Photoreversible Aggregation of the Biliprotein Containing the First and Second GAF Domains of a Cyanobacteriochrome All2699 in Nostoc sp. PCC7120.

As plant photoreceptors, phytochromes are capable of detecting red light and far-red light, thereby governing plant growth. All2699 is a photoreceptor found in Nostoc sp. PCC7120 that specifically responds to red light and far-red light. All2699g1g2 is a truncated protein carrying the first and second GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) domains of All2699. In this study, we found that, upon exposure to red light, the protein underwent aggregation, resulting in the formation of protein aggregates. Conversely, under far-red light irradiation, these protein aggregates dissociated. We delved into the factors that impact the aggregation of All2699g1g2, focusing on the protein structure. Our findings showed that the GAF2 domain contains a low-complexity (LC) loop region, which plays a crucial role in mediating protein aggregation. Specifically, phenylalanine at position 239 within the LC loop region was identified as a key site for the aggregation process. Furthermore, our research revealed that various factors, including irradiation time, temperature, concentration, NaCl concentration, and pH value, can impact the aggregation of All2699g1g2. The aggregation led to variations in Pfr concentration depending on temperature, NaCl concentration, and pH value. In contrast, ΔLC did not aggregate and therefore lacked responses to these factors. Consequently, the LC loop region of All2699g1g2 extended and enhanced sensory properties.

The Orange Carotenoid Protein Triggers Cyanobacterial Photoprotection by Quenching Bilins via a Structural Switch of Its Carotenoid.

Cyanobacteria were the first microorganisms that released oxygen into the atmosphere billions of years ago. To do it safely under intense sunlight, they developed strategies that prevent photooxidation in the photosynthetic membrane, by regulating the light-harvesting activity of their antenna complexes-the phycobilisomes-via the orange-carotenoid protein (OCP). This water-soluble protein interacts with the phycobilisomes and triggers nonphotochemical quenching (NPQ), a mechanism that safely dissipates overexcitation in the membrane. To date, the mechanism of action of OCP in performing NPQ is unknown. In this work, we performed ultrafast spectroscopy on a minimal NPQ system composed of the active domain of OCP bound to the phycobilisome core. The use of this system allowed us to disentangle the signal of the carotenoid from that of the bilins. Our results demonstrate that the binding to the phycobilisomes modifies the structure of the ketocarotenoid associated with OCP. We show that this molecular switch activates NPQ, by enabling excitation-energy transfer from the antenna pigments to the ketocarotenoid.

On the evolution of the plant phytochrome chromophore biosynthesis.

Phytochromes are biliprotein photoreceptors present in plants, algae, certain bacteria, and fungi. Land plant phytochromes use phytochromobilin (PΦB) as the bilin chromophore. Phytochromes of streptophyte algae, the clade within which land plants evolved, employ phycocyanobilin (PCB), leading to a more blue-shifted absorption spectrum. Both chromophores are synthesized by ferredoxin-dependent bilin reductases (FDBRs) starting from biliverdin IXα (BV). In cyanobacteria and chlorophyta, BV is reduced to PCB by the FDBR phycocyanobilin:ferredoxin oxidoreductase (PcyA), whereas, in land plants, BV is reduced to PФB by phytochromobilin synthase (HY2). However, phylogenetic studies suggested the absence of any ortholog of PcyA in streptophyte algae and the presence of only PФB biosynthesis-related genes (HY2). The HY2 of the streptophyte alga Klebsormidium nitens (formerly Klebsormidium flaccidum) has already indirectly been indicated to participate in PCB biosynthesis. Here, we overexpressed and purified a His6-tagged variant of K. nitens HY2 (KflaHY2) in Escherichia coli. Employing anaerobic bilin reductase activity assays and coupled phytochrome assembly assays, we confirmed the product and identified intermediates of the reaction. Site-directed mutagenesis revealed 2 aspartate residues critical for catalysis. While it was not possible to convert KflaHY2 into a PΦB-producing enzyme by simply exchanging the catalytic pair, the biochemical investigation of 2 additional members of the HY2 lineage enabled us to define 2 distinct clades, the PCB-HY2 and the PΦB-HY2 clade. Overall, our study gives insight into the evolution of the HY2 lineage of FDBRs.

