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.

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.

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.

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.

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.

PsbX maintains efficient electron transport in Photosystem II and reduces susceptibility to high light in Synechocystis sp. PCC 6803.

PsbX is a 4.1 kDa intrinsic Photosystem II (PS II) protein, found together with the low-molecular-weight proteins, PsbY and PsbJ, in proximity to cytochrome b559. The function of PsbX is not yet fully characterized but PsbX may play a role in the exchange of the secondary plastoquinone electron acceptor QB with the quinone pool in the thylakoid membrane. To study the role of PsbX, we have constructed a PsbX-lacking strain of Synechocystis sp. PCC 6803. Our studies indicate that the absence of PsbX causes sensitivity to high light and impairs electron transport within PS II. In addition to a change in the QB-binding pocket, PsbX-lacking cells exhibited sensitivity to sodium formate, suggesting altered binding of the bicarbonate ligand to the non-heme iron between the sequential plastoquinone electron acceptors QA and QB. Experiments using 35S-methionine revealed high-light-treated PsbX-lacking cells restore PS II activity during recovery under low light by an increase in the turnover of PS II-associated core proteins. These labeling experiments indicate the recovery after exposure to high light requires both selective removal and replacement of the D1 protein and de novo PS II assembly.

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 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 D1:Ser268 residue of Photosystem II contributes to an alternative pathway for QB protonation in the absence of bound bicarbonate.

Photosystem II catalyses the splitting of water and the reduction in plastoquinone in thylakoid membranes of all oxygenic photosynthetic organisms. The final step in quinol formation is protonation of the reduced secondary quinone electron acceptor QB2-(H+) to give QB H2 . The proton for this step is hypothesized to be provided by a hydrogen-bond network incorporating amino acids from the Photosystem II D1 and D2 reaction center proteins, together with several bound waters and a bicarbonate ion ligated to a non-heme iron found between the primary plastoquinone electron acceptor QA and QB . The aim of this study was to investigate the role of bicarbonate and the D1:Ser268 residue in the formation of QB H2 . Using targeted mutagenesis of the D1 protein in the cyanobacterium Synechocystis sp. PCC 6803, we have created two mutants, S268A and S268T. Our D1:Ser268 mutants exhibited increased sensitivity to formate-induced inhibition of electron transfer between QA and QB and indicate that D1:Ser268 and bicarbonate support the second protonation in the formation of QB H2 via two different pathways that both lead to the protonation of QB2-(H+) by D1:His215.

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.

Stabilization of Photosystem II by the PsbT protein impacts photodamage, repair and biogenesis.

Photosystem II (PS II) catalyzes the light-driven process of water splitting in oxygenic photosynthesis. Four core membrane-spanning proteins, including D1 that binds the majority of the redox-active co-factors, are surrounded by 13 low-molecular-weight (LMW) proteins. We previously observed that deletion of the LMW PsbT protein in the cyanobacterium Synechocystis sp. PCC 6803 slowed electron transfer between the primary and secondary plastoquinone electron acceptors QA and QB and increased the susceptibility of PS II to photodamage. Here we show that photodamaged ∆PsbT cells exhibit unimpaired rates of oxygen evolution if electron transport is supported by HCO3- even though the cells exhibit negligible variable fluorescence. We find that the protein environment in the vicinity of QA and QB is altered upon removal of PsbT resulting in inhibition of QA- oxidation in the presence of 2,5-dimethyl-1,4-benzoquinone, an artificial PS II-specific electron acceptor. Thermoluminescence measurements revealed an increase in charge recombination between the S2 oxidation state of the water-oxidizing complex and QA- by the indirect radiative pathway in ∆PsbT cells and this is accompanied by increased 1O2 production. At the protein level, both D1 removal and replacement, as well as PS II biogenesis, were accelerated in the ∆PsbT strain. Our results demonstrate that PsbT plays a key role in optimizing the electron acceptor complex of the acceptor side of PS II and support the view that repair and biogenesis of PS II share an assembly pathway that incorporates both de novo synthesis and recycling of the assembly modules associated with the core membrane-spanning proteins.

