-type chromophores (biliverdin (BV) vs. phytochromobilin) and their covalent binding sites (PAS vs. GAF domain 7 ) in bacterial and plant PCMs, the photoinduced processes that interconvert the two parent states Pr and Pfr follow similar pathways with spectrally distinguishable Lumi and Meta intermediates 8 , 9 . Fig.…”
Phytochromes are modular photoreceptors of plants, bacteria and fungi that use light as a source of information to regulate fundamental physiological processes. Interconversion between the active and inactive states is accomplished by a photoinduced reaction sequence which couples the sensor with the output module. However, the underlying molecular mechanism is yet not fully understood due to the lack of structural data of functionally relevant intermediate states. Here we report the crystal structure of a Meta-F intermediate state of an Agp2 variant from Agrobacterium fabrum. This intermediate, the identity of which was verified by resonance Raman spectroscopy, was formed by irradiation of the parent Pfr state and displays significant reorientations of almost all amino acids surrounding the chromophore. Structural comparisons allow identifying structural motifs that might serve as conformational switch for initiating the functional secondary structure change that is linked to the (de-)activation of these photoreceptors.
“…1 a). Despite the differences between the bilin-type chromophores (biliverdin (BV) vs. phytochromobilin) and their covalent binding sites (PAS vs. GAF domain 7 ) in bacterial and plant PCMs, the photoinduced processes that interconvert the two parent states Pr and Pfr follow similar pathways with spectrally distinguishable Lumi and Meta intermediates 8 , 9 . Fig.…”
Phytochromes are modular photoreceptors of plants, bacteria and fungi that use light as a source of information to regulate fundamental physiological processes. Interconversion between the active and inactive states is accomplished by a photoinduced reaction sequence which couples the sensor with the output module. However, the underlying molecular mechanism is yet not fully understood due to the lack of structural data of functionally relevant intermediate states. Here we report the crystal structure of a Meta-F intermediate state of an Agp2 variant from Agrobacterium fabrum. This intermediate, the identity of which was verified by resonance Raman spectroscopy, was formed by irradiation of the parent Pfr state and displays significant reorientations of almost all amino acids surrounding the chromophore. Structural comparisons allow identifying structural motifs that might serve as conformational switch for initiating the functional secondary structure change that is linked to the (de-)activation of these photoreceptors.
“…[11,12] The parental and the photoproduct states of canonical and knotless phytochromes are red-light (P r )a nd far-red-light (P fr )a bsorbing, respectively. [5] The photoisomerization reaction and the primary photoproduct (Lumi-R)f ormation in the forward( P r !P fr )p hotoconversion of these phytochromes proceeds with al ifetime of tens [13][14][15][16][17][18][19][20] to hundreds [21][22][23][24][25] of picoseconds. Photoisomerization in other mo-lecular systems (e.g.,a zobenzenes [26,27] andr hodopsins [28][29][30] )i s typicallyu ltrafast, which raises the question about the origin of the remarkablys low photoisomerization rates in phytochromes.…”
Phytochrome photoreceptors operate via photoisomerization of a bound bilin chromophore. Their typical architecture consists of GAF, PAS and PHY domains. Knotless phytochromes lack the PAS domain, while retaining photoconversion abilities, with some being able to photoconvert with just the GAF domain. Therefore, we investigated the ultrafast photoisomerization of the Pr state of a knotless phytochrome to reveal the effect of the PHY domain and its “tongue” region on the transduction of the light signal. We show that the PHY domain does not affect the initial conformational dynamics of the chromophore. However, it significantly accelerates the consecutively induced reorganizational dynamics of the protein, necessary for the progression of the photoisomerization. Consequently, the PHY domain keeps the bilin and its binding pocket in a more reactive conformation, which decreases the extent of protein reorganization required for the chromophore isomerization. Thereby, less energy is lost along nonproductive reaction pathways, resulting in increased efficiency.
“…An obvious Pfr state could not be fully detected in various conditions assayed in this study. We speculate MmBphP may experience a 'fast' Pfr reaction, which evolved on a picosecond or microsecond time scale (Heyne et al 2002;Jaubert et al 2007;Singer et al 2016). This may result from formation of an obvious Pfr state that is too short-lived to be detected in our experiments.…”
Phytochromes are a class of photoreceptors found in plants and in some fungi, cyanobacteria, photoautotrophic, and heterotrophic bacteria. Although phytochromes have been structurally characterized in some bacteria, its biological and ecological roles in magnetotactic bacteria remain unexplored. Here, we describe the biochemical characterization of recombinant bacteriophytochrome (BphP) from magnetotactic bacteria Magnetospirillum magneticum AMB-1 (MmBphP). The recombinant MmBphP displays all the characteristic features, including the property of binding to biliverdin (BV), of a genuine phytochrome. Site-directed mutagenesis identified that cysteine-14 is important for chromophore covalent binding and photoreversibility. Arginine-240 and histidine-246 play key roles in binding to BV. The N-terminal photosensory core domain of MmBphP lacking the C-terminus found in other phytochromes is sufficient to exhibit the characteristic red/far-red-light-induced fast photoreversibility of phytochromes. Moreover, our results showed MmBphP is involved in the phototactic response, suggesting its conservative role as stress-protective. This finding provided us a better understanding of the physiological function of this group of photoreceptors and photoresponse of magnetotactic bacteria.
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