Binding stoichiometry and affinity of the manganese-stabilizing protein affects redox reactions on the oxidizing side of photosystem II

It has been reported previously that the two subunits of PsbO, the photosystem II (PSII) manganese stabilizing protein, have unique functions in relation to the Mn, Ca(2+), and Cl(-) cofactors in eukaryotic PSII [Popelkova; (2008) Biochemistry 47, 12593]. The experiments reported here utilize a set of N-terminal truncation mutants of PsbO, which exhibit altered subunit binding to PSII, to further characterize its role in establishing efficient O(2) evolution activity. The effects of PsbO binding stoichiometry, affinity, and specificity on Q(A)(-) reoxidation kinetics after a single turnover flash, S-state transitions, and O(2) release time have been examined. The data presented here show that weak rebinding of a single PsbO subunit to PsbO-depleted PSII repairs many of the defects in PSII resulting from the removal of the protein, but many of these are not sustainable, as indicated by low steady-state activities of the reconstituted samples [Popelkova; (2003) Biochemistry 42 , 6193]. High affinity binding of PsbO to PSII is required to produce more stable and efficient cycling of the water oxidation reaction. Reconstitution of the second PsbO subunit is needed to further optimize redox reactions on the PSII oxidizing side. Native PsbO and recombinant wild-type PsbO from spinach facilitate PSII redox reactions in a very similar manner, and nonspecific binding of PsbO to PSII has no significance in these reactions.     

Inorganic cofactor stabilization and retention: the unique functions of the two PsbO subunits of eukaryotic photosystem II

Eukaryotic PsbO, the photosystem II (PSII) manganese-stabilizing protein, has two N-terminal sequences that are required for binding of two copies of the protein to PSII [Popelkova, H., et al. (2002) Biochemistry 41, 10038-10045; Popelkova, H., et al. (2003) Biochemistry 42, 6193-6200]. In the work reported here, a set of selected N-terminal truncation mutants of PsbO that affect subunit binding to PSII were used to determine the effects of PsbO stoichiometry on the Mn, Ca(2+), and Cl(-) cofactors and to characterize the roles of each of the PsbO subunits in PSII function. Results of the experiments with the PsbO-depleted PSII membranes reconstituted with the PsbO deletion mutants showed that the presence of PsbO does not affect Ca(2+) retention by PSII in steady-state assays of activity, nor is it required for Ca(2+) to protect the Mn cluster against reductive inhibition in darkness. In contrast to the results with Ca(2+), PsbO increases the affinity of Cl(-) for the active site of the O(2)-evolving complex (OEC) as expected. These results together with other data on activity retention suggest that PsbO can stabilize the Mn cluster by facilitating retention of Cl(-) in the OEC. The data presented here indicate that each of two copies of PsbO has a distinctive function in PSII. Binding of the first PsbO subunit fully stabilizes the Mn cluster and enhances Cl(-) retention, while binding of the second subunit optimizes Cl(-) retention, which in turn maximizes O(2) evolution activity. Nonspecific binding of some PsbO truncation mutants to PSII has no functional significance.     

The double mutation ΔL6MW241F in PsbO, the photosystem II manganese stabilizing protein, yields insights into the evolution of its structure and function

The W241F mutation in spinach manganese-stabilizing protein (PsbO) decreases binding to photosystem II (PSII); its thermostability is increased and reconstituted activity is lower [Wyman et al. (2008) Biochemistry 47, 6490-6498]. The results reported here show that W241F cannot adopt a normal solution structure and fails to reconstitute efficient Cl⁻ retention by PSII. An N-terminal truncation of W241F, producing the ΔL6MW241F double mutant that resembles some features of cyanobacterial PsbO, significantly repairs the defects in W241F. Our data suggest that the C-terminal F → W mutation likely evolved in higher plants and green algae in order to preserve proper PsbO folding and PSII binding and assembly, which promotes efficient Cl⁻ retention in the oxygen-evolving complex.     

