Unlike canonical phytochromes, the GAF domain of cyanobacteriochromes (CBCRs) can bind bilins autonomously and is sufficient for functional photocycles. Despite the astonishing spectral diversity of CBCRs, the GAF1 domain of the three-GAF-domain photoreceptor all2699 from the cyanobacterium Nostoc 7120 is the only CBCR-GAF known that converts from a red-absorbing (Pr) dark state to a far-red-absorbing (Pfr) photoproduct, analogous to the more conservative phytochromes. Here we report a solid-state NMR spectroscopic study of all2699g1 in its Pr state. Conclusive NMR evidence unveils a particular stereochemical heterogeneity at the tetrahedral C31 atom, whereas the crystal structure shows exclusively the R-stereochemistry at this chiral center. Additional NMR experiments were performed on a construct comprising the GAF1 and GAF2 domains of all2699, showing a greater precision in the chromophore–protein interactions in the GAF1-2 construct. A 3D Pr structural model of the all2699g1-2 construct predicts a tongue-like region extending from the GAF2 domain (akin to canonical phytochromes) in the direction of the chromophore, shielding it from the solvent. In addition, this stabilizing element allows exclusively the R-stereochemistry for the chromophore-protein linkage. Site-directed mutagenesis performed on three conserved motifs in the hairpin-like tip confirms the interaction of the tongue region with the GAF1-bound chromophore.

Phytochromes are a diverse family of bilin-binding photoreceptors that regulate a wide range of physiological processes. Their photochemical properties make them attractive for applications in optogenetics and superresolution microscopy. Phytochromes undergo reversible photoconversion triggered by theZ⇄Ephotoisomerization about the double bond in the bilin chromophore. However, it is not fully understood at the molecular level how the protein framework facilitates the complex photoisomerization dynamics. We have studied a single-domain bilin-binding photoreceptor All2699g1 (Nostocsp. PCC 7120) that exhibits photoconversion between the red light-absorbing (Pr) and far red-absorbing (Pfr) states just like canonical phytochromes. We present the crystal structure and examine the photoisomerization mechanism of the Prform as well as the formation of the primary photoproduct Lumi-R using time-resolved spectroscopy and hybrid quantum mechanics/molecular mechanics simulations. We show that the unusually long excited state lifetime (broad lifetime distribution centered at ∼300 picoseconds) is due to the interactions between the isomerizing pyrrole ring D and an adjacent conserved Tyr142. The decay kinetics shows a strongly distributed character which is imposed by the nonexponential protein dynamics. Our findings offer a mechanistic insight into how the quantum efficiency of the bilin photoisomerization is tuned by the protein environment, thereby providing a structural framework for engineering bilin-based optical agents for imaging and optogenetics applications.

The light-harvesting phycobilisome in cyanobacteria and red algae requires the lyase-catalyzed chromophorylation of phycobiliproteins. There are three functionally distinct lyase families known. The heterodimeric E/F type is specific for attaching bilins covalently to α-subunits of phycocyanins and phycoerythrins. Unlike other lyases, the lyase also has chromophore-detaching activity. A subclass of the E/F-type lyases is, furthermore, capable of chemically modifying the chromophore. Although these enzymes were characterized >25 y ago, their structures remained unknown. We determined the crystal structure of the heterodimer of CpcE/F from sp. PCC7120 at 1.89-Å resolution. Both subunits are twisted, crescent-shaped α-solenoid structures. CpcE has 15 and CpcF 10 helices. The inner (concave) layer of CpcE (helices h2, 4, 6, 8, 10, 12, and 14) and the outer (convex) layer of CpcF (h16, 18, 20, 22, and 24) form a cavity into which the phycocyanobilin chromophore can be modeled. This location of the chromophore is supported by mutations at the interface between the subunits and within the cavity. The structure of a structurally related, isomerizing lyase, PecE/F, that converts phycocyanobilin into phycoviolobilin, was modeled using the CpcE/F structure as template. A HC motif critical for the isomerase activity of PecE/F is located at the loop between h20 and h21, supporting the proposal that the nucleophilic addition of Cys-88 to C10 of phycocyanobilin induces the isomerization of phycocyanobilin into phycoviolobilin. Also, the structure of NblB, involved in phycobilisome degradation could be modeled using CpcE as template. Combined with CpcF, NblB shows a low chromophore-detaching activity.

Pink-pigmented facultative methylotrophs from the phyllosphere are extremely rich in photosensors and are here proven to be photochemically functional.