Cyanobacterial PSII Research Articles

The biogenesis and maintenance of PSII: Recent advances and current challenges.

The growth of plants, algae, and cyanobacteria relies on the catalytic activity of the oxygen-evolving PSII complex, which uses solar energy to extract electrons from water to feed into the photosynthetic electron transport chain. PSII is proving to be an excellent system to study how large multi-subunit membrane-protein complexes are assembled in the thylakoid membrane and subsequently repaired in response to photooxidative damage. Here we summarize recent developments in understanding the biogenesis of PSII, with an emphasis on recent insights obtained from biochemical and structural analysis of cyanobacterial PSII assembly/repair intermediates. We also discuss how chlorophyll synthesis is synchronized with protein synthesis and suggest a possible role for PSI in PSII assembly. Special attention is paid to unresolved and controversial issues that could be addressed in future research.

The Ycf48 accessory factor occupies the site of the oxygen-evolving manganese cluster during photosystem II biogenesis

Robust oxygenic photosynthesis requires a suite of accessory factors to ensure efficient assembly and repair of the oxygen-evolving photosystem two (PSII) complex. The highly conserved Ycf48 assembly factor binds to the newly synthesized D1 reaction center polypeptide and promotes the initial steps of PSII assembly, but its binding site is unclear. Here we use cryo-electron microscopy to determine the structure of a cyanobacterial PSII D1/D2 reaction center assembly complex with Ycf48 attached. Ycf48, a 7-bladed beta propeller, binds to the amino-acid residues of D1 that ultimately ligate the water-oxidising Mn4CaO5 cluster, thereby preventing the premature binding of Mn2+ and Ca2+ ions and protecting the site from damage. Interactions with D2 help explain how Ycf48 promotes assembly of the D1/D2 complex. Overall, our work provides valuable insights into the early stages of PSII assembly and the structural changes that create the binding site for the Mn4CaO5 cluster.

Advances in the Understanding of the Lifecycle of Photosystem II.

Photosystem II is a light-driven water-plastoquinone oxidoreductase present in cyanobacteria, algae and plants. It produces molecular oxygen and protons to drive ATP synthesis, fueling life on Earth. As a multi-subunit membrane-protein-pigment complex, Photosystem II undergoes a dynamic cycle of synthesis, damage, and repair known as the Photosystem II lifecycle, to maintain a high level of photosynthetic activity at the cellular level. Cyanobacteria, oxygenic photosynthetic bacteria, are frequently used as model organisms to study oxygenic photosynthetic processes due to their ease of growth and genetic manipulation. The cyanobacterial PSII structure and function have been well-characterized, but its lifecycle is under active investigation. In this review, advances in studying the lifecycle of Photosystem II in cyanobacteria will be discussed, with a particular emphasis on new structural findings enabled by cryo-electron microscopy. These structural findings complement a rich and growing body of biochemical and molecular biology research into Photosystem II assembly and repair.

Binding of ferredoxin NADP+ oxidoreductase (FNR) to plant photosystem I

The binding of FNR to PSI has been postulated long ago, however, a clear evidence is still missing. In this work, using isothermal titration calorimetry (ITC), we found that FNR binds to photosystem I with its light harvesting complex I (PSI-LHCI) from C. reinhardtii with a 1:1 stoichiometry, a Kd of ~0.8 μM and ∆H of −20.7 kcal/mol. Titrations at different temperatures were used to determine the heat capacity change, ∆CP, of the binding, through which the size of the interface area between the proteins was assessed as ~3000 Å2. In a different set of ITC experiments, introduction of various sucrose concentrations was used to estimate that ~95 water molecules are released to the solvent. These observations support the notion of a binding site shared by few of the photosystem I - light harvesting complex I (PSI-LHCI) subunits in addition to PsaE. Based on these results, a hypothetical model was built for the binding site of FNR at PSI, using known crystallographic structures of: cyanobacterial PSI in complex with ferredoxin (Fd), plant PSI-LHCI and Fd:FNR complex from cyanobacteria. FNR binding site location is proposed to be at the foot of the stromal ridge and above the inner LHCI belt. It is expected to form contacts with PsaE, PsaB, PsaF and at least one of the LHCI. In addition, a ~4.5-fold increased affinity between FNR and PSI-LHCI under crowded 1 M sucrose environment led us to conclude that in C. reinhardtii FNR also functions as a subunit of PSI-LHCI.

