Mutagenesis of Ile184 in the cd-loop of the photosystem II D1 protein modifies acceptor-side function via spontaneous mutation of D1-His252 in Synechocystis sp. PCC 6803.

The Photosystem II water-plastoquinone oxidoreductase is a multi-subunit complex which catalyses the light-driven oxidation of water to molecular oxygen in oxygenic photosynthesis. The D1 reaction centre protein exists in multiple forms in cyanobacteria, including D1FR which is expressed under far-red light. We investigated the role of Phe184 that is found in the lumenal cd-loop of D1FR but is typically an isoleucine in other D1 isoforms. The I184F mutant in Synechocystis sp. PCC 6803 was similar to the control strain but accumulated a spontaneous mutation that introduced a Gln residue in place of His252 located on the opposite side of the thylakoid membrane. His252 participates in the protonation of the secondary plastoquinone electron acceptor QB. The I184F:H252Q double mutant exhibited reduced high-light-induced photodamage and an altered QB-binding site that impaired herbicide binding. Additionally, the H252Q mutant had a large increase in the variable fluorescence yield although the number of photochemically active PS II centres was unchanged. In the I184F:H252Q mutant the extent of the increased fluorescence yield decreased. Our data indicates substitution of Ile184 to Phe modulates PS II-specific variable fluorescence in cells with the His252 to Gln substitution by modifying the QB-binding site.

Indirect interactions involving the PsbM or PsbT subunits and the PsbO, PsbU and PsbV proteins stabilize assembly and activity of Photosystem II in Synechocystis sp. PCC 6803.

The low-molecular-weight PsbM and PsbT proteins of Photosystem II (PS II) are both located at the monomer-monomer interface of the mature PS II dimer. Since the extrinsic proteins are associated with the final step of assembly of an active PS II monomer and, in the case of PsbO, are known to impact the stability of the PS II dimer, we have investigated the potential cooperativity between the PsbM and PsbT subunits and the PsbO, PsbU and PsbV extrinsic proteins. Blue-native polyacrylamide electrophoresis and western blotting detected stable PS II monomers in the ∆PsbM:∆PsbO and ∆PsbT:∆PsbO mutants that retained sufficient oxygen-evolving activity to support reduced photoautotrophic growth. In contrast, the ∆PsbM:∆PsbU and ∆PsbT:∆PsbU mutants assembled dimeric PS II at levels comparable to wild type and supported photoautotrophic growth at rates similar to those obtained with the corresponding ∆PsbM and ∆PsbT cells. Removal of PsbV was more detrimental than removal of PsbO. Only limited levels of dimeric PS II were observed in the ∆PsbM:∆PsbV mutant and the overall reduced level of assembled PS II in this mutant resulted in diminished rates of photoautotrophic growth and PS II activity below those obtained in the ∆PsbM:∆PsbO and ∆PsbT:∆PsbO strains. In addition, the ∆PsbT:∆PsbV mutant did not assemble active PS II centers although inactive monomers could be detected. The inability of the ∆PsbT:∆PsbV mutant to grow photoautotrophically, or to evolve oxygen, suggested a stable oxygen-evolving complex could not assemble in this mutant.

International conference on "Photosynthesis and Hydrogen Energy Research for Sustainability-2023": in honor of Robert Blankenship, Gyozo Garab, Michael Grätzel, Norman Hüner and Gunnar Öquist.

The 11th International Photosynthesis Conference on Hydrogen Energy Research and Sustainability 2023 was organized in honor of Robert Blankenship, Gyozo Garab, Michael Grätzel, Norman Hüner, and Gunnar Öquist, in Istanbul, Türkiye at Bahçesehir University Future Campus from 03 to 09 July 2023. It was jointly supported by the International Society of Photosynthesis Research (ISPR) and the International Association for Hydrogen Energy (IAHE). In this article we provide brief details of the conference, its events, keynote speakers, and the scientific contribution of scientists honored at this conference. Further, we also describe the participation of young researchers, their talks, and their awards.

Lys264 of the D2 Protein Performs a Dual Role in Photosystem II Modifying Assembly and Electron Transfer through the Quinone-Iron Acceptor Complex.

