Nanodomains of Cytochrome b6f and Photosystem II Complexes in Spinach Grana Thylakoid Membranes

The cytochrome b₆f (cytb₆f) complex plays a central role in photosynthesis, coupling electron transport between photosystem II (PSII) and photosystem I to the generation of a transmembrane proton gradient used for the biosynthesis of ATP. Photosynthesis relies on rapid shuttling of electrons by plastoquinone (PQ) molecules between PSII and cytb₆f complexes in the lipid phase of the thylakoid membrane. Thus, the relative membrane location of these complexes is crucial, yet remains unknown. Here, we exploit the selective binding of the electron transfer protein plastocyanin (Pc) to the lumenal membrane surface of the cytb₆f complex using a Pc-functionalized atomic force microscope (AFM) probe to identify the position of cytb₆f complexes in grana thylakoid membranes from spinach (Spinacia oleracea). This affinity-mapping AFM method directly correlates membrane surface topography with Pc-cytb₆f interactions, allowing us to construct a map of the grana thylakoid membrane that reveals nanodomains of colocalized PSII and cytb₆f complexes. We suggest that the close proximity between PSII and cytb₆f complexes integrates solar energy conversion and electron transfer by fostering short-range diffusion of PQ in the protein-crowded thylakoid membrane, thereby optimizing photosynthetic efficiency.     

Suppression of Quantum Yield of Photosystem II by Hyperosmotic Stress in Chlamydomonas reinhardtii

Addition of ethylene glycol (EG) or NaCl to cells of Chlamydomonasreinhardtii induced transient non-photochemical quenching ofChl fluorescence correlated with the inhibition of photosyntheticoxygen evolution. The induction of the quenching and subsequentrecovery proceeded not only in the light but also in the dark.The quenching was almost unaffected by the protonophore nigericin,suggesting the involvement of a type of non-photochemical quenchingattributable to a state 2 transition. Higher concentrationsof EG or NaCl caused a delay of the recovery of the maximumfluorescence yield (Fm'). Dark reduction rate of P700⁺ afterthe application of a flash light in the presence of DCMU wasenhanced by the hyperosmotic shock, suggesting a stimulatedreduction of the intersystem electron carriers. It is proposedthat the osmotic stress stimulates electron donation from stromalcomponents via the NAD(P)H dehydrogenase, which results in thereduction of the intersystem chain and triggering of a state2 transition leading to stimulated cyclic PSI activity. (Received May 16, 1995; Accepted July 26, 1995)     

Cr6+胁迫对莱茵衣藻光合作用的影响

以莱茵衣藻(Chlamydomonas reinhardtii)为研究材料,采用氧电极和快速叶绿素a荧光诱导动力学方法研究了不同浓度和时间Cr6+处理对其光合作用的影响.结果表明:当Cr6+浓度大于40 μmol/L时,莱茵衣藻细胞数逐渐下降,而藻细胞变大;表观光合速率成为负值,呼吸作用随Cr6+处理浓度的增加先上升后下降至对照水平;莱茵衣藻有活性放氧复合体比例随Cr6+处理浓度的增加逐渐降低,80 μmol/L Cr6+处理3 d时已下降至13.72%;光合驱动力(DFABS)随Cr6+浓度增加逐步下降,并以DFφPo在DFABS的下降中的贡献最大.研究发现,重金属Cr6+胁迫显著影响莱茵衣藻的光合作用,而对呼吸作用则影响较小;Cr6+主要通过损伤供体侧的放氧复合体以及阻断QA至QB的电子传递而抑制光系统Ⅱ的功能;莱茵衣藻光系统Ⅱ对Cr6+处理比较敏感且存在着多个作用位点,并首先影响反应中心光能捕获效率,其次影响反应中心的活性,最后影响QA-之后的电子传递.     