Crystal structure of a far-red-sensing cyanobacteriochrome reveals an atypical bilin conformation and spectral tuning mechanism.

Cyanobacteriochromes (CBCRs) are small, linear tetrapyrrole (bilin)-binding photoreceptors in the phytochrome superfamily that regulate diverse light-mediated adaptive processes in cyanobacteria. More spectrally diverse than canonical red/far-red-sensing phytochromes, CBCRs were thought to be restricted to sensing visible and near UV light until recently when several subfamilies with far-red-sensing representatives (frCBCRs) were discovered. Two of these frCBCRs subfamilies have been shown to incorporate bilin precursors with larger pi-conjugated chromophores, while the third frCBCR subfamily uses the same phycocyanobilin precursor found in the bulk of the known CBCRs. To elucidate the molecular basis of far-red light perception by this third frCBCR subfamily, we determined the crystal structure of the far-red-absorbing dark state of one such frCBCR Anacy_2551g3 from Anabaena cylindrica PCC 7122 which exhibits a reversible far-red/orange photocycle. Determined by room temperature serial crystallography and cryocrystallography, the refined 2.7-Å structure reveals an unusual all-Z,syn configuration of the phycocyanobilin (PCB) chromophore that is considerably less extended than those of previously characterized red-light sensors in the phytochrome superfamily. Based on structural and spectroscopic comparisons with other bilin-binding proteins together with site-directed mutagenesis data, our studies reveal protein-chromophore interactions that are critical for the atypical bathochromic shift. Based on these analyses, we propose that far-red absorption in Anacy_2551g3 is the result of the additive effect of two distinct red-shift mechanisms involving cationic bilin lactim tautomers stabilized by a constrained all-Z,syn conformation and specific interactions with a highly conserved anionic residue.

Structural basis of the protochromic green/red photocycle of the chromatic acclimation sensor RcaE.

Cyanobacteriochromes (CBCRs) are bilin-binding photosensors of the phytochrome superfamily that show remarkable spectral diversity. The green/red CBCR subfamily is important for regulating chromatic acclimation of photosynthetic antenna in cyanobacteria and is applied for optogenetic control of gene expression in synthetic biology. It is suggested that the absorption change of this subfamily is caused by the bilin C15-Z/C15-E photoisomerization and a subsequent change in the bilin protonation state. However, structural information and direct evidence of the bilin protonation state are lacking. Here, we report a high-resolution (1.63Å) crystal structure of the bilin-binding domain of the chromatic acclimation sensor RcaE in the red-absorbing photoproduct state. The bilin is buried within a "bucket" consisting of hydrophobic residues, in which the bilin configuration/conformation is C5-Z,syn/C10-Z,syn/C15-E,syn with the A- through C-rings coplanar and the D-ring tilted. Three pyrrole nitrogens of the A- through C-rings are covered in the α-face with a hydrophobic lid of Leu249 influencing the bilin pKa, whereas they are directly hydrogen bonded in the ß-face with the carboxyl group of Glu217. Glu217 is further connected to a cluster of waters forming a hole in the bucket, which are in exchange with solvent waters in molecular dynamics simulation. We propose that the "leaky bucket" structure functions as a proton exit/influx pathway upon photoconversion. NMR analysis demonstrated that the four pyrrole nitrogen atoms are indeed fully protonated in the red-absorbing state, but one of them, most likely the B-ring nitrogen, is deprotonated in the green-absorbing state. These findings deepen our understanding of the diverse spectral tuning mechanisms present in CBCRs.