Bicarbonate (HCO3-) binding regulates electron flow between the primary (QA) and secondary (QB) plastoquinone electron acceptors of Photosystem II (PS II). Lys264 of the D2 subunit of PS II contributes to a hydrogen-bond network that stabilizes HCO3- ligation to the non-heme iron in the QA-Fe-QB complex. Using the cyanobacterium Synechocystis sp. PCC 6803, alanine and glutamate were introduced to create the K264A and K264E mutants. Photoautotrophic growth was slowed in K264E cells but not in the K264A strain. Both mutants accumulated an unassembled CP43 precomplex as well as the CP43-lacking RC47 assembly intermediate, indicating weakened binding of the CP43 precomplex to RC47. Assembly was impeded more in K264E cells than in the K264A strain, but K264A cells were more susceptible to high-light-induced photodamage when assayed using PS II-specific electron acceptors. Furthermore, an impaired repair mechanism was observed in the K264A mutant in protein labeling experiments. Unexpectedly, unlike the K264A strain, the K264E mutant displayed inhibited oxygen evolution following high-light exposure when HCO3- was added to support whole chain electron transport. In both mutants, the decay of chlorophyll fluorescence was slowed, indicating impaired electron transfer between QA and QB. Furthermore, the fluorescence decay kinetics in the K264E strain were insensitive to addition of either formate or HCO3-, whereas HCO3--reversible formate-induced inhibition in the K264A mutant was observed. Exchange of plastoquinol with the membrane plastoquinone pool at the QB-binding site was also retarded in both mutants. Hence, D2-Lys264 possesses key roles in both assembly and activity of PS II.

Expression of the far-red D1 protein or introduction of conserved far-red D1 residues into Synechocystis sp. PCC 6803 impairs Photosystem II.

The wavelengths of light harvested in oxygenic photosynthesis are ~400-700 nm. Some cyanobacteria respond to far-red light exposure via a process called far-red light photoacclimation which enables absorption of light at wavelengths >700 nm and its use to support photosynthesis. Far-red-light-induced changes include up-regulation of alternative copies of multiple proteins of Photosystem II (PS II). This includes an alternative copy of the D1 protein, D1FR . Here, we show that D1FR introduced into Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803) can be incorporated into PS II centres that evolve oxygen at low rates but cannot support photoautotrophic growth. Using mutagenesis to modify the psbA2 gene of Synechocystis 6803, we modified residues in helices A, B, and C to be characteristic of D1FR residues. Modification of the Synechocystis 6803 helix A to resemble the D1FR helix A, with modifications in the region of the bound ß-carotene (CarD1 ) and the accessory chlorophyll, ChlZD1 , produced a strain with a similar phenotype to the D1FR strain. In contrast, the D1FR changes in helices B and C had minor impacts on photoautotrophy but impacted the function of PS II, possibly through a change in the equilibrium for electron sharing between the primary and secondary plastoquinone electron acceptors QA and QB in favour of QA - . The addition of combinations of residue changes in helix C indicates compensating effects may occur and highlight the need to experimentally determine the impact of multiple residue changes.

Tyr244 of the D2 Protein Is Required for Correct Assembly and Operation of the Quinone-Iron-Bicarbonate Acceptor Complex of Photosystem II.

Two plastoquinone electron acceptors, QA and QB, are present in Photosystem II (PS II) with their binding sites formed by the D2 and D1 proteins, respectively. A hexacoordinate non-heme iron is bound between QA and QB by D2 and D1, each providing two histidine ligands, and a bicarbonate that is stabilized via hydrogen bonds with D2-Tyr244 and D1-Tyr246. Both tyrosines and bicarbonate are conserved in oxygenic photosynthetic organisms but absent from the corresponding quinone-iron electron acceptor complex of anoxygenic photosynthetic bacteria. We investigated the role of D2-Tyr244 by introducing mutations in the cyanobacterium Synechocystis sp. PCC 6803. Alanine, histidine, and phenylalanine substitutions were introduced creating the Y244A, Y244H, and Y244F mutants. Electron transfer between QA and QB was impaired, the back-reaction with the S2 state of the oxygen-evolving complex was modified, and PS II assembly was disrupted, with the Y244A strain being more affected than the Y244F and Y244H mutants. The strains were also highly susceptible to photodamage in the presence of PS II-specific electron acceptors. Thermoluminescence and chlorophyll a fluorescence decay measurements indicated that the redox potential of the QA/QA- couple became more positive in the Y244F and Y244H mutants, consistent with bicarbonate binding being impacted. The replacement of Tyr244 by alanine also led to an insertion of two amino acid repeats from Gln239 to Ala249 within the DE loop of D2, resulting in an inactive PS II complex that lacked PS II-specific variable fluorescence. The 66 bp insertion giving rise to the inserted amino acids therefore created an obligate photoheterotrophic mutant.