Subunit Organization of a Synechocystis Hetero-Oligomeric Thylakoid FtsH Complex Involved in Photosystem II Repair

FtsH metalloproteases are key components of the photosystem II (PSII) repair cycle, which operates to maintain photosynthetic activity in the light. Despite their physiological importance, the structure and subunit composition of thylakoid FtsH complexes remain uncertain. Mutagenesis has previously revealed that the four FtsH homologs encoded by the cyanobacterium Synechocystis sp PCC 6803 are functionally different: FtsH1 and FtsH3 are required for cell viability, whereas FtsH2 and FtsH4 are dispensable. To gain insights into FtsH2, which is involved in selective D1 protein degradation during PSII repair, we used a strain of Synechocystis 6803 expressing a glutathione S-transferase (GST)–tagged derivative (FtsH2-GST) to isolate FtsH2-containing complexes. Biochemical analysis revealed that FtsH2-GST forms a hetero-oligomeric complex with FtsH3. FtsH2 also interacts with FtsH3 in the wild-type strain, and a mutant depleted in FtsH3, like ftsH2⁻ mutants, displays impaired D1 degradation. FtsH3 also forms a separate heterocomplex with FtsH1, thus explaining why FtsH3 is more important than FtsH2 for cell viability. We investigated the structure of the isolated FtsH2-GST/FtsH3 complex using transmission electron microscopy and single-particle analysis. The three-dimensional structural model obtained at a resolution of 26 Å revealed that the complex is hexameric and consists of alternating FtsH2/FtsH3 subunits.     

Revisiting the Supramolecular Organization of Photosystem II in Chlamydomonas reinhardtii

Photosystem II (PSII) is a multiprotein complex that splits water and initiates electron transfer in photosynthesis. The central part of PSII, the PSII core, is surrounded by light-harvesting complex II proteins (LHCIIs). In higher plants, two or three LHCII trimers are seen on each side of the PSII core whereas only one is seen in the corresponding positions in Chlamydomonas reinhardtii, probably due to the absence of CP24, a minor monomeric LHCII. Here, we re-examined the supramolecular organization of the C. reinhardtii PSII-LHCII supercomplex by determining the effect of different solubilizing detergents. When we solubilized the thylakoid membranes with n-dodecyl-β-d-maltoside (β-DM) or n-dodecyl-α-d-maltoside (α-DM) and subjected them to gel filtration, we observed a clear difference in molecular mass. The α-DM-solubilized PSII-LHCII supercomplex bound twice more LHCII than the β-DM-solubilized supercomplex and retained higher oxygen-evolving activity. Single-particle image analysis from electron micrographs of the α-DM-solubilized and negatively stained supercomplex revealed that the PSII-LHCII supercomplex had a novel supramolecular organization, with three LHCII trimers attached to each side of the core.     

Thylakoid membrane dynamics and state transitions in Chlamydomonas reinhardtii under elevated temperature

Photosynthesis Research - Moderately elevated temperatures can induce state transitions in higher plants by phosphorylation of light-harvesting complex II (LHCII). In this study, we exposed...     

TEF30 Interacts with Photosystem II Monomers and Is Involved in the Repair of Photodamaged Photosystem II in Chlamydomonas reinhardtii