Zinc-Induced Fluorescence Turn-On in Native and Mutant Phycoerythrobilin-Binding Orange Fluorescent Proteins.

Cyanobacteriochrome (CBCR)-derived fluorescent proteins are a class of reporters that can bind bilin cofactors and fluoresce across the ultraviolet to the near-infrared spectrum. Derived from phytochrome-related photoreceptor proteins in cyanobacteria, many of these proteins use a single small GAF domain to autocatalytically bind a bilin and fluoresce. The second GAF domain of All1280 (All1280g2) from Nostoc sp. PCC7120 is a DXCF motif-containing protein that exhibits blue-light-responsive photochemistry when bound to its native cofactor, phycocyanobilin. All1280g2 can also bind non-photoswitching phycoerythrobilin (PEB), resulting in a highly fluorescent protein. Given the small size, high quantum yield, and that unlike green fluorescent proteins, bilin-binding proteins can be used in anaerobic organisms, the orange fluorescent All1280g2-PEB protein is a promising platform for designing new genetically encoded metal ion sensors. Here, we show that All1280g2-PEB undergoes a ∼5-fold reversible zinc-induced fluorescence enhancement with a blue-shifted emission maximum (572 to 517 nm), which is not observed for a related PEB-bound GAF from Synechocystis sp. PCC6803 (Slr1393g3). Zn2+ significantly enhances All1280g2-PEB fluorescence across a biologically relevant pH range from 6.0 to 9.0, with pH-dependent dissociation constants from 1 µM to ∼20-80 nM. Site-directed mutants aiming to sterically decrease and increase access to PEB show a decreased and similar amount of zinc-induced fluorescence enhancement. Mutation of the cysteine residue within the DXCF motif to alanine abolishes the zinc-induced fluorescence enhancement. Collectively, these results support the presence of a unique fluorescence-enhancing Zn2+ binding site in All1280g2-PEB likely involving coordination to the bilin cofactor and requiring a nearby cysteine residue.

The interplay between chromophore and protein determines the extended excited state dynamics in a single-domain phytochrome.

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 the Z ⇄ E photoisomerization 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 (Nostoc sp. 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 Pr form 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.

Expansion of bilin-based red light sensors in the subaerial desert cyanobacterium Nostoc flagelliforme.

Light is the crucial environmental signal for desiccation-tolerant cyanobacteria to activate photosynthesis and prepare for desiccation at dawn. However, the photobiological characteristics of desert cyanobacteria adaptation to one of the harshest habitats on Earth remain unresolved. In this study, we surveyed the genome of a subaerial desert cyanobacterium Nostoc flagelliforme and identified two phytochromes and seven cyanobacteriochromes (CBCRs) with one or more bilin-binding GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) domains. Biochemical and spectroscopic analyses of 69 purified GAF-containing proteins from recombinant phycocyanobilin (PCB), biliverdin or phycoerythrobilin-producing Escherichia coli indicated that nine of these proteins bind chromophores. Further investigation revealed that 11 GAFs form covalent adducts responsive to near-UV and visible light: eight GAFs contained PCB chromophores, three GAFs contained biliverdin chromophores and one contained the PCB isomer, phycoviolobilin. Interestingly, COO91_03972 is the first-ever reported GAF-only CBCR capable of sensing five wavelengths of light. Bioinformatics and biochemical analyses revealed that residue P132 of COO91_03972 is essential for chromophore binding to dual-cysteine CBCRs. Furthermore, the complement of N. flagelliforme CBCRs is enriched in red light sensors. We hypothesize that these sensors are critical for the acclimatization of N. flagelliforme to weak light environments at dawn.

The specificity of the bilin lyase CpcS for chromophore attachment to allophycocyanin in the chlorophyll f-containing cyanobacterium Halomicronima hongdechloris.

