The biogenesis of Photosystem II is a complicated process requiring numerous auxiliary factors to assist in all steps of its assembly. The cyanobacterial protein Ycf39 forms a stress-induced complex with 2 small chlorophyll-binding, High-light-inducible proteins C and D (HliC and HliD), and has been reported to participate in the insertion of chlorophyll molecules into the central D1 subunit of Photosystem II. However, how this process is organized remains unknown. Here, we show that Ycf39 and both HliC and HliD can form distinct complexes with chlorophyll synthase (ChlG) in the model cyanobacterium Synechocystis sp. PCC 6803. We isolated and characterized ChlG complexes from various strains grown under different conditions and provide a mechanistic view of the docking of Ycf39 to ChlG via HliD and the structural role of HliC. In the absence of stress, chlorophyll is produced by the ChlG-HliD2-ChlG complex, which is stabilized by chlorophyll and zeaxanthin molecules bound to the HliD homodimer. The switch to high light leads to stress pressure and greatly elevated synthesis of HliC, resulting in the replacement of HliD homodimers with HliC-HliD heterodimers. Unlike HliD, HliC cannot interact directly with ChlG or Ycf39. Therefore, the original ChlG-HliD2-ChlG complex is converted into a ChlG-HliD-HliC hetero-trimer that presumably binds transiently to Ycf39 and the nascent D1 polypeptide. We speculate that this molecular machinery promotes the delivery of chlorophyll to D1 upon high-light-induced chlorophyll deficiency. The HliD homodimers formed under standard, nonstress growth conditions and attached to ChlG could serve as an emergency chlorophyll reserve.

• MeSH
• bakteriální proteiny * metabolismus   genetika   MeSH
• chlorofyl metabolismus   MeSH
• fotosystém II (proteinový komplex) * metabolismus   MeSH
• ligasy tvořící vazby C-O * metabolismus   genetika   MeSH
• světlo * MeSH
• Synechocystis * metabolismus   účinky záření   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny * MeSH
• chlorofyl MeSH
• chlorophyll synthetase MeSH Prohlížeč
• fotosystém II (proteinový komplex) * MeSH
• high light-inducible protein, cyanobacteria MeSH Prohlížeč
• ligasy tvořící vazby C-O * MeSH
• světlosběrné proteinové komplexy MeSH

The biogenesis of the cyanobacterial photosystem II (PSII) complex requires a number of auxiliary assembly factors that improve efficiency of the process but their precise function is not well understood. To assess a possible synergic action of the Ycf48 and Ycf39 factors acting in early steps of the biogenesis via interaction with the nascent D1 subunit of PSII, we constructed and characterised a double mutant of the cyanobacterium Synechocystis PCC 6803 lacking both these proteins. In addition, we also deleted the ycf39 gene in the double mutant lacking Ycf48 and Pam68, the latter being a ribosomal factor promoting insertion of chlorophyll (Chl) into the CP47 subunit of PSII. The resulting double ΔYcf48/ΔYcf39 and triple ΔYcf48/ΔPam68/ΔYcf39 mutants were deficient in PSII and total Chl, and in contrast to the source mutants, they lost the capacity for autotrophy. Interestingly, autotrophic growth was restored in both of the new multiple mutants by enhancing Chl biosynthesis using a specific ferrochelatase inhibitor. Taking together with the weak radioactive labelling of the D1 protein, these findings can be explained by inhibition of the D1 synthesis caused by the lack and/or incorrect binding of Chl molecules. The results emphasise the key importance of the sufficient Chl supply for the PSII biogenesis and also support the existence of a so far enigmatic regulatory mechanism leading to the reduced overall Chl biosynthesis/accumulation when the PSII assembly is impaired.

• MeSH
• autotrofní procesy MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• chlorofyl metabolismus   MeSH
• delece genu MeSH
• fotosystém II (proteinový komplex) genetika   metabolismus   MeSH
• mutace MeSH
• Synechocystis genetika   růst a vývoj   metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• chlorofyl MeSH
• fotosystém II (proteinový komplex) MeSH

