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 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.

Environmental pH and the Requirement for the Extrinsic Proteins of Photosystem II in the Function of Cyanobacterial Photosynthesis.

In one of the final stages of cyanobacterial Photosystem II (PS II) assembly, binding of up to four extrinsic proteins to PS II stabilizes the oxygen-evolving complex (OEC). Growth of cyanobacterial mutants deficient in certain combinations of these thylakoid-lumen-associated polypeptides is sensitive to changes in environmental pH, despite the physical separation of the membrane-embedded PS II complex from the external environment. In this perspective we discuss the effect of environmental pH on OEC function and photoautotrophic growth in cyanobacteria with reference to pH-sensitive PS II mutants lacking extrinsic proteins. We consider the possibilities that, compared to pH 10.0, pH 7.5 increases susceptibility to PS II-generated reactive oxygen species (ROS) causing photoinhibition and reducing PS II assembly in some mutants, and that perturbations to channels in the lumenal regions of PS II might alter the accessibility of water to the active site as well as egress of oxygen and protons to the thylakoid lumen. Reduced levels of PS II in these mutants, and reduced OEC activity arising from the disruption of substrate/product channels, could reduce the trans-thylakoid pH gradient (ΔpH), leading to the impairment of photosynthesis. Growth of some PS II mutants at pH 7.5 can be rescued by elevating CO2 levels, suggesting that the pH-sensitive phenotype might primarily be an indirect result of back-pressure in the electron transport chain that results in heightened production of ROS by the impaired photosystem.

Functional role of PilA in iron acquisition in the cyanobacterium Synechocystis sp. PCC 6803.

Cyanobacteria require large quantities of iron to maintain their photosynthetic machinery; however, in most environments iron is present in the form of insoluble iron oxides. Whether cyanobacteria can utilize these sources of iron, and the potential molecular mechanisms involved remains to be defined. There is increasing evidence that pili can facilitate electron donation to extracellular electron acceptors, like iron oxides in non-photosynthetic bacteria. In these organisms, the donation of electrons to iron oxides is thought to be crucial for maintaining respiration in the absence of oxygen. Our study investigates if PilA1 (major pilin protein) may also provide a mechanism to convert insoluble ferric iron into soluble ferrous iron. Growth experiments supported by spectroscopic data of a strain deficient in pilA1 indicate that the presence of the pilA1 gene enhances the ability to grow on iron oxides. These observations suggest a novel function of PilA1 in cyanobacterial iron acquisition.

An LED-based fluorometer for chlorophyll quantification in the laboratory and in the field.

The chlorophyll content is an important experimental parameter in agronomy and plant biology research. In this report, we explore the feasibility of determining total concentration of extracts containing chlorophyll a and chlorophyll b by chlorophyll fluorescence. We found that an excitation at 457 nm results in the same integrated fluorescence emission for a molecule of chlorophyll a and a molecule of chlorophyll b. The fluorescence yield induced by 457 nm is therefore proportional to total molar chlorophyll concentration. Based on this observation, we designed an instrument to determine total chlorophyll concentrations. A single light emitting diode (LED) is used to excite chlorophyll extracts. After passing through a long-pass filter, the fluorescence emission is assessed by a photodiode. We demonstrate that this instrument facilitates the determination of total chlorophyll concentrations. We further extended the functionality of the instrument by including LEDs emitting at 435 and 470 nm wavelengths, thereby preferentially exciting chlorophyll a and chlorophyll b. This instrument can be used to determine chlorophyll a and chlorophyll b concentrations in a variety of organisms containing different ratios of chlorophylls. Monte-Carlo simulations are in agreement with experimental data such that a precise determination of chlorophyll concentrations in carotenoid-containing biological samples containing a concentration of less than 5 nmol/mL total chlorophyll can be achieved.

The lipoproteins of cyanobacterial photosystem II.

Photosystem II (PSII) complexes from cyanobacteria and plants perform water splitting and plastoquinone reduction and yet have a different complement of lumenal extrinsic proteins. Whereas PSII from all organisms has the PsbO extrinsic protein, crystal structures of PSII from cyanobacteria have PsbV and PsbU while green algae and higher plants instead contain the extrinsic PsbP and PsbQ subunits. Proteomic studies in Synechocystis sp. PCC 6803 identified three further extrinsic proteins in the thylakoid lumen that are associated with cyanobacterial PSII and these are predicted to attach to the thylakoid membrane via a lipidated N-terminus. These proteins are cyanobacterial homologues to the PsbP and PsbQ subunits as well as to Psb27, an additional extrinsic protein associated with "inactive" photosystems that lack the other extrinsic polypeptides. The PsbQ homologue is not present in Prochlorococcus species but otherwise these proteins have been identified in most cyanobacteria although our phylogenetic analyses identified some strains that lack an apparent motif for lipidation in one or other of these subunits. Over the past decade the physiological function of these additional lipoproteins has been investigated in several cyanobacterial strains and recently the structures for each have been solved. This review will evaluate the physiological and structural results obtained for these lipid-attached extrinsic proteins and in silico protein docking of these proteins to PSII centers will be presented.

The PsbU subunit of photosystem II stabilizes energy transfer and primary photochemistry in the phycobilisome-photosystem II assembly of Synechocystis sp. PCC 6803.

The PsbU subunit of photosystem II (PSII) is one of three extrinsic polypeptides associated with stabilizing the oxygen evolving machinery of photosynthesis in cyanobacteria. We investigated the influence of PsbU on excitation energy transfer and primary photochemistry by spectroscopic analysis of a PsbU-less (or deltaPsbU) mutant. The absence of PsbU was found to have multiple effects on the excited state dynamics of the phycobilisome and PSII. DeltaPsbU cells exhibited decreased variable fluorescence when excited with light absorbed primarily by allophycocyanin but not when excited with light absorbed primarily by chlorophyll a. Fluorescence emission spectra at 77 K showed evidence for impaired energy transfer from the allophycocyanin terminal phycobilisome emitters to PSII. Picosecond fluorescence decay kinetics revealed changes in both allophycocyanin and PSII associated decay components. These changes were consistent with a decrease in the coupling of phycobilisomes to PSII and an increase in the number of closed PSII reaction centers in the dark-adapted deltaPsbU mutant. Our results are consistent with the assumption that PsbU stabilizes both energy transfer and electron transport in the PBS/PSII assembly.

pH-dependent photoautotrophic growth of specific photosystem II mutants lacking lumenal extrinsic polypeptides in Synechocystis PCC 6803.

The removal of either the PsbU or PsbV protein has been investigated in a cyanobacterial DeltaPsbO strain and in mutants carrying deletions or substitutions in lumen-exposed domains of CP47. These experiments have demonstrated a functional interaction between the PsbU protein and photosystem II (PSII) in the absence of the PsbO subunit. The control:DeltaPsbO:DeltaPsbU strain assembled PSII centers at pH 7.5 but did not evolve oxygen; however, photoautotrophic growth was restored at pH 10.0. In addition, several CP47 mutants, lacking extrinsic proteins, were obligate photoheterotrophs at pH 7.5 but photoautotrophic at pH 10.0, whereas other strains remained photoheterotrophs at alkaline pH.