The PsbS protein and low pH are necessary and sufficient to induce quenching in the light-harvesting complex of plants LHCII.

Photosynthesis is tightly regulated in order to withstand dynamic light environments. Under high light intensities, a mechanism known as non-photochemical quenching (NPQ) dissipates excess excitation energy, protecting the photosynthetic machinery from damage. An obstacle that lies in the way of understanding the molecular mechanism of NPQ is the large gap between in vitro and in vivo studies. On the one hand, the complexity of the photosynthetic membrane makes it challenging to obtain molecular information from in vivo experiments. On the other hand, a suitable in vitro system for the study of quenching is not available. Here we have developed a minimal NPQ system using proteoliposomes. With this, we demonstrate that the combination of low pH and PsbS is both necessary and sufficient to induce quenching in LHCII, the main antenna complex of plants. This proteoliposome system can be further exploited to gain more insight into how PsbS and other factors (e.g. zeaxanthin) influence the quenching mechanism observed in LHCII.

The molecular pH-response mechanism of the plant light-stress sensor PsbS.

Plants need to protect themselves from excess light, which causes photo-oxidative damage and lowers the efficiency of photosynthesis. Photosystem II subunit S (PsbS) is a pH sensor protein that plays a crucial role in plant photoprotection by detecting thylakoid lumen acidification in excess light conditions via two lumen-faced glutamates. However, how PsbS is activated under low-pH conditions is unknown. To reveal the molecular response of PsbS to low pH, here we perform an NMR, FTIR and 2DIR spectroscopic analysis of Physcomitrella patens PsbS and of the E176Q mutant in which an active glutamate has been replaced. The PsbS response mechanism at low pH involves the concerted action of repositioning of a short amphipathic helix containing E176 facing the lumen and folding of the luminal loop fragment adjacent to E71 to a 310-helix, providing clear evidence of a conformational pH switch. We propose that this concerted mechanism is a shared motif of proteins of the light-harvesting family that may control thylakoid inter-protein interactions driving photoregulatory responses.

Structural insights into a dimeric Psb27-photosystem II complex from a cyanobacterium Thermosynechococcus vulcanus.

Photosystem II (PSII) is a multisubunit pigment-protein complex and catalyzes light-driven water oxidation, leading to the conversion of light energy into chemical energy and the release of molecular oxygen. Psb27 is a small thylakoid lumen-localized protein known to serve as an assembly factor for the biogenesis and repair of the PSII complex. The exact location and binding fashion of Psb27 in the intermediate PSII remain elusive. Here, we report the structure of a dimeric Psb27-PSII complex purified from a psbV deletion mutant (ΔPsbV) of the cyanobacterium Thermosynechococcus vulcanus, solved by cryo-electron microscopy. Our structure showed that Psb27 is associated with CP43 at the luminal side, with specific interactions formed between Helix 2 and Helix 3 of Psb27 and a loop region between Helix 3 and Helix 4 of CP43 (loop C) as well as the large, lumen-exposed and hydrophilic E-loop of CP43. The binding of Psb27 imposes some conflicts with the N-terminal region of PsbO and also induces some conformational changes in CP43, CP47, and D2. This makes PsbO unable to bind in the Psb27-PSII. Conformational changes also occurred in D1, PsbE, PsbF, and PsbZ; this, together with the conformational changes occurred in CP43, CP47, and D2, may prevent the binding of PsbU and induce dissociation of PsbJ. This structural information provides important insights into the regulation mechanism of Psb27 in the biogenesis and repair of PSII.

Substitution of Deoxycholate with the Amphiphilic Polymer Amphipol A8-35 Improves the Stability of Large Protein Complexes during Native Electrophoresis.