The PsbJ protein is required for photosystem II activity in centers lacking the PsbO and PsbV lumenal subunits.

Photosystem II (PS II) of oxygenic photosynthesis is found in the thylakoid membranes of plastids and cyanobacteria. The mature PS II complex comprises a central core of four membrane proteins that bind the majority of the redox-active cofactors. In cyanobacteria the central core is surrounded by 13 low-molecular-weight (LMW) subunits which each consist of one or two transmembrane helices. Three additional hydrophilic subunits known as PsbO, PsbU and PsbV are found associated with hydrophilic loops belonging to the core proteins protruding into the thylakoid lumen. During biogenesis the majority of the LMW subunits are known to initially associate with individual pre-assembly complexes consisting of one or more of the core proteins; however, the point at which the PsbJ LMW subunit binds to PS II is not known. The majority of models for PS II biogenesis propose that the three extrinsic proteins and PsbJ bind in the final stages of PS II assembly. We have investigated the impact of creating the double mutants ∆PsbJ:∆PsbO, ∆PsbJ:∆PsbU and ∆PsbJ:∆PsbV to investigate potential cooperation between these subunits in the final stages of biogenesis. Our results indicate that PsbJ can bind to PS II in the absence of any one of the extrinsic proteins. However, unlike their respective single mutants, the ∆PsbJ:∆PsbO and ∆PsbJ:∆PsbV strains were not photoautotrophic and were unable to support oxygen evolution suggesting a functional oxygen-evolving complex could not assemble in these strains. In contrast, the PS II centers formed in the ∆PsbJ:∆PsbU strain were capable of photoautotrophic growth and could support oxygen evolution when whole-chain electron transport was supported by the addition of bicarbonate.

The Interaction between PsbT and the DE Loop of D1 in Photosystem II Stabilizes the Quinone-Iron Electron Acceptor Complex.

The X-ray-derived Photosystem II (PS II) structure from the thermophilic cyanobacterium Thermosynechococcus vulcanus (Protein Data Bank entry 4UB6) indicates Phe239 of the DE loop of the D1 protein forms a hydrophobic interaction with Pro27 and Ile29 at the C-terminus of the 5 kDa PsbT protein found at the monomer-monomer interface of the PS II dimer. To investigate the importance of this interaction, we created the F239A and F239L mutants in Synechocystis sp. PCC 6803 through targeted mutagenesis of the D1:Phe239 residue into either an alanine or a leucine. Under moderate-light conditions, the F239A strain displayed reduced rates of oxygen evolution and impaired rates of fluorescence decay following a single-turnover actinic flash, while the F239L strain behaved like the control; however, under high-light conditions, the F239A and F239L strains grew more slowly than the control. Our results indicate the quinone-iron acceptor complex becomes more accessible to exogenously added electron acceptors in the F239A mutant and a ΔPsbT strain when compared with the control and F239L strains. This led to the hypothesis that the interaction between D1:Phe239 and the PsbT subunit is required to restrict movement of the DE loop of the D1 subunit, and we suggest disruption of this interaction may perturb the binding of bicarbonate to the non-heme iron and contribute to the signal for PS II to undergo repair following photodamage.

The hydrophobicity of mutations targeting D1:Val219 modifies formate and diuron binding in the quinone-Fe-acceptor complex of Photosystem II.