The remarkable capability of photosystem II (PSII) to oxidize water comes along with its vulnerability to oxidative damage. Accordingly, organisms harboring PSII have developed strategies to protect PSII from oxidative damage and to repair damaged PSII. Here, we report on the characterization of the THYLAKOID ENRICHED FRACTION30 (TEF30) protein in Chlamydomonas reinhardtii, which is conserved in the green lineage and induced by high light. Fractionation studies revealed that TEF30 is associated with the stromal side of thylakoid membranes. By using blue native/Deriphat-polyacrylamide gel electrophoresis, sucrose density gradients, and isolated PSII particles, we found TEF30 to quantitatively interact with monomeric PSII complexes. Electron microscopy images revealed significantly reduced thylakoid membrane stacking in TEF30-underexpressing cells when compared with control cells. Biophysical and immunological data point to an impaired PSII repair cycle in TEF30-underexpressing cells and a reduced ability to form PSII supercomplexes after high-light exposure. Taken together, our data suggest potential roles for TEF30 in facilitating the incorporation of a new D1 protein and/or the reintegration of CP43 into repaired PSII monomers, protecting repaired PSII monomers from undergoing repeated repair cycles or facilitating the migration of repaired PSII monomers back to stacked regions for supercomplex reassembly.Oxygenic photosynthesis is essential for almost all life on Earth, as it provides the reduced carbon and the oxygen required for respiration. A key enzyme in oxygenic photosynthesis is PSII, which catalyzes the light-driven oxidation of water. The core of PSII in algae and land plants contains D1 (PsbA), D2 (PsbD), CP43 (PsbC), CP47 (PsbB), the α-subunit (PsbE) and β-subunit (PsbF) of cytochrome b₅₅₉, as well as several intrinsic low-molecular-mass subunits. The core monomer is associated with the extrinsic oxygen-evolving complex (OEC) consisting of OEE1 (PSBO), OEE2 (PSBP), and OEE3 (PSBQ), which stabilize the inorganic Mn₄O₅Ca cluster required for water oxidation (for review, see Pagliano et al., 2013). PSII core monomers assemble into dimers to which, at both sides, light-harvesting proteins (LHCII) bind to form PSII supercomplexes. In land plants, each PSII dimer binds two each of the monomeric minor LHCII proteins CP24, CP26, and CP29 in addition to up to four major LHCII trimers (Caffarri et al., 2009; Kouřil et al., 2011). Biochemical evidence suggests that, in the thylakoid membrane, up to eight LHCII trimers can be present per PSII core dimer, presumably because of the existence of a pool of extra LHCII (Kouřil et al., 2013). In Chlamydomonas reinhardtii, lacking CP24, each PSII dimer binds two each of the CP26 and CP29 monomers as well as up to six major LHCII trimers (Tokutsu et al., 2012). The reaction center proteins D1 and D2 bind all the redox-active cofactors required for PSII electron transport (Umena et al., 2011). Light captured by the internal antenna proteins CP43 and CP47 and the outer antenna induces charge separation in PSII, which in turn enables the OEC to oxidize water and provide electrons to the electron transfer chain. In land plants and green algae, PSII supercomplexes are localized to stacked regions of the thylakoid membranes, while the synthesis of PSII cores is considered to take place in stroma lamellae.A particular feature of PSII is its vulnerability to light, with the D1 protein being a target of light-induced damage and the damage being proportional to the photon flux density (PFD) applied (Tyystjärvi and Aro, 1996). To cope with this damage, an elaborate, highly conserved repair mechanism has evolved termed the PSII repair cycle, during which damaged PSII complexes are partially disassembled and the defective D1 protein is replaced by a de novo synthesized copy (for review, see Nixon et al., 2010; Komenda et al., 2012; Mulo et al., 2012; Nath et al., 2013a; Nickelsen and Rengstl, 2013; Tyystjärvi, 2013; Järvi et al., 2015). Photodamage occurs at all light intensities, but when the rate of damage exceeds the capacity for repair, photoinhibition is manifested as a decrease in the proportion of active PSII reaction centers (Aro et al., 1993). While PSII photodamage occurs in the supercomplexes in the stacked membrane regions, the replacement of damaged D1 takes place in stroma lamellae (Aro et al., 2005). Thus, the PSII repair cycle requires the lateral migration of PSII complexes, which is impaired by the macromolecular crowding in stacked thylakoid membranes (Kirchhoff, 2014). Lateral migration of damaged PSII complexes is facilitated by thylakoid membrane unfolding and PSII supercomplex disassembly. Both processes are enhanced by the phosphorylation of the PSII core subunits D1, D2, CP43, and PsbH, which is mainly mediated by the protein kinase STATE TRANSITION8 (STN8; Tikkanen et al., 2008; Fristedt et al., 2009; Herbstová et al., 2012; Nath et al., 2013b; Wunder et al., 2013). Efficient PSII supercomplex disassembly also requires the THYLAKOID FORMATION1 (THF1)/NON-YELLOW COLORING4 (NYC4)/Psb29 protein (Huang et al., 2013; Yamatani et al., 2013). After the migration of PSII monomers to unstacked thylakoid regions, PSII core subunits are dephosphorylated by the PSII core phosphatase PBCP (Samol et al., 2012), which is required for the efficient degradation of D1 (Koivuniemi et al., 1995; Rintamäki et al., 1996; Kato and Sakamoto, 2014). Degradation of D1 is subsequently realized by the membrane-integral FtsH protease (Lindahl et al., 2000; Silva et al., 2003) and by lumenal and stromal Deg proteases (Haussühl et al., 2001; Kapri-Pardes et al., 2007; Sun et al., 2010). Degradation is assisted by the THYLAKOID LUMEN PROTEIN18.3 (TLP18.3), presumably by its phosphatase activity and ability to interact with lumenal Deg1 (Sirpiö et al., 2007; Wu et al., 2011; Zienkiewicz et al., 2012). D1 proteolysis follows the partial disassembly of the PSII complex, during which CP43 and low-molecular-mass subunits are released to generate a CP43-free PSII monomer (Aro et al., 2005). Thereafter, a newly synthesized D1 copy is cotranslationally inserted from a plastidial 70S ribosome into the thylakoid membrane and processed by the CARBOXYL TERMINAL PEPTIDASE A (CTPA; Zhang et al., 1999, 2000; Che et al., 2013). In Arabidopsis (Arabidopsis thaliana), the D1 synthesis rate appears to be negatively regulated by the PROTEIN DISULFIDE ISOMERASE6 (PDI6; Wittenberg et al., 2014). Moreover, yet unknown steps during PSII repair require the stromal cyclophilin ROTAMASE CYP4 and stromal HEAT SHOCK PROTEIN70 (Schroda et al., 1999; Yokthongwattana et al., 2001; Cai et al., 2008). The PSII repair cycle is completed by the reassembly of the CP43 protein, ligation of the OEC, back migration of PSII to stacked membrane regions, and supercomplex formation. Except for CtpA, all mentioned factors appear to be specific for PSII repair, while many more auxiliary factors play roles in PSII de novo synthesis and repair (for review, see Järvi et al., 2015).In this study, we report on the functional characterization of the THYLAKOID ENRICHED FRACTION30 (TEF30) protein in C. reinhardtii. In this organism, TEF30 was first identified in a proteomics study on isolated thylakoid membranes (Allmer et al., 2006). TEF30 attracted our attention because its abundance increased 1.7-fold in membrane-enriched fractions of C. reinhardtii cells that had been shifted from 41 to 145 µmol photons m⁻² s⁻¹ for 8 h (Mettler et al., 2014; Supplemental Fig. S1). The TEF30 ortholog in Arabidopsis M-ENRICHED THYLAKOID PROTEIN1 (MET1; where M stands for mesophyll cells) was functionally characterized only recently (Bhuiyan et al., 2015). Both MET1 and TEF30 interact quantitatively with monomeric PSII core particles at the stroma side of the thylakoid membranes and play a role in the assembly of PSII monomers and/or their migration to stacked membrane regions for supercomplex assembly. While MET1 appears to exert this function during PSII de novo biogenesis and during the PSII repair cycle in Arabidopsis, TEF30 appears to function exclusively during PSII repair in C. reinhardtii.     