Photooxidation and photoreduction of exogenous cytochrome c by photosystem II preparations after various modifications of the water-oxidizing complex

The redox interaction of exogenous cytochrome c550 (Cyt) with PSII isolated from spinach was studied. Illumination of PSII particles in the presence of Cyt led to: (1) Cyt photooxidation by PSII reaction center (demonstrated at the first time), (2) Cyt photoreduction via O2−• photoproduced on the acceptor side of PSII, and (3) Cyt photoreduction by reduced electron carriers of PSII. A step-by-step removal of components of water-oxidizing complex was accompanied by the appearance of Cyt photooxidation, an increase in the superoxide dismutase (SOD)-dependent Cyt photoreduction (related to O2−• formation), and a decrease in the SOD-independent Cyt photoreduction. Re-addition of PsbO protein diminished the Cyt-induced restoration of electron transfer in PSII. Addition of diuron led to inhibition of these photoprocesses, while exogenous Mn2+ inhibited only the Cyt c photooxidation. The results can be important for correct measurements of O2−• photoproduction in PSII and for elucidation of the role of cytochrome c550 in cyanobacterial PSII.

Molecular Mechanisms of Photoadaptation of Photosystem I Supercomplex from an Evolutionary Cyanobacterial/Algal Intermediate.

The monomeric photosystem I-light-harvesting antenna complex I (PSI-LHCI) supercomplex from the extremophilic red alga Cyanidioschyzon merolae represents an intermediate evolutionary link between the cyanobacterial PSI reaction center and its green algal/higher plant counterpart. We show that the C. merolae PSI-LHCI supercomplex is characterized by robustness in various extreme conditions. By a combination of biochemical, spectroscopic, mass spectrometry, and electron microscopy/single particle analyses, we dissected three molecular mechanisms underlying the inherent robustness of the C. merolae PSI-LHCI supercomplex: (1) the accumulation of photoprotective zeaxanthin in the LHCI antenna and the PSI reaction center; (2) structural remodeling of the LHCI antenna and adjustment of the effective absorption cross section; and (3) dynamic readjustment of the stoichiometry of the two PSI-LHCI isomers and changes in the oligomeric state of the PSI-LHCI supercomplex, accompanied by dissociation of the PsaK core subunit. We show that the largest low light-treated C. merolae PSI-LHCI supercomplex can bind up to eight Lhcr antenna subunits, which are organized as two rows on the PsaF/PsaJ side of the core complex. Under our experimental conditions, we found no evidence of functional coupling of the phycobilisomes with the PSI-LHCI supercomplex purified from various light conditions, suggesting that the putative association of this antenna with the PSI supercomplex is absent or may be lost during the purification procedure.

Photoactivation: The Light-Driven Assembly of the Water Oxidation Complex of Photosystem II.

Photosynthetic water oxidation is catalyzed by the Mn4CaO5 cluster of photosystem II. The assembly of the Mn4O5Ca requires light and involves a sequential process called photoactivation. This process harnesses the charge-separation of the photochemical reaction center and the coordination environment provided by the amino acid side chains of the protein to oxidize and organize the incoming manganese ions to form the oxo-bridged metal cluster capable of H2O-oxidation. Although most aspects of this assembly process remain poorly understood, recent advances in the elucidation of the crystal structure of the fully assembled cyanobacterial PSII complex help in the interpretation of the rich history of experiments designed to understand this process. Moreover, recent insights on the structure and stability of the constituent ions of the Mn4CaO5 cluster may guide future experiments. Here we consider the literature and suggest possible models of assembly including one involving single Mn2+ oxidation site for all Mn but requiring ion relocation.

Effect of postmining waters on cyanobacterial photosynthesis.