The D1:Val219 residue of Photosystem II in the cyanobacterium Synechocystis sp. PCC 6803 was mutated to alanine or isoleucine, creating the V219A and V219I mutants, respectively. Oxygen evolution was slowed in these mutants, while chlorophyll a fluorescence induction assays indicated slowed electron transfer. As previously observed [Erickson J.M., Rahire, M., Rochaix, J.-D. and Mets. L. (1985) Science, 228, 204-207], the V219I mutant was resistant to 3,4-dichloro-1,1-dimethyl urea (DCMU); however, the V219A strain displayed no DCMU resistance. Additionally, the V219A strain was less sensitive to the addition of formate than the control, while the V219I strain was more sensitive to formate. Both mutant strains were susceptible to photodamage and required protein synthesis for recovery. We hypothesize that the sensitivity to DCMU and the extent of bicarbonate-reversible formate-induced inhibition, as well as the capacity for recovery in cells following photodamage, are influenced by the hydrophobicity of the environment associated with the Val219 residue in D1.

The diversity and distribution of D1 proteins in cyanobacteria.

The psbA gene family in cyanobacteria encodes different forms of the D1 protein that is part of the Photosystem II reaction centre. We have identified a phylogenetically distinct D1 group that is intermediate between previously identified G3-D1 and G4-D1 proteins (Cardona et al. Mol Biol Evol 32:1310-1328, 2015). This new group contained two subgroups: D1INT, which was frequently in the genomes of heterocystous cyanobacteria and D1FR that was part of the far-red light photoacclimation gene cluster of cyanobacteria. In addition, we have identified subgroups within G3, the micro-aerobically expressed D1 protein. There are amino acid changes associated with each of the subgroups that might affect the function of Photosystem II. We show a phylogenetically broad range of cyanobacteria have these D1 types, as well as the genes encoding the G2 protein and chlorophyll f synthase. We suggest identification of additional D1 isoforms and the presence of multiple D1 isoforms in phylogenetically diverse cyanobacteria supports the role of these proteins in conferring a selective advantage under specific conditions.

International conference on "Photosynthesis and Hydrogen Energy Research for Sustainability-2019": in honor of Tingyun Kuang, Anthony Larkum, Cesare Marchetti, and Kimiyuki Satoh.

The 10th International Conference on «Photosynthesis and Hydrogen Energy Research for Sustainability-2019¼ was held in honor of Tingyun Kuang (China), Anthony Larkum (Australia), Cesare Marchetti (Italy), and Kimiyuki Satoh (Japan), in St. Petersburg (Russia) during June 23-28, 2019. The official conference organizers from the Russian side were from the Institute of Basic Biological Problems of the Russian Academy of Sciences (IBBP RAS), Russian Society for Photobiology (RSP), and the Komarov Botanical Institute of the Russian Academy of Sciences ([K]BIN RAS). This conference was organized with the help of Monomax Company, a member of the International Congress Convention Association (ICCA), and was supported by the Ministry of Education and Science of the Russian Federation. Here, we provide a brief description of the conference, its scientific program, as well as a brief introduction and key contributions of the four honored scientists. Further, we emphasize the recognition given, at this conference, to several outstanding young researchers, from around the World, for their research in the area of our conference. A special feature of this paper is the inclusion of photographs provided by one of us (Tatsuya Tomo). Lastly, we urge the readers to watch for information on the next 11th conference on "Photosynthesis and Hydrogen Energy Research for Sustainability-2021," to be held in Bulgaria in 2021.

Manganese Compounds as Water-Oxidizing Catalysts: From the Natural Water-Oxidizing Complex to Nanosized Manganese Oxide Structures.

All cyanobacteria, algae, and plants use a similar water-oxidizing catalyst for water oxidation. This catalyst is housed in Photosystem II, a membrane-protein complex that functions as a light-driven water oxidase in oxygenic photosynthesis. Water oxidation is also an important reaction in artificial photosynthesis because it has the potential to provide cheap electrons from water for hydrogen production or for the reduction of carbon dioxide on an industrial scale. The water-oxidizing complex of Photosystem II is a Mn-Ca cluster that oxidizes water with a low overpotential and high turnover frequency number of up to 25-90 molecules of O2 released per second. In this Review, we discuss the atomic structure of the Mn-Ca cluster of the Photosystem II water-oxidizing complex from the viewpoint that the underlying mechanism can be informative when designing artificial water-oxidizing catalysts. This is followed by consideration of functional Mn-based model complexes for water oxidation and the issue of Mn complexes decomposing to Mn oxide. We then provide a detailed assessment of the chemistry of Mn oxides by considering how their bulk and nanoscale properties contribute to their effectiveness as water-oxidizing catalysts.