Activation of a Reserve Pool of Photosystem II in Chlamydomonas reinhardtii Counteracts Photoinhibition

The effect of strong irradiance (2000 micromole photons per square meter per second) on PSII heterogeneity in intact cells of Chlamydomonas reinhardtii was investigated. Low light (LL, 15 micromole photons per square meter per second) grown C. reinhardtii are photoinhibited upon exposure to strong irradiance, and the loss of photosynthetic functioning is due to damage to PSII. Under physiological growth conditions, PSII is distributed into two pools. The large antenna size (PSII_α) centers account for about 70% of all PSII in the thylakoid membrane and are responsible for plastoquinone reduction (Q_b-reducing centers). The smaller antenna (PSII_β) account for the remainder of PSII and exist in a state not yet able to photoreduce plastoquinone (Q_b-nonreducing centers). The exposure of C. reinhardtii cells to 60 minutes of strong irradiance disabled about half of the primary charge separation between P680 and pheophytin. The PSII_β content remained the same or slightly increased during strong-irradiance treatment, whereas the photochemical activity of PSII_α decreased by 80%. Analysis of fluorescence induction transients displayed by intact cells indicated that strong irradiance led to a conversion of PSII_β from a Q_b-nonreducing to a Q_b-reducing state. Parallel measurements of the rate of oxygen evolution revealed that photosynthetic electron transport was maintained at high rates, despite the loss of activity by a majority of PSII_α. The results suggest that PSII_β in C. reinhardtii may serve as a reserve pool of PSII that augments photosynthetic electron-transport rates during exposure to strong irradiance and partially compensates for the adverse effect of photoinhibition on PSII_α.     