Subsequent events to GTP binding by the plant PsbO protein: structural changes, GTP hydrolysis and dissociation from the photosystem II complex

Besides an essential role in optimizing water oxidation in photosystem II (PSII), it has been reported that the spinach PsbO protein binds GTP [C. Spetea, T. Hundal, B. Lundin, M. Heddad, I. Adamska, B. Andersson, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 1409-1414]. Here we predict four GTP-binding domains in the structure of spinach PsbO, all localized in the beta-barrel domain of the protein, as judged from comparison with the 3D-structure of the cyanobacterial counterpart. These domains are not conserved in the sequences of the cyanobacterial or green algae PsbO proteins. MgGTP induces specific changes in the structure of the PsbO protein in solution, as detected by circular dichroism and intrinsic fluorescence spectroscopy. Spinach PsbO has a low intrinsic GTPase activity, which is enhanced fifteen-fold when the protein is associated with the PSII complex in its dimeric form. GTP stimulates the dissociation of PsbO from PSII under light conditions known to also release Mn(2+) and Ca(2+) ions from the oxygen-evolving complex and to induce degradation of the PSII reaction centre D1 protein. We propose the occurrence in higher plants of a PsbO-mediated GTPase activity associated with PSII, which has consequences for the function of the oxygen-evolving complex and D1 protein turnover.     

Functional dissection of two Arabidopsis PsbO proteins: PsbO1 and PsbO2

PsbO protein is an extrinsic subunit of photosystem II (PSII) and has been proposed to play a central role in stabilization of the catalytic manganese cluster. Arabidopsis thaliana has two psbO genes that express two PsbO proteins; PsbO1 and PsbO2. We reported previously that a mutant plant that lacked PsbO1 (psbo1) showed considerable growth retardation despite the presence of PsbO2 [Murakami, R., Ifuku, K., Takabayashi, A., Shikanai, T., Endo, T., and Sato, F. (2002) FEBS Lett523, 138-142]. In the present study, we characterized the functional differences between PsbO1 and PsbO2. We found that PsbO1 is the major isoform in the wild-type, and the amount of PsbO2 in psbo1 was significantly less than the total amount of PsbO in the wild-type. The amount of PsbO as well as the efficiency of PSII in psbo1 increased as the plants grew; howeVER, it neVER reached the total PsbO level observed in the wild-type, suggesting that the poor activity of PSII in psbo1 was caused by a shortage of PsbO. In addition, an in vitro reconstitution experiment using recombinant PsbOs and urea-washed PSII particles showed that oxygen evolution was better recoVERed by PsbO1 than by PsbO2. Further analysis using chimeric and mutated PsbOs suggested that the amino acid changes Val186-->Ser, Leu246-->Ile, and Val204-->Ile could explain the functional difference between the two PsbOs. Therefore we concluded that both the lower expression level and the inferior functionality of PsbO2 are responsible for the phenotype observed in psbo1.     

Subsequent events to GTP binding by the plant PsbO protein: Structural changes, GTP hydrolysis and dissociation from the photosystem II complex

Besides an essential role in optimizing water oxidation in photosystem II (PSII), it has been reported that the spinach PsbO protein binds GTP [C. Spetea, T. Hundal, B. Lundin, M. Heddad, I. Adamska, B. Andersson, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 1409-1414]. Here we predict four GTP-binding domains in the structure of spinach PsbO, all localized in the β-barrel domain of the protein, as judged from comparison with the 3D-structure of the cyanobacterial counterpart. These domains are not conserved in the sequences of the cyanobacterial or green algae PsbO proteins. MgGTP induces specific changes in the structure of the PsbO protein in solution, as detected by circular dichroism and intrinsic fluorescence spectroscopy. Spinach PsbO has a low intrinsic GTPase activity, which is enhanced fifteen-fold when the protein is associated with the PSII complex in its dimeric form. GTP stimulates the dissociation of PsbO from PSII under light conditions known to also release Mn²⁺ and Ca²⁺ ions from the oxygen-evolving complex and to induce degradation of the PSII reaction centre D1 protein. We propose the occurrence in higher plants of a PsbO-mediated GTPase activity associated with PSII, which has consequences for the function of the oxygen-evolving complex and D1 protein turnover.     