Phycobilisomes are light-harvesting antenna complexes of cyanobacteria and red algae that are comprised of chromoproteins called phycobiliproteins. PBS core structures are made up of allophycocyanin subunits. Halomicronema hongdechloris (H. hongdechloris) is one of the cyanobacteria that produce chlorophyll f (Chl f) under far-red light and is regulated by the Far-Red Light Photoacclimation gene cluster. There are five genes encoding APC in this specific gene cluster, and they are responsible for assembling the red-shifted PBS in H. hongdechloris grown under far-red light. In this study, the five apc genes located in the FaRLiP gene cluster were heterologously expressed in an Escherichia coli reconstitution system. The canonical APC-encoding genes were also constructed in the same system for comparison. Additionally, five annotated phycobiliprotein lyase-encoding genes (cpcS) from the H. hongdechloris genome were phylogenetically classified and experimentally tested for their catalytic properties including their contribution to the shifted absorption of PBS. Through analysis of recombinant proteins, we determined that the heterodimer of CpcS-I and CpcU are able to ligate a chromophore to the APC-α/APC-ß subunits. We discuss some hypotheses towards understanding the roles of the specialised APC and contributions of PBP lyases.

Light- and pH-dependent structural changes in cyanobacteriochrome AnPixJg2.

Cyanobacteriochromes (CBCRs) are phytochrome-related photosensory proteins that play an essential role in regulating phototaxis, chromatic acclimation, and cell aggregation in cyanobacteria. Here, we apply solid-state NMR spectroscopy to the red/green GAF2 domain of the CBCR AnPixJ assembled in vitro with a uniformly 13C- and 15N-labeled bilin chromophore, tracking changes in electronic structure, geometry, and structural heterogeneity of the chromophore as well as intimate contacts between the chromophore and protein residues in the photocycle. Our data confirm that the bilin ring D is strongly twisted with respect to the B-C plane in both dark and photoproduct states. We also identify a greater structural heterogeneity of the bilin chromophore in the photoproduct than in the dark state. In addition, the binding pocket is more hydrated in the photoproduct. Observation of interfacial 1H contacts of the photoproduct chromophore, together with quantum mechanics/molecular mechanics (QM/MM)-based structural models for this photoproduct, clearly suggests the presence of a biprotonated (cationic) imidazolium side-chain for a conserved histidine residue (322) at a distance of ~2.7 Å, generalizing the recent theoretical findings that explicitly link the structural heterogeneity of the dark-state chromophore to the protonation of this specific residue. Moreover, we examine pH effects on this in vitro assembled holoprotein, showing a substantially altered electronic structure and protonation of the photoproduct chromophore even with a small pH drop from 7.8 to 7.2. Our studies provide further information regarding the light- and pH-induced changes of the chromophore and the rearrangements of the hydrogen-bonding and electrostatic interaction network around it. Possible correlations between structural heterogeneity of the chromophore, protonation of the histidine residue nearby, and hydration of the pocket in both photostates are discussed.

The Arabidopsis CRUMPLED LEAF protein, a homolog of the cyanobacterial bilin lyase, retains the bilin-binding pocket for a yet unknown function.

The photosynthetic bacterial phycobiliprotein lyases, also called CpcT lyases, catalyze the biogenesis of phycobilisome, a light-harvesting antenna complex, through the covalent attachment of chromophores to the antenna proteins. The Arabidopsis CRUMPLED LEAF (CRL) protein is a homolog of the cyanobacterial CpcT lyase. Loss of CRL leads to multiple lesions, including localized foliar cell death, constitutive expression of stress-related nuclear genes, abnormal cell cycle, and impaired plastid division. Notwithstanding the apparent phenotypes, the function of CRL still remains elusive. To gain insight into the function of CRL, we examined whether CRL still retains the capacity to bind with the bacterial chromophore phycocyanobilin (PCB) and its plant analog phytochromobilin (PΦB). The revealed structure of the CpcT domain of CRL is comparable to that of the CpcT lyase, despite the low sequence identity. The subsequent in vitro biochemical assays found, as shown for the CpcT lyase, that PCB/PΦB binds to the CRL dimer. However, some mutant forms of CRL, substantially compromised in their bilin-binding ability, still restore the crl-induced multiple lesions. These results suggest that although CRL retains the bilin-binding pocket, it seems not functionally associated with the crl-induced multiple lesions.