Oxygenic photosynthesis relies on accessory factors to promote the assembly and maintenance of the photosynthetic apparatus in the thylakoid membranes. The highly conserved membrane-bound rubredoxin-like protein RubA has previously been implicated in the accumulation of both PSI and PSII, but its mode of action remains unclear. Here, we show that RubA in the cyanobacterium Synechocystis sp PCC 6803 is required for photoautotrophic growth in fluctuating light and acts early in PSII biogenesis by promoting the formation of the heterodimeric D1/D2 reaction center complex, the site of primary photochemistry. We find that RubA, like the accessory factor Ycf48, is a component of the initial D1 assembly module as well as larger PSII assembly intermediates and that the redox-responsive rubredoxin-like domain is located on the cytoplasmic surface of PSII complexes. Fusion of RubA to Ycf48 still permits normal PSII assembly, suggesting a spatiotemporal proximity of both proteins during their action. RubA is also important for the accumulation of PSI, but this is an indirect effect stemming from the downregulation of light-dependent chlorophyll biosynthesis induced by PSII deficiency. Overall, our data support the involvement of RubA in the redox control of PSII biogenesis.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• biologické pigmenty izolace a purifikace   MeSH
• chlorofyl biosyntéza   MeSH
• fotosyntéza fyziologie   MeSH
• fotosystém I (proteinový komplex) metabolismus   MeSH
• fotosystém II (proteinový komplex) metabolismus   MeSH
• mutace MeSH
• rubredoxiny chemie   genetika   metabolismus   MeSH
• Synechocystis genetika   růst a vývoj   metabolismus   MeSH
• tylakoidy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• biologické pigmenty MeSH
• chlorofyl MeSH
• fotosystém I (proteinový komplex) MeSH
• fotosystém II (proteinový komplex) MeSH
• rubredoxiny MeSH

Ferrochelatase (FeCh) is an essential enzyme catalyzing the synthesis of heme. Interestingly, in cyanobacteria, algae, and plants, FeCh possesses a conserved transmembrane chlorophyll a/b binding (CAB) domain that resembles the first and the third helix of light-harvesting complexes, including a chlorophyll-binding motif. Whether the FeCh CAB domain also binds chlorophyll is unknown. Here, using biochemical and radiolabeled precursor experiments, we found that partially inhibited activity of FeCh in the cyanobacterium Synechocystis PCC 6803 leads to overproduction of chlorophyll molecules that accumulate in the thylakoid membrane and, together with carotenoids, bind to FeCh. We observed that pigments bound to purified FeCh are organized in an energy-dissipative conformation and further show that FeCh can exist in vivo as a monomer or a dimer depending on its own activity. However, pigmented FeCh was purified exclusively as a dimer. Separately expressed and purified FeCH CAB domain contained a pigment composition similar to that of full-length FeCh and retained its quenching properties. Phylogenetic analysis suggested that the CAB domain was acquired by a fusion between FeCh and a single-helix, high light-inducible protein early in the evolution of cyanobacteria. Following this fusion, the FeCh CAB domain with a functional chlorophyll-binding motif was retained in all currently known cyanobacterial genomes except for a single lineage of endosymbiotic cyanobacteria. Our findings indicate that FeCh from Synechocystis exists mostly as a pigment-free monomer in cells but can dimerize, in which case its CAB domain creates a functional pigment-binding segment organized in an energy-dissipating configuration.

• Klíčová slova
• Synechocystis, carotenoid, chlorophyll, chloroplast, ferrochelatase, heme, light harvesting complex (LHC)-like proteins, membrane protein, photosynthesis, photosynthetic pigment, pigment binding, plant biochemistry,
• MeSH
• chlorofyl a metabolismus   MeSH
• chlorofyl metabolismus   MeSH
• dimerizace MeSH
• ferrochelatasa chemie   metabolismus   MeSH
• fylogeneze MeSH
• karotenoidy metabolismus   MeSH
• konformace proteinů MeSH
• světlosběrné proteinové komplexy metabolismus   MeSH
• Synechocystis enzymologie   MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chlorofyl a MeSH
• chlorofyl MeSH
• chlorophyll b MeSH Prohlížeč
• ferrochelatasa MeSH
• karotenoidy MeSH
• světlosběrné proteinové komplexy MeSH

Robust photosynthesis in chloroplasts and cyanobacteria requires the participation of accessory proteins to facilitate the assembly and maintenance of the photosynthetic apparatus located within the thylakoid membranes. The highly conserved Ycf48 protein acts early in the biogenesis of the oxygen-evolving photosystem II (PSII) complex by binding to newly synthesized precursor D1 subunit and by promoting efficient association with the D2 protein to form a PSII reaction center (PSII RC) assembly intermediate. Ycf48 is also required for efficient replacement of damaged D1 during the repair of PSII. However, the structural features underpinning Ycf48 function remain unclear. Here we show that Ycf48 proteins encoded by the thermophilic cyanobacterium Thermosynechococcus elongatus and the red alga Cyanidioschyzon merolae form seven-bladed beta-propellers with the 19-aa insertion characteristic of eukaryotic Ycf48 located at the junction of blades 3 and 4. Knowledge of these structures has allowed us to identify a conserved "Arg patch" on the surface of Ycf48 that is important for binding of Ycf48 to PSII RCs but also to larger complexes, including trimeric photosystem I (PSI). Reduced accumulation of chlorophyll in the absence of Ycf48 and the association of Ycf48 with PSI provide evidence of a more wide-ranging role for Ycf48 in the biogenesis of the photosynthetic apparatus than previously thought. Copurification of Ycf48 with the cyanobacterial YidC protein insertase supports the involvement of Ycf48 during the cotranslational insertion of chlorophyll-binding apopolypeptides into the membrane.