New waterbodies have been created in a postmining area of the brown-coal basin in Sokolov, Czech Republic. The former open-cast quarry, Medard, has been filling with water from a local river, the surrounding catchment spoil heaps, and acid mine drainages. The effect of acidic (pH down to 2.5) and high-conductivity water (up to 1400 mS/m) on selected cyanobacteria and the possibility of cyanobacterial water bloom in the newly formed Lake Medard were studied by means of chlorophyll fluorometry (actual photosystem II [PSII] quantum yield, ΔF/Fm', and relative electron transport rate, rETR). The acidic spoil-heap waters caused a decrease in cyanobacterial photosynthetic activity of 52 to 100% of the initial ΔF/Fm' value. The Dolichospermum strains were about 10 times more sensitive than Microcystis viridis. The high concentration of dissolved ions appeared to have less effect on cyanobacterial PSII. Although the bottom meta- and hypolimnion layers were proven to negatively influence the cyanobacteria, the perennial stratification of the lake does not enable the water characteristics of the upper layers to change extensively and thus possibly suppresses the undesirable cyanobacterial bloom. The response of cyanobacteria to spoil-heap waters appears to be species-specific and can promote selection of those resistant to postmining environments.

A Novel Approach for Efficient Photosynthetic Hydrogen Production

Despite its enormous complexity, the plant PSI is arguably the most efficient nano-photochemical machine in Nature. It emerged as a homodimeric structure containing several chlorophyll molecules over 3.5 billion years ago, and has perfected its photoelectric properties ever since. Based on the structure of cyanobacterial and plant PSI together with the information gained from recently discovered PSI encoded by marine viruses we suggested a holistic solution for reasonable and efficient photosynthetic hydrogen production.

Genetic analysis of the Photosystem I subunits from the red alga, Galdieria sulphuraria

Currently, there are very little data available regarding the photosynthetic apparatus of red algae. We have analyzed the genes for Photosystem I in the recently sequenced genome of the red alga Galdieria sulphuraria. All subunits that are conserved between plants and cyanobacteria were unambiguously identified in the Galdieria genome: PsaA, PsaB, PsaC, PsaD, PsaE, PsaF, PsaI, PsaJ, PsaK and PsaL. From the plant specific subunits, PsaN and PsaO were identified but the sequence homology was much lower than for the subunits that are present in plants and cyanobacteria. The subunit PsaX, which is specific for thermophilic cyanobacteria, is not present in the Galdieria genome, whereas PsaM is a plastid-encoded protein as in other red algae. The sequences of the core subunits of PSI were further analyzed by mapping of the conserved areas in the crystal structures of cyanobacterial and plant PSI. The structural comparison shows that PSI from the red alga Galdieria may represent a common ancestral structure at the interface between cyanobacterial and plant PSI. Some subunits have a “zwitter” structure that contains structural elements that show similarities with either plant or cyanobacterial PSI. The structure of PsaL, which is responsible for the trimerization of PSI in cyanobacteria, lacks a short helix and the Ca 2+ binding site, which are essential for trimer formation indicating that the Galdieria PSI is a monomer. However the sequence homology to plant PsaL is low and lacks strong conservation of the interaction sites with PsaH. Furthermore, the sites for interaction of plant PSI with the LHCI complex are not well conserved between plants and Galdieria, which may indicate that Galdieria may contain a PSI that is evolutionarily much more ancient than PSI from green algae, plants and the current cyanobacteria.

Structure of Plant Photosystem I Revealed by Theoretical Modeling

Photosystem (PS) I is a large membrane protein complex vital for oxygenic photosynthesis, one of the most important biological processes on the planet. We present an "atomic" model of higher plant PSI, based on theoretical modeling using the recent 4.4 angstroms x-ray crystal structure of PSI from pea. Because of the lack of information on the amino acid side chains in the x-ray structural model and the high cofactor content in this system, novel modeling techniques were developed. Our model reveals some important structural features of plant PSI that were not visible in the crystal structure, and our model sheds light on the evolutionary relationship between plant and cyanobacterial PSI.