Transient Deprotonation of the Chromophore Affects Protein Dynamics Proximal and Distal to the Linear Tetrapyrrole Chromophore in Phytochrome Cph1.

Phytochromes are biological red/far-red light sensors found in many organisms. Prototypical phytochromes, including Cph1 from the cyanobacterium Synechocystis 6803, act as photochemical switches that interconvert between stable red (Pr)- and metastable far-red (Pfr)-absorbing states induced by photoisomerization of the bilin chromophore. The connection between photoconversion and the cellular output signal involves light-mediated global structural changes in the interaction between the photosensory module (PAS-GAF-PHY) and the C-terminal transmitter (output) module, usually a histidine kinase, as in the case of Cph1. The chromophore deprotonates transiently during the Pr → Pfr photoconversion in association with extensive global structural changes required for signal transmission. Here, we performed equilibrium studies in the Pr state, involving pH titration of the linear tetrapyrrole chromophore in different Cph1 constructs, and measurement of pH-dependent structural changes at various positions in the protein using picosecond time-resolved fluorescence anisotropy. The fluorescent reporter group was attached at positions 371 (PHY domain), 305 (GAF domain), and 120 (PAS domain), as well as at sites in the PAS-GAF bidomain. We show direct correlation of chromophore deprotonation with pH-dependent conformational changes in the various domains. Our results suggest that chromophore deprotonation is closely associated with a higher protein mobility (conformational space) both in proximal and in distal protein sites, implying a causal relationship that might be important for the global large protein arrangements and thus intramolecular signal transduction.

Protochromic absorption changes in the two-cysteine photocycle of a blue/orange cyanobacteriochrome.

Cyanobacteriochromes (CBCRs) are phytochrome-related photosensors with diverse spectral sensitivities spanning the entire visible spectrum. They covalently bind bilin chromophores via conserved cysteine residues and undergo 15Z/15E bilin photoisomerization upon light illumination. CBCR subfamilies absorbing violet-blue light use an additional cysteine residue to form a second bilin-thiol adduct in a two-Cys photocycle. However, the process of second thiol adduct formation is incompletely understood, especially the involvement of the bilin protonation state. Here, we focused on the Oscil6304_2705 protein from the cyanobacterium Oscillatoria acuminata PCC 6304, which photoconverts between a blue-absorbing 15Z state ( 15Z Pb) and orange-absorbing 15E state ( 15E Po). pH titration analysis revealed that 15Z Pb was stable over a wide pH range, suggesting that bilin protonation is stabilized by a second thiol adduct. As revealed by resonance Raman spectroscopy, 15E Po harbored protonated bilin at both acidic and neutral pH, but readily converted to a deprotonated green-absorbing 15Z state ( 15Z Pg) at alkaline pH. Site-directed mutagenesis revealed that the conserved Asp-71 and His-102 residues are required for second thiol adduct formation in 15Z Pb and bilin protonation in 15E Po, respectively. An Oscil6304_2705 variant lacking the second cysteine residue, Cys-73, photoconverted between deprotonated 15Z Pg and protonated 15E Pr, similarly to the protochromic photocycle of the green/red CBCR subfamily. Time-resolved spectroscopy revealed 15Z Pg formation as an intermediate in the 15E Pr-to- 15Z Pg conversion with a significant solvent-isotope effect, suggesting the sequential occurrence of 15EP-to-15Z photoisomerization, deprotonation, and second thiol adduct formation. Our findings uncover the details of protochromic absorption changes underlying the two-Cys photocycle of violet-blue-absorbing CBCR subfamilies.