Comparison of D1´- and D1-containing PS II reaction centre complexes under different environmental conditions in Synechocystis sp. PCC 6803.

In oxygenic photosynthesis, the D1 protein of Photosystem II is the primary target of photodamage and environmental stress can accelerate this process. The cyanobacterial response to stress includes transcriptional regulation of genes encoding D1, including low-oxygen-induction of psbA1 encoding the D1´ protein in Synechocystis sp. PCC 6803. The psbA1 gene is also transiently up-regulated in high light, and its deletion has been reported to increase ammonium-induced photoinhibition. Therefore we investigated the role of D1´-containing PS II centres under different environmental conditions. A strain containing only D1´-PS II centres under aerobic conditions exhibited increased sensitivity to ammonium chloride and high light compared to a D1-containing strain. Additionally a D1´-PS II strain was outperformed by a D1-PS II strain under normal conditions; however, a strain containing low-oxygen-induced D1´-PS II centres was more resilient under high light than an equivalent D1 strain. These D1´-containing centres had chlorophyll a fluorescence characteristics indicative of altered forward electron transport and back charge recombination with the donor side of PS II. Our results indicate D1´-PS II centres are important in the reconfiguration of thylakoid electron transport in response to high light and low oxygen.

The importance of the hydrophilic region of PsbL for the plastoquinone electron acceptor complex of Photosystem II.

The PsbL protein is a 4.5kDa subunit at the monomer-monomer interface of Photosystem II (PS II) consisting of a single membrane-spanning domain and a hydrophilic stretch of ~15 residues facing the cytosolic (or stromal) side of the photosystem. Deletion of conserved residues in the N-terminal region has been used to investigate the importance of this hydrophilic extension. Using Synechocystis sp. PCC 6803, three deletion strains: ∆(N6-N8), ∆(P11-V12) and ∆(E13-N15), have been created. The ∆(N6-N8) and ∆(P11-V12) strains remained photoautotrophic but were more susceptible to photodamage than the wild type; however, the ∆(E13-N15) cells had the most severe phenotype. The Δ(E13-N15) mutant showed decreased photoautotrophic growth, a reduced number of PS II centers, impaired oxygen evolution in the presence of PS II-specific electron acceptors, and was highly susceptible to photodamage. The decay kinetics of chlorophyll a variable fluorescence after a single turnover saturating flash and the sensitivity to low concentrations of PS II-directed herbicides in the Δ(E13-N15) strain indicate that the binding of plastoquinone to the QB-binding site had been altered such that the affinity of QB is reduced. In addition, the PS II-specific electron acceptor 2,5-dimethyl-p-benzoquinone was found to inhibit electron transfer through the quinone-acceptor complex of the ∆(E13-N15) strain. The PsbL Y20A mutant was also investigated and it exhibited increased susceptibility to photodamage and increased herbicide sensitivity. Our data suggest that the N-terminal hydrophilic region of PsbL influences forward electron transfer from QA through indirect interactions with the D-E loop of the D1 reaction center protein. Our results further indicate that disruption of interactions between the N-terminal region of PsbL and other PS II subunits or lipids destabilizes PS II dimer formation. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.

Water exchange in manganese-based water-oxidizing catalysts in photosynthetic systems: from the water-oxidizing complex in photosystem II to nano-sized manganese oxides.

The water-oxidizing complex (WOC), also known as the oxygen-evolving complex (OEC), of photosystem II in oxygenic photosynthetic organisms efficiently catalyzes water oxidation. It is, therefore, responsible for the presence of oxygen in the Earth's atmosphere. The WOC is a manganese-calcium (Mn4CaO5(H2O)4) cluster housed in a protein complex. In this review, we focus on water exchange chemistry of metal hydrates and discuss the mechanisms and factors affecting this chemical process. Further, water exchange rates for both the biological cofactor and synthetic manganese water splitting are discussed. The importance of fully unveiling the water exchange mechanism to understand the chemistry of water oxidation is also emphasized here. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.

Dynamics of Photosynthesis in a Glycogen-Deficient glgC Mutant of Synechococcus sp. Strain PCC 7002.