PSII-Tc Protein Plays an Important Role in Dimerization of Photosystem II

We cloned and determined the nucleotide sequence of PSII genes, psbB and psbTc, from the thermophilic cyanobacterium, Thermosynechococcus elongatus strain BP-1. PSII-Tc, encoded by psbTc, is a small membrane-spanning subunit of the PSII core complex of cyanobacteria and plants. However, its role has not been fully elucidated. We generated an insertional disruptant of psbTc and studied the role of the PSII-Tc protein in cyanobacterial PSII. The following observations were made: (i) The psbTc disruptant could grow photoautotrophically at a rate similar to that of wild-type T. elongatus under a wide range of light conditions. (ii) Thylakoids and oxygen-evolving PSII complexes were successfully isolated from the psbTc disruptant as well as the wild type. There was no significant difference in the oxygen evolution activities of cells, thylakoids or PSII complexes between the psbTc disruptant and the wild type. This is in contrast to the lower activities in the other PSII mutants of T. elongatus. (iii) Chromatographic separation of monomeric and dimeric PSII revealed that recovery of dimeric PSII was dramatically reduced in the psbTc disruptant. (iv) SDS-urea-PAGE showed a complete loss of the 4.7-kDa band in the mutant PSII. Since this band in wild-type PSII consists of PSII-M and PSII-Tc, we assume that PSII-Tc is critical for the binding of PSII-M in the PSII complex and is involved directly and indirectly in the dimerization of PSII. These results appear to be in good agreement with the recent structural model of the dimeric PSII complex.

Gene expression profiling reflects physiological processes in salt acclimation of Synechocystis sp. strain PCC 6803.

The kinetics of genome-wide responses of gene expression during the acclimation of cells of Synechocystis sp. PCC 6803 to salt stress were followed by DNA-microarray technique and compared to changes in main physiological parameters. During the first 30 min of salt stress, about 240 genes became induced higher than 3-fold, while about 140 genes were repressed. However, most changes in gene expression were only transient and observed among genes for hypothetical proteins. At 24 h after onset of salt stress conditions, the expression of only 39 genes remained significantly enhanced. Among them, many genes that encode proteins essential for salt acclimation were detected, while only a small number of genes for hypothetical proteins remained activated. Following the expression of genes for main functions of the cyanobacterial cell, i.e. PSI, PSII, phycobilisomes, and synthesis of compatible solutes, such as ion homeostasis, distinct kinetic patterns were found. While most of the genes for basal physiological functions were transiently repressed during the 1st h after the onset of salt stress, genes for proteins specifically related to salt acclimation were activated. This gene expression pattern reflects well the changes in main physiological processes in salt-stressed cells, i.e. transient inhibition of photosynthesis and pigment synthesis as well as immediate activation of synthesis of compatible solutes. The results clearly document that following the kinetics of genome-wide expression, profiling can be used to envisage physiological changes in the cyanobacterial cell after certain changes in growth conditions.

A REVISED ESTIMATE OF THE IRON USE EFFICIENCY OF NITROGEN FIXATION, WITH SPECIAL REFERENCE TO THE MARINE CYANOBACTERIUM TRICHODESMIUM SPP. (CYANOPHYTA)1

Estimates of the iron use efficiency (IUE) for diazotrophic plant growth have been used to suggest iron limitation of marine N2 fixation. However, in the course of these inferences, neither the physiological complexity of these estimates nor the specific physiological parameters of marine diazotrophs were evaluated. Here, a semiempirical prediction of the IUE of diazotrophic growth for Trichodesmium was computed from considerations of the Fe content and reaction rates of the nitrogenase complex and PSI:PSII ratios, as well as field measurements of Mehler activity, cellular Fe‐superoxide dismutase activity, and diel variability in C and N2 fixation. With a PSI:PSII ratio of 1 and 48% Mehler activity, the instantaneous IUE (0.33 mol C fixed·mol cellular Fe−1·s−1) was only 4‐fold lower than that calculated for a phytoplankter growing on reduced N. We computed a range of daily integrated IUE values from 2900 to 7700 mol C·mol Fe−1·d−1, accounting for the diel variability in C and N2 fixation as well as the uncertainties in cyanobacterial nitrogenase biochemistry and PSI:II ratios of field‐collected Trichodesmium. The lowest observed Fe‐superoxide dismutase:C quota of 2.9 (μmol:mol) suggests a maintenance requirement for this enzyme. The maintenance Fe:C requirement of 13.5 μmol:mol (derived from cultures of Trichodesmium IMS 101) and values of the IUE yielded an Fe requirement ranging from 27 to 48 Fe:C (μmol:mol) to achieve a diazotrophic growth rate of 0.1 d−1. Based on these predicted requirements, the Fe:C contents of Caribbean Sea and most North Atlantic Ocean populations sampled thus far exceed that required to support the observed rates of N2 fixation.