Native polyacrylamide gel electrophoresis (PAGE) is a powerful technique for protein complex separation that retains both their activity and structure. In photosynthetic research, native-PAGE is particularly useful given that photosynthetic complexes are generally large in size, ranging from 200 kD to 1 MD or more. Recently, it has been reported that the addition of amphipol A8-35 to solubilized protein samples improved protein complex stability. In a previous study, we found that amphipol A8-35 could substitute sodium deoxycholate (DOC), a conventional electrophoretic carrier, in clear-native (CN)-PAGE. In this study, we present the optimization of amphipol-based CN-PAGE. We found that the ratio of amphipol A8-35 to α-dodecyl maltoside, a detergent commonly used to solubilize photosynthetic complexes, was critical for resolving photosynthetic machinery in CN-PAGE. In addition, LHCII dissociation from PSII-LHCII was effectively prevented by amphipol-based CN-PAGE compared with that of DOC-based CN-PAGE. Our data strongly suggest that majority of the PSII-LHCII in vivo forms C2S2M2 at least in Arabidopsis and Physcomitrella. The other forms might appear owing to the dissociation of LHCII from PSII during sample preparation and electrophoresis, which could be prevented by the addition of amphipol A8-35 after solubilization from thylakoid membranes. These results suggest that amphipol-based CN-PAGE may be a better alternative to DOC-based CN-PAGE for the study of labile protein complexes.

Proton Release Process during the S2-to-S3 Transition of Photosynthetic Water Oxidation As Revealed by the pH Dependence of Kinetics Monitored by Time-Resolved Infrared Spectroscopy.

Photosynthetic water oxidation takes place at the Mn4CaO5 cluster in photosystem II via a light-driven cycle of intermediates called S states (S0-S4). Clarifying how electron and proton transfer reactions are coupled with each other in the S2 → S3 transition, which occurs just before O-O bond formation, is crucial for understanding the water oxidation mechanism. Here, we investigated the pH dependence of the kinetics of the S2 → S3 transition using time-resolved infrared (TRIR) spectroscopy to identify the proton release phase in this transition. TRIR measurements of YD-less PSII core complexes from the D2-Y160F mutant of Thermosynechococcus elongatus showed that the last phase in this transition (τ ∼ 350 µs at pH 6) was strongly dependent on pH, and its time constant at pH 5 was larger than that at pH 8 by a factor of >3. In contrast, the earlier phase with a time constant of ∼100 µs was virtually independent of pH. These results strongly support the view that proton release is a rate-limiting step of the proton-coupled electron transfer in the last phase of the S2 → S3 transition. This proton release enables electron transfer by removing an excessive positive charge from the catalytic center and hence decreasing its redox potential.

A novel chlorophyll protein complex in the repair cycle of photosystem II.

In oxygenic photosynthetic organisms, photosystem II (PSII) is a unique membrane protein complex that catalyzes light-driven oxidation of water. PSII undergoes frequent damage due to its demanding photochemistry. It must undergo a repair and reassembly process following photodamage, many facets of which remain unknown. We have discovered a PSII subcomplex that lacks 5 key PSII core reaction center polypeptides: D1, D2, PsbE, PsbF, and PsbI. This pigment-protein complex does contain the PSII core antenna proteins CP47 and CP43, as well as most of their associated low molecular mass subunits, and the assembly factor Psb27. Immunoblotting, mass spectrometry, and ultrafast spectroscopic results support the absence of a functional reaction center in this complex, which we call the "no reaction center" complex (NRC). Analytical ultracentrifugation and clear native PAGE analysis show that NRC is a stable pigment-protein complex and not a mixture of free CP47 and CP43 proteins. NRC appears in higher abundance in cells exposed to high light and impaired protein synthesis, and genetic deletion of PsbO on the PSII luminal side results in an increased NRC population, indicative that NRC forms in response to photodamage as part of the PSII repair process. Our finding challenges the current model of the PSII repair cycle and implies an alternative PSII repair strategy. Formation of this complex may maximize PSII repair economy by preserving intact PSII core antennas in a single complex available for PSII reassembly, minimizing the risk of randomly diluting multiple recycling components in the thylakoid membrane following a photodamage event.

Amphipol-assisted purification method for the highly active and stable photosystem II supercomplex of Chlamydomonas reinhardtii.