Site-directed mutagenesis of conserved C-terminal tyrosine and tryptophan residues of PsbO, the photosystem II manganese-stabilizing protein, alters its activity and fluorescence properties

The extrinsic photosystem II PsbO subunit (manganese-stabilizing protein) contains near-UV CD signals from its complement of aromatic amino acid residues (one Trp, eight Tyr, and 13 Phe residues). Acidification, N-bromosuccinimide modification of Trp, reduction or elimination of a disulfide bond, or deletion of C-terminal amino acids abolishes these signals. Site-directed mutations that substitute Phe for Trp241 and Tyr242, near the C-terminus of PsbO, were used to examine the contribution of these residues to the activity and spectral properties of the protein. Although this substitution is, in theory, conservative, neither mutant binds efficiently to PSII, even though these proteins appear to retain wild-type solution structures. Removal of six residues from the N-terminus of the W241F mutant restores activity to near-wild-type levels. The near-UV CD spectra of the mutants are modified; well-defined Tyr and Trp peaks are lost. Characterizations of the fluorescence spectra of the full-length WF and YF mutants indicate that Y242 contributes significantly to PsbO's Tyr fluorescence emission and that an excited-state tyrosinate could be present in PsbO. Deletion of W241 shows that this residue is a major contributor to PsbO's fluorescence emission. Loss of function is consistent with the proposal that a native C-terminal domain is required for PsbO binding and activity, and restoration of activity by deletion of N-terminal amino acids may provide some insights into the evolution of this important photosynthetic protein.     

Towards understanding the functional difference between the two PsbO isoforms in Arabidopsis thaliana—insights from phenotypic analyses of psbo knockout mutants

The extrinsic PsbO subunit of the water-oxidizing photosystem II (PSII) complex is represented by two isoforms in Arabidopsis thaliana, namely PsbO1 and PsbO2. Recent analyses of psbo1 and psbo2 knockout mutants have brought insights into their roles in photosynthesis and light stress. Here we analyzed the two psbo mutants in terms of PsbOs expression pattern, organization of PSII complexes and GTPase activity. Both PsbOs are present in wild-type plants, and their expression is mutually controlled in the mutants. Almost all PSII complexes are in the monomeric form not only in the psbo1 but also in the psbo2 mutant grown under high-light conditions. This results either from an enhanced susceptibility of PSII to photoinactivation or from malfunction of the repair cycle. Notably, the psbo1 mutant displays such problems even under growth-light conditions. These results together with the finding that PsbO2 has a threefold higher GTPase activity than PsbO1 have significance for the turnover of the PSII D1 subunit in Arabidopsis.     

pH optimum of the photosystem II H₂O oxidation reaction: effects of PsbO, the manganese-stabilizing protein, Cl- retention, and deprotonation of a component required for O₂ evolution activity

Hydroxide ion inhibits Photosystem II (PSII) activity by extracting Cl(-) from its binding site in the O(2)-evolving complex (OEC) under continuous illumination [Critchley, C., et al. (1982) Biochim. Biophys. Acta 682, 436]. The experiments reported here examine whether two subunits of PsbO, the manganese-stabilizing protein, bound to eukaryotic PSII play a role in protecting the OEC against OH(-) inhibition. The data show that the PSII binding properties of PsbO affect the pH optimum for O(2) evolution activity as well as the Cl(-) affinity of the OEC that decreases with an increasing pH. These results suggest that PsbO functions as a barrier against inhibition of the OEC by OH(-). Through facilitation of efficient retention of Cl(-) in PSII [Popelkova, H., et al. (2008) Biochemistry 47, 12593], PsbO influences the ability of Cl(-) to resist OH(-)-induced release from its site in the OEC. Preventing inhibition by OH(-) allows for normal (short) lifetimes of the S(2) and S(3) states in darkness [Roose, J. L., et al. (2011) Biochemistry 50, 5988] and for maximal steady-state activity by PSII. The data presented here indicate that activation of H(2)O oxidation occurs with a pK(a) of ～6.5, which could be a function of deprotonation of one or more amino acid residues that reside near the OEC active site on the D1 and CP43 intrinsic subunits of the PSII reaction center.     