Photosystem II peripheral accessory chlorophyll mutants in Chlamydomonas reinhardtii. Biochemical characterization and sensitivity to photo-inhibition

In addition to the four chlorophylls (Chls) involved in primary charge separation, the photosystem II (PSII) reaction center polypeptides, D1 and D2, coordinate a pair of symmetry-related, peripheral accessory Chls. These Chls are axially coordinated by the D1-H118 and D2-H117 residues and are in close association with the proximal Chl antennae proteins, CP43 and CP47. To gain insight into the function(s) of each of the peripheral Chls, we generated site-specific mutations of the amino acid residues that coordinate these Chls and characterized their energy and electron transfer properties. Our results demonstrate that D1-H118 and D2-H117 mutants differ with respect to: (a) their relative numbers of functional PSII complexes, (b) their relative ability to stabilize charge-separated states, (c) light-harvesting efficiency, and (d) their sensitivity to photo-inhibition. The D2-H117N and D2-H117Q mutants had reduced levels of functional PSII complexes and oxygen evolution capacity as well as reduced light-harvesting efficiencies relative to wild-type cells. In contrast, the D1-H118Q mutant was capable of near wild-type rates of oxygen evolution at saturating light intensities. The D1-H118Q mutant also was substantially more resistant to photo-inhibition than wild type. This reduced sensitivity to photo-inhibition is presumably associated with a reduced light-harvesting efficiency in this mutant. Finally, it is noted that the PSII peripheral accessory Chls have similarities to a to a pair of Chls also present in the PSI reaction center complex.     

Chlorophyll triplet states associated with Photosystem I and Photosystem II in thylakoids of the green alga Chlamydomonas reinhardtii

The analysis of FDMR spectra, recorded at multiple emission wavelengths, by a global decomposition technique, has allowed us to characterise the triplet populations associated with Photosystem I and Photosystem II of thylakoids in the green alga Chlamydomonas reinhardtii. Three triplet populations are observed at fluorescence emissions characteristic of Photosystem II, and their zero field splitting parameters have been determined. These are similar to the zero field parameters for the three Photosystem II triplets previously reported for spinach thylakoids, suggesting that they have a widespread occurrence in nature. None of these triplets have the zero field splitting parameters characteristic of the Photosystem II recombination triplet observed only under reducing conditions. Because these triplets are generated under non-reducing redox conditions, when the recombination triplet is undetectable, it is suggested that they may be involved in the photoinhibition of Photosystem II. At emission wavelengths characteristic of Photosystem I, three triplet populations are observed, two of which are attributed to the P(700) recombination triplet frozen in two different conformations, based on the microwave-induced fluorescence emission spectra and the triplet minus singlet difference spectra. The third triplet population detected at Photosystem I emission wavelengths, which was previously unresolved, is proposed to originate from the antenna chlorophyll of the core or the unusually blue-shifted outer antenna complexes of this organism.     

Low molecular weight subunits associated with the cytochrome b 6 f complexes from spinach and Chlamydomonas reinhardtii

Cytochrome b ₆ f complexes, prepared from spinach and Chlamydomonas thylakoids, have been examined for their content of low molecular weight subunits. The spinach complex contains two prominent low molecular weight subunits of 3.7 and 4.1 kD while a single prominent component of 4.5 kD was present in the Chlamydomonas complex. An estimation of the relative stoichiometry of these subunits suggests several are present at levels approximating one copy per cytochrome complex. The low molecular weight subunits were purified by reversed phase HPLC and N-terminal sequences obtained. Both the spinach and Chlamydomonas cytochrome complexes contain a subunit that is identified as the previously characterized petG gene product (4.8 kD in spinach and 4.1 kD in Chlamydomonas). A second subunit (3.8 kD in spinach and 3.7 kD in Chlamydomonas) appears to be homologous in the two complexes and is likely to be a nuclear gene product. The possible presence of other low molecular weight subunits in these complexes is also considered.     

