Cyanobacteriochromes are cyanobacterial tetrapyrrole-binding photoreceptors that share a bilin-binding GAF domain with photoreceptors of the phytochrome family. Cyanobacteriochromes are divided into many subclasses with distinct spectral properties. Among them, putative phototaxis regulators PixJs of Anabaena sp. PCC 7120 and Thermosynechococcus elongatus BP-1 (denoted as AnPixJ and TePixJ, respectively) are representative of subclasses showing red-green-type and blue/green-type reversible photoconversion, respectively. Here, we determined crystal structures for the AnPixJ GAF domain in its red-absorbing 15 Z state (Pr) and the TePixJ GAF domain in its green-absorbing 15 E state (Pg). The overall structure of these proteins is similar to each other and also similar to known phytochromes. Critical differences found are as follows: ( i ) the chromophore of AnPixJ Pr is phycocyanobilin in a C5- Z ,syn/C10- Z ,syn/C15- Z ,anti configuration and that of TePixJ Pg is phycoviolobilin in a C10- Z ,syn/C15- E ,anti configuration, ( ii ) a side chain of the key aspartic acid is hydrogen bonded to the tetrapyrrole rings A, B and C in AnPixJ Pr and to the pyrrole ring D in TePixJ Pg, ( iii ) additional protein-chromophore interactions are provided by subclass-specific residues including tryptophan in AnPixJ and cysteine in TePixJ. Possible structural changes following the photoisomerization of the chromophore between C15- Z and C15- E are discussed based on the X-ray structures at 1.8 and 2.0-Å resolution, respectively, in two distinct configurations.

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Cyanobacteriochromes are a newly recognized group of photoreceptors that are distinct relatives of phytochromes but are found only in cyanobacteria. A putative cyanobacteriochrome, CcaS, is known to chromatically regulate the expression of the phycobilisome linker gene (cpcG2) in Synechocystis sp. PCC 6803. In this study, we isolated the chromophore-binding domain of CcaS from Synechocystis as well as from phycocyanobilin-producing Escherichia coli. Both preparations showed the same reversible photoconversion between a green-absorbing form (Pg, max ‫؍‬ 535 nm) and a red-absorbing form (Pr, max ‫؍‬ 672 nm). Mass spectrometry and denaturation analyses suggested that Pg and Pr bind phycocyanobilin in a double-bond configuration of C15-Z and C15-E, respectively. Autophosphorylation activity of the histidine kinase domain in nearly full-length CcaS was up-regulated by preirradiation with green light. Similarly, phosphotransfer to the cognate response regulator, CcaR, was higher in Pr than in Pg. From these results, we conclude that CcaS phosphorylates CcaR under green light and induces expression of cpcG2, leading to accumulation of CpcG2-phycobilisome as a chromatic acclimation system. CcaS is the first recognized green light receptor in the expanded phytochrome superfamily, which includes phytochromes and cyanobacteriochromes.chromatic adaptation ͉ phycocyanobilin ͉ phytochrome ͉ cyanobacteria ͉ photoreceptor P hytochromes (Phys) are photoreceptors that typically perceive red and far-red light and regulate a wide range of physiological responses in plants, bacteria, cyanobacteria, and fungi (1). They exhibit reversible photoconversion between two distinct forms: the red-absorbing form (Pr) and the far-redabsorbing form (Pfr). Their N-terminal photosensory region, which consists of Per-ARNT-Sim (PAS), cGMP phosphodiesterase/adenylyl cyclase/FhlA (GAF), and phytochrome domains, is highly conserved, but there are variations in the chromophore of the linear tetrapyrrole, such as phytochromobilin, phycocyanobilin (PCB), and biliverdin. It is reported that phytochromobilin or PCB is covalently anchored at a conserved cysteine residue in the GAF domain (2, 3), whereas biliverdin is anchored at another conserved cysteine residue in the N terminus of the PAS domain (4). The perception of light by Phys triggers a Z to E isomerization of the C15-C16 double bond between the C and D pyrrole rings as well as subsequent conformational changes of the chromophore and the apoprotein [supporting information (SI) Fig. S1] (5) which signal to downstream processes. Recent crystallographic analyses of bacterial Phys (DrBphP and RpBphP3) have revealed the three-dimensional structure of PAS and GAF domains in the Pr form (6-8). The biliverdin chromophore is buried deep within a pocket in the GAF domain with a configuration of C5-Z,syn/C10-Z,syn/C15-Z,anti. Because the residues in the chromophore-binding pocket are highly conserved, it was proposed that Phys share a common photoconversion mechanism, albeit with certain variations.''Cyanobac...