Structural investigation of PsbO from plant and cyanobacterial photosystem II

The manganese-stabilizing protein PsbO is associated with the luminal side of thylakoids close to the redox-active Mn₄Ca cluster at the catalytically active site of photosystem II (PSII). PsbO is believed to increase the efficiency of oxygen evolution and to stabilize the Mn₄Ca cluster against photoinhibition. Using small-angle X-ray scattering, we investigated the low-resolution structure of wild-type spinach PsbO and that of chimeric spinach PsbO fused with maltose-binding protein. Small-angle X-ray scattering data revealed that both proteins are monomeric in solution, and that plant and cyanobacterial PsbO have similar structures. We show a highly efficient expression system that allows recombinant production of the active wild type and the chimeric PsbO from spinach and cyanobacteria, with yields compatible with biophysical and structural studies. The binding of spinach PsbO fused with maltose-binding protein to PSII depleted of extrinsic subunits (PSII-ΔpsbO,P,Q) was confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The reconstituted PSII was shown to have similar oxygen evolution rates as obtained with wild-type spinach PsbO.     

Binding of manganese stabilizing protein to photosystem II: identification of essential N-terminal threonine residues and domains that prevent nonspecific binding

The N-terminus of spinach photosystem II manganese stabilizing protein (MSP) contains two amino acid sequences, (4)KRLTYD(10)E and (15)TYL(18)E, that are necessary for binding of two copies of this subunit to the enzyme [Popelkova et al. (2002) Biochemistry 41, 10038-10045]. To better understand the basis of MSP-photosystem II interactions, the role of threonine residues in the highly conserved motifs T(Y/F)DE and TY has been characterized. Deletion mutants lacking the first 5, 6, 7, and 15 amino acid residues at the N-terminus of the protein were examined for their ability to reconstitute activity in MSP-depleted photosystem II. The results reported here show that truncations of five and six amino acid residues (mutants DeltaR5M and DeltaL6M mutants) have no negative effect on recovery of oxygen evolution activity or on binding of MSP to photosystem II. Deletion of seven residues (mutant DeltaT7M) decreases reconstitution activity to 40% of the control value and reduces functional binding of the mutant protein to photosystem II from two to one copy. Deletion of 15 amino acid residues (mutant DeltaT15M) severely impairs functional binding of MSP, and lowers O(2) evolution activity to less than 20% of the control. DeltaT7M is the only mutant that exhibited neither nonspecific binding to photosystem II nor changes in tertiary structure. These, and previous results, show that the highly conserved Thr7 and Thr15 residues of MSP are required for functional binding of two copies of the eukaryotic protein to photosystem II. Although the N-terminal domains, (1)EGGKR(6)L, (8)YDEIQS(14)K, and (16)YL(18)E of spinach MSP are unnecessary for specific, functional binding interactions, these sequences are necessary to prevent nonspecific binding of the protein to photosystem II.     

N-terminal truncations of manganese stabilizing protein identify two amino acid sequences required for binding of the eukaryotic protein to photosystem II and reveal the absence of one binding-related sequence in cyanobacteria

Manganese stabilizing protein (MSP) is an intrinsically disordered extrinsic subunit of photosystem II that regulates the stability and kinetic performance of the tetranuclear manganese cluster that oxidizes water to oxygen. An earlier study showed that deletion of the (1)E-(3)G domain of MSP caused no loss of activity reconstitution, whereas deletion of the (4)K-(10)E domain reduced binding of the protein from 2 to 1 mol of MSP/mol of photosystem II and lowered activity reconstitution to about 50% of the control value [Popelkova et al. (2002) Biochemistry 41, 2702-2711]. In this work we present evidence that deletion of 13 or 14 amino acid residues from the MSP N-terminus (mutants DeltaS13M and DeltaK14M) does not interfere either with functional binding of one copy of MSP to photosystem II or with reconstitution of oxygen evolution activity to 50% of the control level. Both of these mutants exhibit nonspecific binding to photosystem II at higher protein concentrations. Truncation of the MSP sequence by 18 amino acids (mutant DeltaE18M), however, causes a loss of protein binding and activity reconstitution. This result demonstrates that the N-terminal domain (15)T-(18)E is required for binding of at least one copy of MSP to photosystem II. Analyses of CD spectra reveal changes in the structure of DeltaE18M (loss of beta-sheet, gain of unordered structure). Use of the information gained from these experiments in analyses of N-terminal sequences of MSP from a number of species indicates that higher plants and algae possess two recognition domains that are required for MSP binding to PSII, whereas cyanobacteria lack the first N-terminal domain found in eukaryotes. This may explain the absence of a second copy of MSP in the crystal structure of PSII from Synechococcus elongatus [Zouni et al. (2001) Nature 409, 739-743].     