Crystal structure of the first eukaryotic bilin reductase GtPEBB reveals a flipped binding mode of dihydrobiliverdin.

Phycobilins are light-harvesting pigments of cyanobacteria, red algae, and cryptophytes. The biosynthesis of phycoerythrobilin (PEB) is catalyzed by the subsequent action of two ferredoxin-dependent bilin reductases (FDBRs). Although 15,16-dihydrobiliverdin (DHBV):ferredoxin oxidoreductase (PebA) catalyzes the two-electron reduction of biliverdin IXα to 15,16-DHBV, PEB:ferredoxin oxidoreductase (PebB) reduces this intermediate further to PEB. Interestingly, marine viruses encode the FDBR PebS combining both activities within one enzyme. Although PebA and PebS share a canonical fold with similar substrate-binding pockets, the structural determinants for the stereo- and regiospecific modification of their tetrapyrrole substrates are incompletely understood, also because of the lack of a PebB structure. Here, we solved the X-ray crystal structures of both substrate-free and -bound PEBB from the cryptophyte Guillardia theta at 1.90 and 1.65 Å, respectively. The structures of PEBB exhibit the typical α/ß/α-sandwich fold. Interestingly, the open-chain tetrapyrrole substrate DHBV is bound in an unexpected flipped orientation within the canonical FDBR active site. Biochemical analyses of the WT enzyme and active site variants identified two central aspartate residues Asp-99 and Asp-219 as essential for catalytic activity. In addition, the conserved Arg-215 plays a critical role in substrate specificity, binding orientation, and active site integrity. Because these critical residues are conserved within certain FDBRs displaying A-ring reduction activity, we propose that they present a conserved mechanism for this reaction. The flipped substrate-binding mode indicates that two-electron reducing FDBRs utilize the same primary site within the binding pocket and that substrate orientation is the determinant for A- or D-ring regiospecificity.

Decomposition of cyanobacterial light harvesting complexes: NblA-dependent role of the bilin lyase homolog NblB.

Phycobilisomes, the macromolecular light harvesting complexes of cyanobacteria are degraded under nutrient-limiting conditions. This crucial response is required to adjust light excitation to the metabolic status and avoid damage by excess excitation. Phycobilisomes are comprised of phycobiliproteins, apo-proteins that covalently bind bilin chromophores. In the cyanobacterium Synechococcus elongatus, the phycobiliproteins allophycocyanin and phycocyanin comprise the core and the rods of the phycobilisome, respectively. Previously, NblB was identified as an essential component required for phycocyanin degradation under nutrient starvation. This protein is homologous to bilin-lyases, enzymes that catalyze the covalent attachment of bilins to apo-proteins. However, the nblB-inactivated strain is not impaired in phycobiliprotein synthesis, but rather is characterized by aberrant phycocyanin degradation. Here, using a phycocyanin-deficient strain, we demonstrate that NblB is required for degradation of the core pigment, allophycocyanin. Furthermore, we show that the protein NblB is expressed under nutrient sufficient conditions, but during nitrogen starvation its level decreases about two-fold. This finding is in contrast to an additional component essential for degradation, NblA, the expression of which is highly induced under starvation. We further identified NblB residues required for phycocyanin degradation in vivo. Finally, we demonstrate phycocyanin degradation in a cell-free system, thereby providing support for the suggestion that NblB directly mediates pigment degradation by chromophore detachment. The dependence of NblB function on NblA revealed using this system, together with the results indicating presence of NblB under nutrient sufficient conditions, suggests a rapid mechanism for induction of pigment degradation, which requires only the expression of NblA.

Characterization of the pheophorbide a oxygenase/phyllobilin pathway of chlorophyll breakdown in grasses.