Cross-reconstitution of extrinsic cytochrome c-550 and 12 kDa protein between cyanobacterial and red algal PSII

Red algal and cyanobacterial PSII contain two similar extrinsic proteins, namely, cytochrome c-550 and a 12 kDa protein. Their amino acid sequences showed a relatively high homology of about 40% between red algae and cyanobacteria. In this study, cross-reconstitution experiments of the extrinsic proteins with red algal (Cyanidium caldarium) or cyanobacterial (Synechococcus vulcanus) PSII were performed in order to compare the binding and functional properties of the extrinsic proteins between red algae and cyanobacteria. Red algal cytochrome c-550, as well as cyanobacterial cytochrome c-550, bound directly to cyanobacterial PSII. However, none of them bound directly to red algal PSII. This indicates that direct binding of cytochrome c-550 to PSII depends upon the structure of intrinsic proteins of PSII but not cytochrome c-550 itself. Red algal 12 kDa protein functionally bound to cyanobacterial PSII, while cyanobacterial 12 kDa protein did not bind to red algal PSII. The antibodies raised against cyanobacterial or red alga 12 kDa protein did not cross-react with red algal or cyanobacterial 12 kDa protein, respectively. These results imply that, while the structure and function of cytochrome c-550 were rather conserved, those of the 12 kDa protein have been changed during evolution from cyanobacteria to red algae.

Structural analysis of the photosynthetic reaction center from the green sulfur bacterium Chlorobium tepidum

The reaction center (RC) core complex was isolated from the green sulfur bacterium Chlorobium tepidum and characterized by gel electrophoresis, gel filtration, and analytical ultracentrifugation. The purified complex contained the PscA and PscC subunits and a small amount of the Fenna–Matthews–Olson protein (FMO protein) as an impurity. The mass of the core complexes was found to be 248 kDa by scanning transmission electron microscopy (STEM). This is compatible with the presence of two copies of the PscA subunit and at least one copy of the PscC subunit, and provides evidence that the isolated complex exists as a monomer. Digital images of negatively stained RC complexes were recorded by STEM and analyzed by single-particle averaging. The complex had a length of 14 nm and a width of 8 nm, comparable to the length and width of the monomeric cyanobacterial PSI complex. The averages revealed a pseudo two-fold symmetry axis, which is a prominent structural element of the monomeric form.

Mechanisms of Electron Flow through the QB Site in Photosystem II. 3. Effects of the Presence of Membrane Structure on the Redox Reactions at the QB Site

Reduction kinetics of chloro- or methyl-substi tuted benzoquinones (BQs) in spinach PSII membrane fragments or /!-heptyl-/7- D-thioglucoside-extracted PSII core complexes (HTG-PSII) was studied and compared to that in cyanobacterial PSII core complexes [Satoh et al. (1995) Plant Cell Physiol. 36: 597]. It was found that the BQs accept electrons at two sites (the QB and PQ sites) in the both spinach preparations as in the cyanobacterial preparation. Maximum turnover rates (Vmax) and binding affinities (Km) of the two sites were estimated. Comparison of the values in PSII membrane fragments with those in HTG-PSII showed that removal of the membrane structure or lightharvesting chlorophyll a/b protein complexes from the PSII core complexes had little effect on the characteristics of the Q,, site, indicating that the HTG-PSII have the intact QB site and are good materials to study the site. The Km and Vmn values were comparable to those in the cyanobacterial preparation. Low affinity (high Km values) and high Vmtx values of methyl-substituted BQs to the QB site and almost the same rate of intrinsic electron flow through the QB site in the both spinach preparations further support the hypothesis that the plastoquinone (PQ) molecule at the QB site is not replaced by another PQ molecule but, after reduction, only its head group goes out of the QB pocket, and comes back after transferring the electrons and protons to a free PQ molecule [Satoh et al. (1993) Z. Naturforsch. 48c: 174].