Photosystem II (PSII) splits water and drives electron transfer to plastoquinone via photochemical reactions using light energy. It is surrounded by light-harvesting complex II (LHCII) to form the PSII-LHCII supercomplex. Complete characterization of its structure and function has, however, been hampered due to instability of the complex in the presence of detergent. To overcome this problem, we developed a new procedure for purifying the PSII-LHCII supercomplexes of Chlamydomonas reinhardtii employing amphipol A8-35. The obtained supercomplexes showed little LHCII dissociation even 4 days after purification. Oxygen-evolving activity was retained within amphipol if the extrinsic polypeptides were kept associated by betaine. Electron microscopy revealed that this method also improved structural uniformity and that the major organization was C2 S2 M2 L2 .

Isolation and characterization of a photosystem II preparation from thylakoid membranes of the extreme halophyte Salicornia veneta Pignatti et Lausi.

Salicornia veneta (Pignatti et Lausi) is an extreme halophyte living in salt marsh where NaCl concentration may be as high as 1 M. Here we report on the isolation and characterization of a PSII preparation obtained by Triton X-100 solubilisation of the thylakoid membrane. By a combination of gel electrophoresis, immunoblotting and mass spectrometry, the depletion of a number of PSII proteins such as PsbQ, PsbM and PsbT was highlighted. Moreover, the requirement of Cl- and Ca2+ for optimal oxygen evolution was determined, showing that in absence of PsbQ a higher level of these ions are required. At high Cl- concentrations, oxygen evolution was inhibited in the same way in Salicornia veneta and spinach. Reconstitution of Salicornia veneta PSII preparation with partially purified spinach PsbP and PsbQ restored oxygen evolution activity at low Cl- and Ca2+ concentrations. Adaptation to high salt makes several PSII proteins dispensable.

Isolation of Intact Thylakoid Membranes from Heterocysts of Filamentous, Nitrogen-Fixing Cyanobacteria.

The isolation of thylakoid membranes, including intact membrane protein complexes, from heterocysts of filamentous cyanobacteria such as Nostoc punctiforme, is described. Protocols for BN-PAGE/SDS-PAGE 2-D electrophoresis are not included. However, the chapter ends with advisory notes on sample preparation for blue-native PAGE of thylakoid membrane proteins, which can then be used together with any standard protocol.

Use of Nicotiana tabacum transplastomic plants engineered to express a His-tagged CP47 for the isolation of functional photosystem II core complexes.

This work focuses on the development of a molecular tool for purification of Photosystem II (PSII) from Nicotiana tabacum (L.). To this end, the chloroplast psbB gene encoding the CP47 PSII subunit was replaced with an engineered version of the same gene containing a C-terminal His-tag. Molecular analyses assessed the effective integration of the recombinant gene and its expression. Despite not exhibiting any obvious phenotype, the transplastomic plants remained heteroplasmic even after three rounds of regeneration under antibiotic selection. However, the recombinant His-tagged CP47 protein associated in vivo to the other PSII subunits allowing the isolation of a functional PSII core complex, although with low yield of extraction. These results will open up possible perspectives for further spectroscopic and structural studies.

Bicarbonate-induced redox tuning in Photosystem II for regulation and protection.

The midpoint potential (Em) of [Formula: see text], the one-electron acceptor quinone of Photosystem II (PSII), provides the thermodynamic reference for calibrating PSII bioenergetics. Uncertainty exists in the literature, with two values differing by ∼80 mV. Here, we have resolved this discrepancy by using spectroelectrochemistry on plant PSII-enriched membranes. Removal of bicarbonate (HCO3-) shifts the Em from ∼-145 mV to -70 mV. The higher values reported earlier are attributed to the loss of HCO3- during the titrations (pH 6.5, stirred under argon gassing). These findings mean that HCO3- binds less strongly when QA-• is present. Light-induced QA-• formation triggered HCO3- loss as manifest by the slowed electron transfer and the upshift in the Em of QA HCO3--depleted PSII also showed diminished light-induced 1O2 formation. This finding is consistent with a model in which the increase in the Em of [Formula: see text] promotes safe, direct [Formula: see text] charge recombination at the expense of the damaging back-reaction route that involves chlorophyll triplet-mediated 1O2 formation [Johnson GN, et al. (1995) Biochim Biophys Acta 1229:202-207]. These findings provide a redox tuning mechanism, in which the interdependence of the redox state of QA and the binding by HCO3- regulates and protects PSII. The potential for a sink (CO2) to source (PSII) feedback mechanism is discussed.