The chlorophyll-a/b proteins of photosystem II in Chlamydomonas reinhardtii

We have adapted the procedure for the isolation of PSII membranes from higher plants (D.A. Berthold et al., 1981, FEBS Lett. 134, 231–234) to the green algae Chlamydomonas reinhardtii. The chlorophyll (Chl)-binding proteins from this PSII preparation have been further separated into single Chl-binding polypeptides and characterized spectroscopically. Seven single polypeptides were shown to bind Chl a and Chl b. In particular, we demonstrate that polypeptides p9, p10 and p22, which had not been previously shown to bind Chl a and b, have characteristics similar to those of CP29, CP26 and CP24 from higher plants. We note, however, that p9 and p10 are phosphorylatable in C. reinhardtii, at variance with CP29 and CP26 from higher plants. Our data support the notion that the PSII antenna systems in C. reinhardtii and in higher plants are very similar. Therefore, studies on the organization and regulation of light-harvesting processes in C. reinhardtii may provide information of general relevance for both green algae and higher plants.Abbreviations Chl chlorophyll - IEF isoelectrofocusing - LHC light harvesting complex - MW molecular weight - PAGE polyacrylamide gel electrophoresis - PS photosystem - RC reaction centre - SDS sodium dodecylsulfate We thank Dr. J. Olive (Institut Jacques Monod, Paris, France) for the electron-microscopy analysis, C. de Vitry (Institut de Biologie Physico-Chimique, Paris, France) for the kind gift of a PSII RC preparation and P. Dainese and M.L. Di Paolo (Universitá di Padova, Padova, Italy) for helpfull discussions. Professor Strasser and Elizbeth Scwartz (Université de Genova, Genova, Switzerland) are thanked for assistance in taking low-temperature fluorescence emission spectra. Roberto Bassi was recipient of a short-term fellowship from the European Molecular Biology Organization fellowship, during the early phases of the work.     

Cross-reconstitution of the extrinsic proteins and Photosystem II complexes from Chlamydomonas reinhardtii and Spinacia oleracea

Cross-reconstitution of the extrinsic proteins and Photosystem II (PS II) from a green alga, Chlamydomonas reinhardtii, and a higher plant,Spinacia oleracea, was performed to clarify the differences of binding properties of the extrinsic proteins between these two species of organisms. (1) Chlamydomonas PsbP and PsbQ directly bound to Chlamydomonas PS II independent of the other extrinsic proteins but not to spinach PS II. (2) Chlamydomonas PsbP and PsbQ directly bound to the functional sites of Chlamydomonas PS II independent of the origins of PsbO, while spinach PsbP and PsbQ only bound to non-functional sites on Chlamydomonas PS II. (3) Both Chlamydomonas PsbP and spinach PsbP functionally bound to spinach PS II in the presence of spinach PsbO. (4) While Chlamydomonas PsbP functionally bound to spinach PS II in the presence of Chlamydomonas PsbO, spinach PsbP bound loosely to spinach PS II in the presence of Chlamydomonas PsbO with no concomitant restoration of oxygen evolution. (5) Chlamydomonas PsbQ bound to spinach PS II in the presence of Chlamydomonas PsbP and PsbO or spinach PsbO but not to spinach PS II in the presence of spinach PsbP and Chlamydomonas PsbO or spinach PsbO. (6) Spinach PsbQ did not bind to spinach PS II in the presence of Chlamydomonas PsbO and PsbP. On the basis of these results, we showed a simplified scheme for binding patterns of the green algal and higher plant extrinsic proteins with respective PS II.     

Characterization of a novel Photosystem I-LHCI supercomplex isolated from Chlamydomonas reinhardtii under anaerobic (State II) conditions

A novel supercomplex of Photosystem I (PSI) with light harvesting complex I (LHCI) was isolated from the green alga Chlamydomonas reinhardtii. This novel supercomplex is unique as it is the first stable supercomplex of PSI together with its external antenna. The supercomplex contains 256 chlorophylls per reaction center. The supercomplex was isolated under anaerobic conditions and may represent the State II form of the photosynthetic unit. In contrast to previously reported supercomplexes isolated in State I, which contain only 4 LHC I proteins, this supercomplex contains 10-11 LHC I proteins tightly bound to the PSI core. In contrast to plants, no LHC II is tightly bound to the PSI-LHCI supercomplex in State II. Investigation of the energy transfer from the antenna system to the reaction center core shows that the LHC supercomplexes are tightly coupled to the PSI core, not only structurally but also energetically. The excitation energy transfer kinetics are completely dominated by the fast phase, with a near-complete lack of long-lived fluorescence. This tight coupling is in contrast to all reports of energy transfer in PSI-LHCI supercomplexes (in State I), which have so far been described as weakly coupled supercomplexes with low efficiency for excitation energy transfer. These results indicate that there are large and dynamic changes of the PSI-LHCI supercomplex during the acclimation from aerobic (State I) to anaerobic (State II) conditions in Chlamydomonas.     