Chemical crosslinking studies of extrinsic proteins in cyanobacterial photosystem II

Chemical crosslinking with a zero-length crosslinker, 1-ethyl-3-(3-dimethylaminopropyl) carbodimide hydrochloride, was applied to a cyanobacterial photosystem II complex retaining three extrinsic proteins, the 33 kDa manganese-stabilizing protein, cytochrome (cyt) c-550 and the 12 kDa protein. Three major crosslinked products were obtained in addition to the crosslinked product between the extrinsic 33 kDa and the intrinsic CP47 proteins. They were identified to be: cyt c-550-12 kDa; cyt c-550-12 kDa-33 kDa; D2-cyt c-550-12 kDa. These results indicate that the three extrinsic proteins are closely located with each other in cyanobacterial PSII, supporting the previous proposal that, like the 33 kDa protein, cyt c-550 and the 12 kDa protein are associated with PSII at the lumenal side of thylakoids. The results also suggested that the D2 reaction center protein provides a direct binding site for the extrinsic cyt c-550.

Evolution of photosynthetic reaction centers and light harvesting chlorophyll proteins

It is proposed that there is a single evolutionary origin for photosynthetic reaction centers and also for most light-harvesting chlorophyll proteins. It is generally accepted that the purple bacterial reaction center (quinone-reducing photosystem) and the plant and cyanobacterial PSII (oxygen-evolving photosystem) are homologous. It is also apparent that the green sulfur bacterial reaction center is homologous to cyanobacterial PSI (the pyridine nucleotide reducing photosystem). However, it is less obvious that PSI is related to the purple bacterial reaction center. It is herein proposed that PSI represents a gene fusion of the precursors of small light harvesting bacteriochlorophyll proteins from purple bacteria and purple bacterial reaction centers. Furthermore, it is proposed that reaction centers evolved from the membrane-spanning cytochrome b of the cytochrome bc 1 complex and that most membrane-spanning cytochromes may have a common origin.

Cellular localization of cytochrome c550. Its specific association with cyanobacterial photosystem II.

Cellular localization of cytochrome (cyt) c550, a low potential, c-type monoheme cytochrome, in a thermophilic cyanobacterium Synechococcus vulcanus was investigated by systematic fractionation of the cells followed by its enzymatic and immunological detection. While cyt c-553, a soluble cyt that donates an electron to P700, was detected in the supernatant of osmotic disruption of lysozyme-treated cells, cyt c550 was detected only in the thylakoid membrane fraction, being tightly bound to thylakoids, and its removal required sonication in the presence of 1 M CaCl2. Upon further fractionation of the thylakoids into photosystem (PS) I and PSII by lauryl dimethylamine N-oxide solubilization, cyt c550 was exclusively concentrated in the crude PSII fraction together with cyt f, with no significant amount being detected in any of the soluble and PSI fractions. Upon further fractionation of the crude PSII by n-dodecyl beta-D-maltoside solubilization followed by column chromatography, cyt c550 was detected exclusively in the purified PSII core complex fraction but not in any other fractions. A 12-kDa protein, one of the extrinsic components of cyanobacterial PSII, exhibited completely the same behavior as that of cyt c550 during these fractionation procedures. These results, coupled with our previous results that cyt c550 binds stoichiometrically to the cyanobacterial PSII core complex and enhances O2 evolution (Shen, J.-R., and Inoue, Y. (1993) Biochemistry 32, 1825-1832), indicate that there is only one species of cyt c550 in cyanobacterial cells and that this cyt is exclusively associated with PSII as a functional component for O2 evolution.