Degradation of PsbO by the Deg Protease HhoA Is Thioredoxin Dependent

The widely distributed members of the Deg/HtrA protease family play an important role in the proteolysis of misfolded and damaged proteins. Here we show that the Deg protease rHhoA is able to degrade PsbO, the extrinsic protein of the Photosystem II (PSII) oxygen-evolving complex in Synechocystis sp. PCC 6803 and in spinach. PsbO is known to be stable in its oxidized form, but after reduction by thioredoxin it became a substrate for recombinant HhoA (rHhoA). rHhoA cleaved reduced eukaryotic (specifically, spinach) PsbO at defined sites and created distinct PsbO fragments that were not further degraded. As for the corresponding prokaryotic substrate (reduced PsbO of Synechocystis sp. PCC 6803), no PsbO fragments were observed. Assembly to PSII protected PsbO from degradation. For Synechocystis sp. PCC 6803, our results show that HhoA, HhoB, and HtrA are localized in the periplasma and/or at the thylakoid membrane. In agreement with the idea that PsbO could be a physiological substrate for Deg proteases, part of the cellular fraction of the three Deg proteases of Synechocystis sp. PCC 6803 (HhoA, HhoB, and HtrA) was detected in the PSII-enriched membrane fraction.     

Isolation of a novel heterodimeric PSII complex via strep-tagged PsbO

The multi-subunit membrane protein complex photosystem II (PSII) catalyzes the light-driven oxidation of water and with this the initial step of photosynthetic electron transport in plants, algae, and cyanobacteria. Its biogenesis is coordinated by a network of auxiliary proteins that facilitate the stepwise assembly of individual subunits and cofactors, forming various intermediate complexes until fully functional mature PSII is present at the end of the process. In the current study, we purified PSII complexes from a mutant line of the thermophilic cyanobacterium Thermosynechococcus vestitus BP-1 in which the extrinsic subunit PsbO, characteristic for active PSII, was fused with an N-terminal Twin-Strep-tag. Three distinct PSII complexes were separated by ion-exchange chromatography after the initial affinity purification. Two complexes differ in their oligomeric state (monomeric and dimeric) but share the typical subunit composition of mature PSII. They are characterized by the very high oxygen evolving activity of approx. 6000 μmol O₂·(mg Chl·h)^?1. Analysis of the third (heterodimeric) PSII complex revealed lower oxygen evolving activity of approx. 3000 μmol O₂·(mg Chl·h)^?1 and a manganese content of 2.7 (±0.2) per reaction center compared to 3.7 (±0.2) of fully active PSII. Mass spectrometry and time-resolved fluorescence spectroscopy further indicated that PsbO is partially replaced by Psb27 in this PSII fraction, thus implying a role of this complex in PSII repair.     

Construction and characterization of a functional mutant of Synechocystis 6803 harbouring a eukaryotic PSII-H subunit.

A Synechocystis 6803 mutant carrying a chimaeric photosystem II (PSII), in which the Zea mays PsbH subunit (7.7 kDa calculated molecular mass) replaces the cyanobacterial copy (7.0 kDa), was constructed. With the exception of the N-terminal 12 amino acid extension, which has a phosphorylatable threonine, the eukaryotic polypeptide is 78% homologous to its bacterial counterpart. Biochemical characterization of this mutant shows that it expresses the engineered gene correctly and is competent for photoautotrophic growth. Fluorescence analysis and oxygen evolution measurements in the presence of exogenous acceptors indicate that the observed phenotype results from a chimaeric PSII rather than from the absence of function associated with PsbH, suggesting that the heterologous protein is assembled into a functional PSII. Inhibition of oxygen evolution by herbicides belonging to different classes shows that the sensitivity of the mutant PSII is changed only towards phenolic compounds. This result indicates slight conformational modification of the QB/herbicide binding pocket of the D1 polypeptide caused by the bulky PsbH protein in the mutant, and also suggests close structural interaction of the D1 and PsbH subunits in the topological arrangement of PSII.     