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.

D1:Glu244 and D1:Tyr246 of the bicarbonate-binding environment of Photosystem II moderate high light susceptibility and electron transfer through the quinone-Fe-acceptor complex.

In cyanobacteria, Glu-244 and Tyr-246 of the Photosystem II (PS II) D1 protein are hydrogen bonded to two water molecules that are part of a hydrogen-bond network between the bicarbonate ligand to a non-heme iron and the cytosol. Ala substitutions were introduced in Synechocystis sp. PCC 6803 to investigate the roles of these residues and the hydrogen-bond network on electron transfer between the primary plastoquinone acceptor, QA, and the secondary plastoquinone acceptor, QB, of the quinone-Fe-acceptor complex. All mutants assembled PS II; however, an increase in the PS II to PS I ratio was apparent, particularly in the E244A:Y246A double mutant. The mutants also showed impaired oxygen evolution and retarded chlorophyll a fluorescence decays following single turnover actinic flashes, which appeared to be primarily due to reduced QB binding in the E244A strain and an enhanced back reaction with the S2 state of the oxygen-evolving complex in the Y246A mutant. Impaired PS II in the Y246A and E244A:Y246A mutants resulted in inactivation of the psbA gene encoding D1. The Y246A and E244A:Y246A mutants also showed high light sensitivity whereas the E244A mutant showed enhanced resilience towards photodamage. Unlike the control strain, all of the mutants were insensitive to the addition of formate or bicarbonate in assays following chlorophyll decay kinetics that reflect electron transfer between QA and QB, suggesting the bicarbonate binding environment was perturbed. Our data also indicate that waters W582 and W622 (PDB: 4UB6) have essential roles in maintaining the architecture of the acceptor side of PS II.

Environmental pH and a Glu364 to Gln mutation in the chlorophyll-binding CP47 protein affect redox-active TyrD and charge recombination in Photosystem II.

In Photosystem II, loop E of the chlorophyll-binding CP47 protein is located near a redox-active tyrosine, YD , forming a symmetrical analog to loop E in CP43, which provides a ligand to the oxygen-evolving complex (OEC). A Glu364 to Gln substitution in CP47, near YD , does not affect growth in the cyanobacterium Synechocystis sp. PCC 6803; however, deletion of the extrinsic protein PsbV in this mutant leads to a strain displaying a pH-sensitive phenotype. Using thermoluminescence, chlorophyll fluorescence, and flash-induced oxygen evolution analyses, we demonstrate that Glu364 influences the stability of YD and the redox state of the OEC, and highlight the effects of external pH on photosynthetic electron transfer in intact cyanobacterial cells.

Mutation of Gly195 of the ChlH Subunit of Mg-chelatase Reduces Chlorophyll and Further Disrupts PS II Assembly in a Ycf48-Deficient Strain of Synechocystis sp. PCC 6803.

Biogenesis of the photosystems in oxygenic phototrophs requires co-translational insertion of chlorophyll a. The first committed step of chlorophyll a biosynthesis is the insertion of a Mg(2+) ion into the tetrapyrrole intermediate protoporphyrin IX, catalyzed by Mg-chelatase. We have identified a Synechocystis sp. PCC 6803 strain with a spontaneous mutation in chlH that results in a Gly195 to Glu substitution in a conserved region of the catalytic subunit of Mg-chelatase. Mutant strains containing the ChlH Gly195 to Glu mutation were generated using a two-step protocol that introduced the chlH gene into a putative neutral site in the chromosome prior to deletion of the native gene. The Gly195 to Glu mutation resulted in strains with decreased chlorophyll a. Deletion of the PS II assembly factor Ycf48 in a strain carrying the ChlH Gly195 to Glu mutation did not grow photoautotrophically. In addition, the ChlH-G195E:ΔYcf48 strain showed impaired PS II activity and decreased assembly of PS II centers in comparison to a ΔYcf48 strain. We suggest decreased chlorophyll in the ChlH-G195E mutant provides a background to screen for the role of assembly factors that are not essential under optimal growth conditions.