Cyanobacteriochromes (CBCRs) are cyanobacterial members of the phytochrome superfamily of photosensors. Like phytochromes, CBCRs convert between two photostates by photoisomerization of a covalently bound linear tetrapyrrole (bilin) chromophore. Although phytochromes are red/far-red sensors, CBCRs exhibit diverse photocycles spanning the visible spectrum and the near-UV (330-680 nm). Two CBCR subfamilies detect near-UV to blue light (330-450 nm) via a "two-Cys photocycle" that couples bilin 15Z/15E photoisomerization with formation or elimination of a second bilincysteine adduct. On the other hand, mechanisms for tuning the absorption between the green and red regions of the spectrum have not been elucidated as of yet. CcaS and RcaE are members of a CBCR subfamily that regulates complementary chromatic acclimation, in which cyanobacteria optimize light-harvesting antennae in response to green or red ambient light. CcaS has been shown to undergo a green/red photocycle: reversible photoconversion between a green-absorbing 15Z state ( 15Z P g ) and a red-absorbing 15E state ( 15E P r ). We demonstrate that RcaE from Fremyella diplosiphon undergoes the same photocycle and exhibits light-regulated kinase activity. In both RcaE and CcaS, the bilin chromophore is deprotonated as 15Z P g but protonated as 15E P r . This change of bilin protonation state is modulated by three key residues that are conserved in green/red CBCRs. We therefore designate the photocycle of green/red CBCRs a "protochromic photocycle," in which the dramatic change from green to red absorption is not induced by initial bilin photoisomerization but by a subsequent change in bilin protonation state.light sensing | phycobiliprotein | signal transduction | spectral tuning | two-component signaling P hytochrome photosensors initially were discovered in plants and later found in cyanobacteria, nonoxygenic photosynthetic bacteria, nonphotosynthetic bacteria, fungi, and algae (1, 2). These photoreceptors bind linear tetrapyrrole (bilin) chromophores within a conserved GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) domain via a covalent thioether linkage to a conserved Cys residue (Fig. S1A)(3-6). Upon illumination, phytochromes reversibly convert between a red-absorbing dark state and a far-red-absorbing photoproduct. This red/far-red photocycle is triggered by photoisomerization of the bilin 15,16-double bond between the 15Z and 15E configurations (7,8), with 15Z giving red absorption and 15E far-red absorption (4, 6, 9). In phytochromes, the conjugated π system of the bilin is protonated in both photostates, and this protonation is necessary to maintain the red and far-red absorption (10-12). Conserved GAF residues supply a hydrogen bond network to tune the chemical and spectral properties of the bilin (Fig. S1B).* Cyanobacteriochromes (CBCRs) are widespread cyanobacterial photosensors with phytochrome-related GAF domains (1,2,13,14). Although CBCRs also convert between two photostates via bilin photoisomerization at C15, they exhibit much more spe...

The cyanobacterial phototaxis regulator protein, TePixJ, is a member of the subfamily of cyanobacteriochromes that binds phycoviolobilin (PVB) as a chromophore and exhibits reversible photoconversion between blue light-absorbing (Pb) and green light-absorbing (Pg) forms. We reconstituted the PVB-binding photoactive holocomplex in vivo and in vitro. Coexpression of the apoprotein and phycocyanobilin (PCB) in Escherichia coli (in vivo reconstitution) produced a mixture of the PCB-bound and PVB-bound holoproteins. Reconstitution in vitro of the apoprotein and synthetic PCB quickly generated a photoactive complex, which covalently bound PCB and exhibited partially reversible photoconversion between two species by UV-vis spectroscopy (with a λ(max) values of 430 and 545 nm). Further incubation produced slow isomerization of PCB to PVB with concomitant improvement of photoreactivity. Site-directed mutagenesis confirmed that Cys522, and a second conserved Cys (Cys494), are both essential for the assembly of the photoactive complex. Fourier transform infrared (FTIR) spectroscopy revealed green light-induced cross-linking, and blue light-induced release, of a thiol group, possibly that of Cys494. These results suggest that the Pb/Pg-type cyanobacteriochrome TePixJ is assembled in at least three steps: (i) rapid and stable chromophorylation of PCB, (ii) additional photoreversible chromophorylation, and (iii) subsequent slow isomerization of PCB to PVB. In addition to its known autolyase activity with Cys522 and photoreversible isomerase activity (of the Z and E isomers at C15 and C16 of PCB), the GAF domain of TePixJ therefore appears to have other roles: as an isomerase (converting PCB to PVB) and as a photoreversible autolyase with a second conserved Cys residue.