Light-dependent chlorophyll f synthase is a highly divergent paralog of PsbA of photosystem II.

Chlorophyll f (Chl f) permits some cyanobacteria to expand the spectral range for photosynthesis by absorbing far-red light. We used reverse genetics and heterologous expression to identify the enzyme for Chl f synthesis. Null mutants of "super-rogue" psbA4 genes, divergent paralogs of psbA genes encoding the D1 core subunit of photosystem II, abolished Chl f synthesis in two cyanobacteria that grow in far-red light. Heterologous expression of the psbA4 gene, which we rename chlF, enables Chl f biosynthesis in Synechococcus sp. PCC 7002. Because the reaction requires light, Chl f synthase is probably a photo-oxidoreductase that employs catalytically useful Chl a molecules, tyrosine YZ, and plastoquinone (as does photosystem II) but lacks a Mn4Ca1O5 cluster. Introduction of Chl f biosynthesis into crop plants could expand their ability to use solar energy.

Spatially Separated Photosystem II and a Silicon Photoelectrochemical Cell for Overall Water Splitting: A Natural-Artificial Photosynthetic Hybrid.

Integrating natural and artificial photosynthetic platforms is an important approach to developing solar-driven hybrid systems with exceptional function over the individual components. A natural-artificial photosynthetic hybrid platform is formed by wiring photosystem II (PSII) and a platinum-decorated silicon photoelectrochemical (PEC) cell in a tandem manner based on a photocatalytic-PEC Z-scheme design. Although the individual components cannot achieve overall water splitting, the hybrid platform demonstrated the capability of unassisted solar-driven overall water splitting. Moreover, H2 and O2 evolution can be separated in this system, which is ascribed to the functionality afforded by the unconventional Z-scheme design. Furthermore, the tandem configuration and the spatial separation between PSII and artificial components provide more opportunities to develop efficient natural-artificial hybrid photosynthesis systems.

Isolation of novel PSII-LHCII megacomplexes from pea plants characterized by a combination of proteomics and electron microscopy.

In higher plants, photosystem II (PSII) is a multi-subunit pigment-protein complex embedded in the thylakoid membranes of chloroplasts, where it is present mostly in dimeric form within the grana. Its light-harvesting antenna system, LHCII, is composed of trimeric and monomeric complexes, which can associate in variable number with the dimeric PSII core complex in order to form different types of PSII-LHCII supercomplexes. Moreover, PSII-LHCII supercomplexes can laterally associate within the thylakoid membrane plane, thus forming higher molecular mass complexes, termed PSII-LHCII megacomplexes (Boekema et al. 1999a, in Biochemistry 38:2233-2239; Boekema et al. 1999b, in Eur J Biochem 266:444-452). In this study, pure PSII-LHCII megacomplexes were directly isolated from stacked pea thylakoid membranes by a rapid single-step solubilization, using the detergent n-dodecyl-α-D-maltoside, followed by sucrose gradient ultracentrifugation. The megacomplexes were subjected to biochemical and structural analyses. Transmission electron microscopy on negatively stained samples, followed by single-particle analyses, revealed a novel form of PSII-LHCII megacomplexes, as compared to previous studies (Boekema et al.1999a, in Biochemistry 38:2233-2239; Boekema et al. 1999b, in Eur J Biochem 266:444-452), consisting of two PSII-LHCII supercomplexes sitting side-by-side in the membrane plane, sandwiched together with a second copy. This second copy of the megacomplex is most likely derived from the opposite membrane of a granal stack. Two predominant forms of intact sandwiched megacomplexes were observed and termed, according to (Dekker and Boekema 2005 Biochim Biophys Acta 1706:12-39), as (C2S2)4 and (C2S2 + C2S2M2)2 megacomplexes. By applying a gel-based proteomic approach, the protein composition of the isolated megacomplexes was fully characterized. In summary, the new structural forms of isolated megacomplexes and the related modeling performed provide novel insights into how PSII-LHCII supercomplexes may bind to each other, not only in the membrane plane, but also between granal stacks within the chloroplast.