MAIN CONCLUSION: Although the PAO/phyllobilin pathway of chlorophyll breakdown is active in grass leaf senescence, the abundance of phyllobilins is far below the amount of degraded chlorophyll. The yellowing of fully developed leaves is the most prominent visual symptom of plant senescence. Thereby, chlorophyll is degraded via the so-called pheophorbide a oxygenase (PAO)/phyllobilin pathway to a species-specific set of phyllobilins, linear tetrapyrrolic products of chlorophyll breakdown. Here, we investigated the diversity and abundance of phyllobilins in cereal and forage crops, i.e. barley, rice, ryegrass, sorghum and wheat, using liquid chromatography-mass spectrometry. A total of thirteen phyllobilins were identified, among them four novel, not yet described ones, pointing to a rather high diversity of phyllobilin-modifying activities present in the Gramineae. Along with these phyllobilins, barley orthologs of known Arabidopsis thaliana chlorophyll catabolic enzymes were demonstrated to localize in the chloroplast, and two of them, i.e. PAO and pheophytin pheophorbide hydrolase, complemented respective Arabidopsis mutants. These data confirm functionality of the PAO/phyllobilin pathway in grasses. Interestingly, when comparing phyllobilin abundance with amounts of degraded chlorophyll in senescent leaves, in most analyzed grass species only minor fractions of chlorophyll were recovered as phyllobilins, opposite to A. thaliana where phyllobilin quantities match degraded chlorophyll rather well. These data show that, despite the presence and activity of the PAO/phyllobilin pathway in barley (and other cereals), phyllobilins do not accumulate stoichiometrically, implying possible degradation of chlorophyll beyond the phyllobilin level.

Light-Regulated Synthesis of Cyclic-di-GMP by a Bidomain Construct of the Cyanobacteriochrome Tlr0924 (SesA) without Stable Dimerization.

Phytochromes and cyanobacteriochromes (CBCRs) use double-bond photoisomerization of their linear tetrapyrrole (bilin) chromophores within cGMP-specific phosphodiesterases/adenylyl cyclases/FhlA (GAF) domain-containing photosensory modules to regulate activity of C-terminal output domains. CBCRs exhibit photocycles that are much more diverse than those of phytochromes and are often found in large modular proteins such as Tlr0924 (SesA), one of three blue light regulators of cell aggregation in the cyanobacterium Thermosynechococcus elongatus. Tlr0924 contains a single bilin-binding GAF domain adjacent to a C-terminal diguanylate cyclase (GGDEF) domain whose catalytic activity requires formation of a dimeric transition state presumably supported by a multidomain extension at its N-terminus. To probe the structural basis of light-mediated signal propagation from the photosensory input domain to a signaling output domain for a representative CBCR, these studies explore the properties of a bidomain GAF-GGDEF construct of Tlr0924 (Tlr0924Δ) that retains light-regulated diguanylate cyclase activity. Surprisingly, circular dichroism spectroscopy and size exclusion chromatography data do not support formation of stable dimers in either the blue-absorbing 15ZPb dark state or the green-absorbing 15EPg photoproduct state of Tlr0924Δ. Analysis of variants containing site-specific mutations reveals that proper signal transmission requires both chromophorylation of the GAF domain and individual residues within the amphipathic linker region between GAF and GGDEF domains. On the basis of these data, we propose a model in which bilin binding and light signals are propagated from the GAF domain via the linker to alter the equilibrium and interconversion dynamics between active and inactive conformations of the GGDEF domain to favor or disfavor formation of catalytically competent dimers.

Tryptophan-Rich Sensory Protein/Translocator Protein (TSPO) from Cyanobacterium Fremyella diplosiphon Binds a Broad Range of Functionally Relevant Tetrapyrroles.