Two wild-type substrains of a motile cyanobacterium Synechocystis sp. PCC 6803 show positive phototaxis toward a light source (PCC-P) and negative phototaxis away from light (PCC-N). In this study, we found that a novel two-component system of photoresponse is involved in the phototactic regulation. Inactivation of slr1212 (pixA), which encodes a photoreceptor histidine kinase, reverted the positive phototaxis of PCC-P to negative phototaxis, and inactivation of the downstream slr1213 (nixB) and slr1214 (nixC), which encode AraC-like transcription factor-type and PatA-type response regulators, respectively, reverted the negative phototaxis of PCC-N to positive phototaxis. Opposite effects of pixA and nixBC disruption implies an unexpected signal transduction pathway in the switching of positive and negative phototaxis. The blue/green-type cyanobacteriochrome GAF domain of PixA was expressed in Synechocystis and phycocyanobilin-producing Escherichia coli. The holoprotein covalently bound a chromophore phycoviolobilin and showed reversible photoconversion between the violet- (Pv, λ(peak) = 396 nm) and green-absorbing (Pg, λ(peak) = 533 nm) forms, although the protein from E. coli partially bound a precursor phycocyanobilin. These results were discussed with regard to an idea that PixA serves as a violet light receptor for switching of positive and negative phototaxis by transcriptional and functional regulation.

Oxygenic photosynthesis is driven by photosystems I and II (PSI and PSII, respectively). Both have specific antenna complexes and the phycobilisome (PBS) is the major antenna protein complex in cyanobacteria, typically consisting of a core from which several rod-like subcomplexes protrude. PBS preferentially transfers light energy to PSII, whereas a PSI-specific antenna has not been identified. The cyanobacterium Anabaena sp. PCC 7120 has rodcore linker genes (cpcG1-cpcG2-cpcG3-cpcG4). Their products, except CpcG3, have been detected in the conventional PBS. Here we report the isolation of a supercomplex that comprises a PSI tetramer and a second, unique type of a PBS, specific to PSI. This rodshaped PBS includes phycocyanin (PC) and CpcG3 (hereafter renamed "CpcL"), but no allophycocyanin or CpcGs. Fluorescence excitation showed efficient energy transfer from PBS to PSI. The supercomplex was analyzed by electron microscopy and singleparticle averaging. In the supercomplex, one to three rod-shaped CpcL-PBSs associate to a tetrameric PSI complex. They are mostly composed of two hexameric PC units and bind at the periphery of PSI, at the interfaces of two monomers. Structural modeling indicates, based on 2D projection maps, how the PsaI, PsaL, and PsaM subunits link PSI monomers into dimers and into a rhombically shaped tetramer or "pseudotetramer." The 3D model further shows where PBSs associate with the large subunits PsaA and PsaB of PSI. It is proposed that the alternative form of CpcL-PBS is functional in harvesting energy in a wide number of cyanobacteria, partially to facilitate the involvement of PSI in nitrogen fixation. I n photosynthesis, light-harvesting antennas are essential to efficiently collect solar energy. Photosynthetic organisms have diverse antenna protein-pigment complexes, which are specifically associated with photosystems I or II (PSI or PSII, respectively) (1). Light-harvesting chlorophyll a/b-binding proteins form the peripheral antenna of PSI or PSII in green plants (2) and light-harvesting chlorophyll a/c-binding proteins are present in brown algae and related organisms (3, 4). In cyanobacteria the phycobilisome (PBS) serves as a major antenna for PSII. No specific antenna has been isolated for PSI in cyanobacteria, although PBS transfers light energy to PSI under conditions of state transition (5), a temporal energy redistribution mechanism between PSII and PSI (6, 7).Generally, the PBS is a supercomplex of rod and core subcomplexes, which consist of various phycobilin-binding proteins connected by several classes of colorless linker proteins (8). Whereas phycocyanin (PC) is the major phycobiliprotein of the rod, allophycocyanin (APC) is the major phycobiliprotein of the core cylinders. The rod-core linker cyanobacterial phycocyanin protein G (CpcG), which connects the rod to the core, plays a key role in the assembly of the PBS (9). The chromosome of the filamentous, N 2 -fixing cyanobacterium Anabaena sp. PCC 7120 (hereafter "Anabaena") bears tandem repeats of rod-core linker gene...

Cyanobacteria have several putative photoreceptors (designated cyanobacteriochromes) that are related to but distinct from the established phytochromes. The GAF domain of the phototaxis regulator, PixJ, from a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TePixJ_GAF) is a cyanobacteriochrome which exhibits reversible photoconversion between a blue light-absorbing form (max = 433 nm) and a green light-absorbing form (max = 531 nm). To study the chromophore, we prepared TePixJ_GAF chromoprotein from heterologously expressed Synechocystis and performed spectral analysis after denaturation by comparing it with the cyanobacterial phytochrome Cph1 which harbors phycocyanobilin (PCB) as a chromophore. The results indicated that the chromophore of TePixJ is not PCB, but its isomer, phycoviolobilin (PVB). It is suggested that the GAF domain of TePixJ has auto-lyase and auto-isomerase activities.