High Yield Non-detergent Isolation of Photosystem I-Light-harvesting Chlorophyll II Membranes from Spinach Thylakoids: IMPLICATIONS FOR THE ORGANIZATION OF THE PS I ANTENNAE IN HIGHER PLANTS.

Styrene-maleic acid copolymer was used to effect a non-detergent partial solubilization of thylakoids from spinach. A high density membrane fraction, which was not solubilized by the copolymer, was isolated and was highly enriched in the Photosystem (PS) I-light-harvesting chlorophyll (LHC) II supercomplex and depleted of PS II, the cytochrome b6/f complex, and ATP synthase. The LHC II associated with the supercomplex appeared to be energetically coupled to PS I based on 77 K fluorescence, P700 photooxidation, and PS I electron transport light saturation experiments. The chlorophyll (Chl) a/b ratio of the PS I-LHC II membranes was 3.2 ± 0.9, indicating that on average, three LHC II trimers may associate with each PS I. The implication of these findings within the context of higher plant PS I antenna organization is discussed.

Is It Beneficial for the Major Photosynthetic Antenna Complex of Plants To Form Trimers?

The process of primary electric charge separation in photosynthesis takes place in the reaction centers, but photosynthesis can operate efficiently and fluently due to the activity of several pigment-protein complexes called antenna, which absorb light quanta and transfer electronic excitations toward the reaction centers. LHCII is the major photosynthetic pigment-protein antenna complex of plants and appears in the trimeric form. Several recent reports point to trimeric organization of LHCII as a key factor responsible for the chloroplast architecture via stabilization of granal organization of the thylakoid membranes. In the present work, we address the question of whether such an organization could also directly influence the antenna properties of this pigment-protein complex. Chlorophyll fluorescence analysis reveals that excitation energy transfer in LHCII is substantially more efficient in trimers and dissipative energy losses are higher in monomers. It could be concluded that trimers are exceptionally well suited to perform the antenna function. Possibility of fine regulation of the photosynthetic antenna function via the LHCII trimer-monomer transition is also discussed, based on the fluorescence lifetime analysis in a single chloroplast.

Crystallization of Photosystem II for Time-Resolved Structural Studies Using an X-ray Free Electron Laser.

Photosystem II (PSII) is a membrane protein supercomplex that executes the initial reaction of photosynthesis in higher plants, algae, and cyanobacteria. It captures the light from the sun to catalyze a transmembrane charge separation. In a series of four charge separation events, utilizing the energy from four photons, PSII oxidizes two water molecules to obtain dioxygen, four protons, and four electrons. The light reactions of photosystems I and II (PSI and PSII) result in the formation of an electrochemical transmembrane proton gradient that is used for the production of ATP. Electrons that are subsequently transferred from PSI via the soluble protein ferredoxin to ferredoxin-NADP(+) reductase that reduces NADP(+) to NADPH. The products of photosynthesis and the elemental oxygen evolved sustain all higher life on Earth. All oxygen in the atmosphere is produced by the oxygen-evolving complex in PSII, a process that changed our planet from an anoxygenic to an oxygenic atmosphere 2.5 billion years ago. In this chapter, we provide recent insight into the mechanisms of this process and methods used in probing this question.

Effects of extrinsic proteins on the protein conformation of the oxygen-evolving center in cyanobacterial photosystem II as revealed by Fourier transform infrared spectroscopy.