Tryptophan-rich sensory protein/translocator protein (TSPO) is a membrane protein involved in stress adaptation in the cyanobacterium Fremyella diplosiphon. Characterized mammalian and proteobacterial TSPO homologues bind tetrapyrroles and cholesterol ligands. We investigated the ligand binding properties of TSPO from F. diplosiphon (FdTSPO1), which was functionally characterized in prior genetic studies. Two additional TSPO proteins (FdTSPO2 and FdTSPO3) are present in F. diplosiphon; they are similar in size to reported bacterial TSPOs and smaller than FdTSPO1. The longer cyanobacterial TSPO1 is found almost exclusively in filamentous cyanobacteria and has a relatively low degree of homology to bacterial and mammalian TSPO homologues with confirmed tetrapyrrole binding. To probe distinctions of long-form TSPOs, we tested the binding of porphyrin and bilin to FdTSPO1 and measured binding affinities in the low micromolar range, with the highest binding affinity detected for heme. Although tetrapyrrole ligands bound FdTSPO1 with affinities similar to those previously reported for proteobacterial TSPO, binding of cholesterol to FdTSPO1 was particularly poor and was not improved by introducing an amino acid motif known to enhance cholesterol binding in other bacterial TSPO homologues. Additionally, we detected limited binding of bacterial hopanoids to FdTSPO1. Cyanobacterial TSPO1 from the oxygenic photosynthetic F. diplosiphon, thus, binds a range of tetrapyrroles of functional relevance with efficiencies similar to those of mammalian and proteobacterial homologues, but the level of cholesterol binding is greatly reduced compared to that of mammalian TSPO. Furthermore, the ΔFdTSPO1 mutant exhibits altered growth in the presence of biliverdin compared to that of wild-type cells under green light. Together, these results suggest that TSPO molecules may play roles in bilin homeostasis or trafficking in cyanobacteria.

[Clinicopathologic features of three cases of erythropoietic protoporphyria with liver involvement].

Objective: To investigate the clinicopathologic features of the erythropoietic protoporphyria (EPP) with liver involvement. Methods: The clinical findings and hepatic biopsy of 3 cases of EPP diagnosed between July, 2011 to August, 2014 with liver involvement were reviewed, with relevant literature review. Results: All patients presented with persistent and refractory abdominal pain, with obvious jaundice and deranged liver function. Imaging showed homogeneous hepatomegaly in all patients. Histologically, the hepatocytes were edematous, and contained numerous cytoplasmic globular brown pigments and bile pigments, which were also found in Kupffer cells, in the bile canaliculi and in some of dilated sinusoid. The pigments were of different sizes and showed uneven distribution. Some pigments showed bright red or yellow birefringence with a distinctive central maltese cross configuration on polarizing microscopy. Furthermore, some hepatocytes showed piecemeal necrosis and steatosis, the portal tracts were usually infiltrated by lymphocytes, with fibroplasia and biliary ductular reaction. There was no dilatation of intrahepatic bile ducts. Conclusion: Full understanding of the clinical and pathological features of EPP with liver involvement can help to recognize this small group of patients, and to offer proper effective treatments.

Structural basis for the photoconversion of a phytochrome to the activated Pfr form.

Phytochromes are a collection of bilin-containing photoreceptors that regulate numerous photoresponses in plants and microorganisms through their ability to photointerconvert between a red-light-absorbing, ground state (Pr) and a far-red-light-absorbing, photoactivated state (Pfr). Although the structures of several phytochromes as Pr have been determined, little is known about the structure of Pfr and how it initiates signalling. Here we describe the three-dimensional solution structure of the bilin-binding domain as Pfr, using the cyanobacterial phytochrome from Synechococcus OSB'. Contrary to predictions, light-induced rotation of the A pyrrole ring but not the D ring is the primary motion of the chromophore during photoconversion. Subsequent rearrangements within the protein then affect intradomain and interdomain contact sites within the phytochrome dimer. On the basis of our models, we propose that phytochromes act by propagating reversible light-driven conformational changes in the bilin to altered contacts between the adjacent output domains, which in most phytochromes direct differential phosphotransfer.