• Klíčová slova
• chlorophyll-binding proteins, photosynthesis, photosystem II,
• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• fotosystém I (proteinový komplex) biosyntéza   genetika   MeSH
• fotosystém II (proteinový komplex) biosyntéza   genetika   MeSH
• sinice genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• fotosystém I (proteinový komplex) MeSH
• fotosystém II (proteinový komplex) MeSH

Photosystem II (PSII) is a large enzyme complex embedded in the thylakoid membrane of oxygenic phototrophs. The biogenesis of PSII requires the assembly of more than 30 subunits, with the assistance of a number of auxiliary proteins. In plants and cyanobacteria, the photosynthesis-affected mutant 68 (Pam68) is important for PSII assembly. However, its mechanisms of action remain unknown. Using a Synechocystis PCC 6803 strain expressing Flag-tagged Pam68, we purified a large protein complex containing ribosomes, SecY translocase, and the chlorophyll-binding PSII inner antenna CP47. Using 2D gel electrophoresis, we identified a pigmented Pam68-CP47 subcomplex and found Pam68 bound to ribosomes. Our results show that Pam68 binds to ribosomes even in the absence of CP47 translation. Furthermore, Pam68 associates with CP47 at an early phase of its biogenesis and promotes the synthesis of this chlorophyll-binding polypeptide until the attachment of the small PSII subunit PsbH. Deletion of both Pam68 and PsbH nearly abolishes the synthesis of CP47, which can be restored by enhancing chlorophyll biosynthesis. These results strongly suggest that ribosome-bound Pam68 stabilizes membrane segments of CP47 and facilitates the insertion of chlorophyll molecules into the translated CP47 polypeptide chain.

• MeSH
• 2D gelová elektroforéza MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• chlorofyl metabolismus   MeSH
• fosfoproteiny genetika   metabolismus   MeSH
• fotosystém II (proteinový komplex) genetika   metabolismus   MeSH
• mutace MeSH
• ribozomy metabolismus   MeSH
• světlosběrné proteinové komplexy genetika   metabolismus   MeSH
• Synechocystis genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• chlorofyl MeSH
• fosfoproteiny MeSH
• fotosystém II (proteinový komplex) MeSH
• photosystem II, chlorophyll-binding protein, CP-47 MeSH Prohlížeč
• photosystem II, psbH subunit MeSH Prohlížeč
• světlosběrné proteinové komplexy MeSH

In oxygenic phototrophs, chlorophylls, hemes, and bilins are synthesized by a common branched pathway. Given the phototoxic nature of tetrapyrroles, this pathway must be tightly regulated, and an important regulatory role is attributed to magnesium chelatase enzyme at the branching between the heme and chlorophyll pathway. Gun4 is a porphyrin-binding protein known to stimulate in vitro the magnesium chelatase activity, but how the Gun4-porphyrin complex acts in the cell was unknown. To address this issue, we first performed simulations to determine the porphyrin-docking mechanism to the cyanobacterial Gun4 structure. After correcting crystallographic loop contacts, we determined the binding site for magnesium protoporphyrin IX. Molecular modeling revealed that the orientation of α6/α7 loop is critical for the binding, and the magnesium ion held within the porphyrin is coordinated by Asn-211 residue. We also identified the basis for stronger binding in the Gun4-1 variant and for weaker binding in the W192A mutant. The W192A-Gun4 was further characterized in magnesium chelatase assay showing that tight porphyrin binding in Gun4 facilitates its interaction with the magnesium chelatase ChlH subunit. Finally, we introduced the W192A mutation into cells and show that the Gun4-porphyrin complex is important for the accumulation of ChlH and for channeling metabolites into the chlorophyll biosynthetic pathway.