Extrinsic proteins of photosystem II (PSII) play an important role in optimizing oxygen-evolving reactions in all oxyphototrophs. The currently available crystal structures of cyanobacterial PSII core complexes show the binding structures of the extrinsic proteins, PsbO, PsbV, and PsbU; however, how the individual extrinsic proteins affect the structure and the function of the oxygen-evolving center (OEC) in cyanobacterial PSII remains unknown. In this study, we have investigated the effects of the binding of the extrinsic proteins on the protein conformation of the OEC in PSII core complexes from the thermophilic cyanobacterium Thermosynechococcus elongatus, using light-induced Fourier transform infrared (FTIR) difference spectroscopy. Upon removal of the three extrinsic proteins, an S2-minus-S1 FTIR difference spectrum measured in the presence of a high CaCl2 concentration showed a drastic change in amide I bands, reflecting perturbation of the secondary structures of polypeptides, whereas the overall spectral intensity was lost at a low CaCl2 concentration, indicative of inactivation of the Mn4CaO5 cluster. The amide I features as well as the overall intensity were recovered mainly by binding of PsbO, while complete amide I recovery was achieved by further binding of PsbV and PsbU. We thus concluded that PsbO, together with smaller contributions of PsbV and PsbU, plays a role in the maintenance of the proper protein conformation of the OEC in cyanobacterial PSII, which provides the stability of the Mn4CaO5 cluster via the enhanced retention capability of Ca²âº and Cl⁻ ions.

Crystal structure of CyanoQ from the thermophilic cyanobacterium Thermosynechococcus elongatus and detection in isolated photosystem II complexes.

The PsbQ-like protein, termed CyanoQ, found in the cyanobacterium Synechocystis sp. PCC 6803 is thought to bind to the lumenal surface of photosystem II (PSII), helping to shield the Mn4CaO5 oxygen-evolving cluster. CyanoQ is, however, absent from the crystal structures of PSII isolated from thermophilic cyanobacteria raising the possibility that the association of CyanoQ with PSII might not be a conserved feature. Here, we show that CyanoQ (encoded by tll2057) is indeed expressed in the thermophilic cyanobacterium Thermosynechococcus elongatus and provide evidence in support of its assignment as a lipoprotein. Using an immunochemical approach, we show that CyanoQ co-purifies with PSII and is actually present in highly pure PSII samples used to generate PSII crystals. The absence of CyanoQ in the final crystal structure is possibly due to detachment of CyanoQ during crystallisation or its presence in sub-stoichiometric amounts. In contrast, the PsbP homologue, CyanoP, is severely depleted in isolated PSII complexes. We have also determined the crystal structure of CyanoQ from T. elongatus to a resolution of 1.6 Å. It lacks bound metal ions and contains a four-helix up-down bundle similar to the ones found in Synechocystis CyanoQ and spinach PsbQ. However, the N-terminal region and extensive lysine patch that are thought to be important for binding of PsbQ to PSII are not conserved in T. elongatus CyanoQ.

Light-harvesting ability of the fucoxanthin chlorophyll a/c-binding protein associated with photosystem II from the Diatom Chaetoceros gracilis as revealed by picosecond time-resolved fluorescence spectroscopy.

The fucoxanthin chlorophyll a/c-binding protein (FCP) is a unique antenna complex possessed by diatoms. Although FCP complexes have been isolated from various diatoms, there is no direct evidence for the existence of FCP associated with photosystem II (FCPII). Here, we report the isolation and spectroscopic characterization of FCPII complex from the diatom Chaetoceros gracilis. The FCPII complex was purified using sucrose centrifugation and anion-exchange chromatography. Clear-native PAGE and SDS-PAGE analyses revealed that the FCPII complex was composed of FCP-A oligomer and FCP-B/C trimer. Time-resolved fluorescence spectra of the FCPII complex were measured at 77 K. The characteristic lifetimes and fluorescence components were determined using global fitting analysis, followed by the construction of fluorescence decay-associated spectra (FDAS). FDAS exhibited fluorescence rises and decays, reflecting excitation energy transfer, with the time constants of 150 ps, 800 ps, and 2.9 ns. The long time constants are most likely attributed to the intercomplex excitation energy transfer between FCP-A oligomer and FCP-B/C trimer in the FCPII complex. The 5.6 ns FDAS likely originates from the final energy traps. In contrast, the FDAS exhibited no quenching component with any time constant. These results indicate that the FCPII complex is efficient in light harvesting and excitation energy transfer.