Mutation of Residue Arginine~(18)of Cytochrome b_(559)a-Subunit and its Effects on Photosystem II Activities in Chlamydomonas reinhardtii

It has been known that arginine is used as the basic amino acid in the a-subunit of cytochrome b_(559)(Cyt b_(559)) excepthistidine. However, previous studies have focused on the function of histidine in the activities of photosystem (PS) II andthere are no reports regarding the structural and/or functional roles of arginine in PSII complexes. In the present study,two arginine (R18) mutants of Chlamydomonas reinhardtii were constructed using site-directed mutagenesis, in whichR18 was replaced by glutamic acid (E) and glycine (G). The results show that the oxygen evolution of the PSII complexin the R18G and R18E mutants was approximately 60% of wild-type (WT) levels and that, after irradiation at high lightintensity, oxygen evolution for the PSII of mutants was reduced to zero compared with 40% in WT cells. The efficiency oflight capture by PSII (F_v/F_m) of R18G and R18E mutants was approximately 42%-46% that of WT cells. Furthermore, levelsof the a-subunit of Cyt b_(559) and PsbO proteins were reduced in thylakoid membranes compared with WT. Overall, thesedata suggest that R18 plays a significant role in helping Cyt b_559 maintain the structure of the PSII complex and its activity,although it is not directly bound to the heme group.     

A highly active histidine-tagged Chlamydomonas reinhardtii Photosystem II preparation for structural and biophysical analysis.

We describe a genetically engineered strain of Chlamydomonas reinhardtii where the PsbH subunit of Photosystem II (PSII) has been modified to include a C-terminal polyhistidine tag. The strain was generated by the rescue to photoautotrophic growth of a psbH insertional mutant following chloroplast transformation with the modified gene. This selection strategy confirms that the addition of the tag to PsbH does not prevent the assembly of functional PSII, and results in an engineered strain with tagged PSII but no antibiotic-resistance markers in the chloroplast genome. Consequently, the strain is suitable for subsequent genetic manipulation of chloroplast PSII genes. We also describe a rapid PSII isolation procedure that gives a preparation capable of high rates of oxygen evolution. This preparation is suitable for spectroscopic analysis as shown by EPR analysis of the S2 state of the water oxidation cycle. Furthermore, electron microscopy, coupled to single particle analysis, has revealed the isolated PSII to be structurally homogeneous core dimers that are ideally suited for higher resolution structural studies.     

Characterization of New Strains of Nonphotosynthetic Mutants of Chlamydomonas reinhardtii III. Photosystem II-Related Thylakoid Proteins in Five Mutants and Double Mutants

PS II-enriched particles of the wild type, of three mutantsand of two double mutants of Chlamydomonas reinhardtii wereanalyzed by lithium dodecylsulfate polyacrylamide gel electrophoresisat 4°C. The mutant Pg 27 was devoid of light-harvestingChl-protein complex (CP) CP II, but had normal cytochrome b-559and displayed all wild type photochemical activities. The mutantFl 50 lacked a pool of cytochrome b-559 photooxidizable at 77K but was able to photooxidize a second pool at 293 K in thepresence of FCCP; it showed some weak PS II activity. The mutantFl 39 lacked both these cytochrome b-559 pools and did not displayany PS II activity. The double mutants Fl 39 Pg 28 and Fl 50Pg 27 had defects similar to those of their respective parentsFl 39 or Fl 50 but, in addition, they were devoid of Chi b andof CP II. In these four mutants having impaired PS II function,five proteins of M_r=50,000, 47,000, 33,000, 27,000 and 19,000were totally (Fl 39, Fl 39 Pg 28) or partly (Fl 50, Fl 50 Pg27) missing. The first two of these proteins corresponded tothe apoproteins of CP III and IV. These results pointed out a strong correlation between thesefive proteins, cytochrome b-559 and PS II primary photochemistry.In mutation and cross experiments, these five PS II-associatedproteins and cytochrome b-559 appeared to be linked characterscontrolled by nuclear gene(s), but they behaved independentlyof CP II. (Received January 24, 1983; Accepted July 20, 1983)