Cyanobacterial glycogen-deficient mutants display impaired degradation of light-harvesting phycobilisomes under nitrogen-limiting growth conditions and secrete a suite of organic acids as a putative reductant-spilling mechanism. This genetic background, therefore, represents an important platform to better understand the complex relationships between light harvesting, photosynthetic electron transport, carbon fixation, and carbon/nitrogen metabolisms. In this study, we conducted a comprehensive analysis of the dynamics of photosynthesis as a function of reductant sink manipulation in a glycogen-deficient glgC mutant of Synechococcus sp. strain PCC 7002. The glgC mutant showed increased susceptibility to photoinhibition during the initial phase of nitrogen deprivation. However, after extended periods of nitrogen deprivation, glgC mutant cells maintained higher levels of photosynthetic activity than the wild type, supporting continuous organic acid secretion in the absence of biomass accumulation. In contrast to the wild type, the glgC mutant maintained efficient energy transfer from phycobilisomes to photosystem II (PSII) reaction centers, had an elevated PSII/PSI ratio as a result of reduced PSII degradation, and retained a nitrogen-replete-type ultrastructure, including an extensive thylakoid membrane network, after prolonged nitrogen deprivation. Together, these results suggest that multiple global signals for nitrogen deprivation are not activated in the glgC mutant, allowing the maintenance of active photosynthetic complexes under conditions where photosynthesis would normally be abolished.

Characterization of a Synechocystis sp. PCC 6803 double mutant lacking the CyanoP and Ycf48 proteins of Photosystem II.

Homologs of the Photosystem II (PS II) subunit PsbP are found in plants, algae, and cyanobacteria. In higher plants, PsbP is associated with mature PS II centers, but in cyanobacteria, the homologous CyanoP protein appears sub-stoichiometric to PS II. We have investigated the role of CyanoP by characterizing knockout mutants of the cyanobacterium Synechocystis sp. PCC 6803. Removal of CyanoP resulted in changes to phycobilisome coupling and energy transfer to PS II, but the function of PS II itself remained similar to wild type. We therefore investigated the hypothesis that CyanoP is involved in the biogenesis or repair of PS II by creating a double mutant lacking both CyanoP and the PS II assembly factor Ycf48. This strain exhibited an additive reduction in the amplitude of variable chlorophyll a fluorescence induction relative to either of the single mutants but displayed increased oxygen evolution, slight increases in PS II monomer and dimer levels, and a reduction in accumulation of an early PS II assembly complex containing CP47, compared to the ΔYcf48 strain.

Arg24 and 26 of the D2 protein are important for photosystem II assembly and plastoquinol exchange in Synechocystis sp. PCC 6803.

Photosystem II (PS II) assembly is a stepwise process involving preassembly complexes or modules focused around four core PS II proteins. The current model of PS II assembly in cyanobacteria is derived from studies involving the deletion of one or more of these core subunits. Such deletions may destabilize other PS II assembly intermediates, making constructing a clear picture of the intermediate events difficult. Information on plastoquinone exchange pathways operating within PS II is also unclear and relies heavily on computer-aided simulations. Deletion of PsbX in [S. Biswas, J.J. Eaton-Rye, Biochim. Biophys. Acta - Bioenerg. 1863 (2022) 148519] suggested modified QB binding in PS II lacking this subunit. This study has indicated the phenotype of the ∆PsbX mutant arose by disrupting a conserved hydrogen bond between PsbX and the D2 (PsbD) protein. We mutated two conserved arginine residues (D2:Arg24 and D2:Arg26) to further understand the observations made with the ∆PsbX mutant. Mutating Arg24 disrupted the interaction between PsbX and D2, replicating the high-light sensitivity and altered fluorescence decay kinetics observed in the ∆PsbX strain. The Arg26 residue, on the other hand, was more important for either PS II assembly or for stabilizing the fully assembled complex. The effects of mutating both arginine residues to alanine or aspartate were severe enough to render the corresponding double mutants non-photoautotrophic. Our study furthers our knowledge of the amino-acid interactions stabilizing plastoquinone-exchange pathways while providing a platform to study PS II assembly and repair without the actual deletion of any proteins.