• Klíčová slova
• Gun4, Mgprotoporphyrin IX, Synechocystis 6803, chlorophyll, cyanobacteria, docking, molecular modeling, porphyrin,
• MeSH
• bakteriální proteiny chemie   metabolismus   MeSH
• chlorofyl biosyntéza   MeSH
• cirkulární dichroismus MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• mutace MeSH
• porfyriny metabolismus   MeSH
• simulace molekulární dynamiky MeSH
• Synechocystis genetika   růst a vývoj   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• chlorofyl MeSH
• porfyriny MeSH

Efficient assembly and repair of the oxygen-evolving photosystem II (PSII) complex is vital for maintaining photosynthetic activity in plants, algae, and cyanobacteria. How chlorophyll is delivered to PSII during assembly and how vulnerable assembly complexes are protected from photodamage are unknown. Here, we identify a chlorophyll and β-carotene binding protein complex in the cyanobacterium Synechocystis PCC 6803 important for formation of the D1/D2 reaction center assembly complex. It is composed of putative short-chain dehydrogenase/reductase Ycf39, encoded by the slr0399 gene, and two members of the high-light-inducible protein (Hlip) family, HliC and HliD, which are small membrane proteins related to the light-harvesting chlorophyll binding complexes found in plants. Perturbed chlorophyll recycling in a Ycf39-null mutant and copurification of chlorophyll synthase and unassembled D1 with the Ycf39-Hlip complex indicate a role in the delivery of chlorophyll to newly synthesized D1. Sequence similarities suggest the presence of a related complex in chloroplasts.

• MeSH
• fotosystém II (proteinový komplex) metabolismus   MeSH
• proteiny vázající chlorofyl metabolismus   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• Synechocystis metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fotosystém II (proteinový komplex) MeSH
• proteiny vázající chlorofyl MeSH

Chlorophyll (Chl) is an essential component of the photosynthetic apparatus. Embedded into Chl-binding proteins, Chl molecules play a central role in light harvesting and charge separation within the photosystems. It is critical for the photosynthetic cell to not only ensure the synthesis of a sufficient amount of new Chl-binding proteins but also avoids any misbalance between apoprotein synthesis and the formation of potentially phototoxic Chl molecules. According to the available data, Chl-binding proteins are translated on membrane bound ribosomes and their integration into the membrane is provided by the SecYEG/Alb3 translocon machinery. It appears that the insertion of Chl molecules into growing polypeptide is a prerequisite for the correct folding and finishing of Chl-binding protein synthesis. Although the Chl biosynthetic pathway is fairly well-described on the level of enzymatic steps, a link between Chl biosynthesis and the synthesis of apoproteins remains elusive. In this review, I summarize the current knowledge about this issue putting emphasis on protein-protein interactions. I present a model of the Chl biosynthetic pathway organized into a multi-enzymatic complex and physically attached to the SecYEG/Alb3 translocon. Localization of this hypothetical large biosynthetic centre in the cyanobacterial cell is also discussed as well as regulatory mechanisms coordinating the rate of Chl and apoprotein synthesis.

• MeSH
• bakteriální proteiny metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• chlorofyl metabolismus   MeSH
• fotosyntéza MeSH
• proteiny vázající chlorofyl biosyntéza   MeSH
• sinice cytologie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• bakteriální proteiny MeSH
• chlorofyl MeSH
• proteiny vázající chlorofyl MeSH

Ferrochelatase (FeCH) catalyzes the insertion of Fe(2+) into protoporphyrin, forming protoheme. In photosynthetic organisms, FeCH and magnesium chelatase lie at a biosynthetic branch point where partitioning down the heme and chlorophyll (Chl) pathways occurs. Unlike their mammalian, yeast, and other bacterial counterparts, cyanobacterial and algal FeCHs as well as FeCH2 isoform from plants possess a carboxyl-terminal Chl a/b-binding (CAB) domain with a conserved Chl-binding motif. The CAB domain is connected to the FeCH catalytic core by a proline-rich linker sequence (region II). In order to dissect the regulatory, catalytic, and structural roles of the region II and CAB domains, we analyzed a FeCH ΔH347 mutant that retains region II but lacks the CAB domain and compared it with the ΔH324-FeCH mutant that lacks both these domains. We found that the CAB domain is not required for catalytic activity but is essential for dimerization of FeCH; its absence causes aberrant accumulation of Chl-protein complexes under high light accompanied by high levels of the Chl precursor chlorophyllide. Thus, the CAB domain appears to serve mainly a regulatory function, possibly in balancing Chl biosynthesis with the synthesis of cognate apoproteins. Region II is essential for the catalytic function of the plastid-type FeCH enzyme, although the low residual activity of the ΔH324-FeCH is more than sufficient to furnish the cellular demand for heme. We propose that the apparent surplus of FeCH activity in the wild type is critical for cell viability under high light due to a regulatory role of FeCH in the distribution of Chl into apoproteins.

• MeSH
• aklimatizace MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• chlorofyl biosyntéza   MeSH
• ferrochelatasa genetika   metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• multimerizace proteinu MeSH
• mutace MeSH
• světlo MeSH
• Synechocystis enzymologie   genetika   růst a vývoj   MeSH
• tetrapyrroly biosyntéza   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• chlorofyl MeSH
• ferrochelatasa MeSH
• tetrapyrroly MeSH