Isolation and characterization of oxygen-evolving photosystem II complexes retaining the PsbO, P and Q proteins from Euglena gracilis

Oxygen-evolving photosystem II (PSII) complexes of Euglena gracilis were isolated and characterized. (1) The PSII complexes contained three extrinsic proteins of 33 kDa (PsbO), 23 kDa (PsbP) and 17 kDa (PsbQ), and showed oxygen-evolving activity of around 700 micromol O2 (mg Chl)(-1) h(-1) even in the absence of Cl- and Ca2+ ions. (2) NaCl-treatment removed not only PsbP and PsbQ but also a part of PsbO from Euglena PSII, indicating that PsbO binds to Euglena PSII more loosely than those of other organisms. Treatments by urea/NaCl, alkaline Tris or CaCl2 completely removed the three extrinsic proteins from Euglena PSII. (3) Each of the Euglena extrinsic proteins bound directly to PSII independent of the other extrinsic proteins, which is similar to the binding properties of the extrinsic proteins in a green alga, Chlamydomonas reinhardtii. (4) One of the significant features of Euglena PSII is that the oxygen evolution was not enhanced by Ca2+. When CaCl2-treated Euglena PSII was reconstituted with PsbO, the oxygen-evolving activity was stimulated by the addition of NaCl, but no further stimulation was observed by CaCl2. (5) Oxygen evolution of Euglena PSII reconstituted with PsbO from C. reinhardtii or spinach instead of that from Euglena also showed no enhancement by Ca2+, whereas a significant enhancement of oxygen evolution was observed by Ca2+ when the green algal or higher plant PSII was reconstituted with Euglena PsbO instead of their own PsbO. These results indicate that the PSII intrinsic proteins instead of the extrinsic PsbO protein, are responsible for the stimulation of oxygen evolution by Ca2+. Sequence comparison of major PSII intrinsic proteins revealed that PsbI of Euglena PSII is remarkably different from other organisms in that Euglena PsbI possesses extra 16-17 residues exposed to the luminal side. This may be related to the loss of enhancement of oxygen evolution by Ca2+ ion.     

The Arabidopsis PsbO2 protein regulates dephosphorylation and turnover of the photosystem II reaction centre D1 protein

The extrinsic photosystem II (PSII) protein of 33 kDa (PsbO), which stabilizes the water-oxidizing complex, is represented in Arabidopsis thaliana (Arabidopsis) by two isoforms. Two T-DNA insertion mutant lines deficient in either the PsbO1 or the PsbO2 protein were retarded in growth in comparison with the wild type, while differing from each other phenotypically. Both PsbO proteins were able to support the oxygen evolution activity of PSII, although PsbO2 was less efficient than PsbO1 under photoinhibitory conditions. Prolonged high light stress led to reduced growth and fitness of the mutant lacking PsbO2 as compared with the wild type and the mutant lacking PsbO1. During a short period of treatment of detached leaves or isolated thylakoids at high light levels, inactivation of PSII electron transport in the PsbO2-deficient mutant was slowed down, and the subsequent degradation of the D1 protein was totally inhibited. The steady-state levels of in vivo phosphorylation of the PSII reaction centre proteins D1 and D2 were specifically reduced in the mutant containing only PsbO2, in comparison with the mutant containing only PsbO1 or with wild-type plants. Phosphorylation of PSII proteins in vitro proceeded similarly in thylakoid membranes from both mutants and wild-type plants. However, dephosphorylation of the D1 protein occurred much faster in the thylakoids containing only PsbO2. We conclude that the function of PsbO1 in Arabidopsis is mostly in support of PSII activity, whereas the interaction of PsbO2 with PSII regulates the turnover of the D1 protein, increasing its accessibility to the phosphatases and proteases involved in its degradation.     

In vivo system for analyzing the function of the PsbP protein using <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

The PsbP protein is an extrinsic subunit of photosystem II (PSII) specifically developed in green-plant species including land plants and green algae. The protein–protein interactions involving PsbP and its effect on oxygen evolution have been investigated in vitro using isolated PSII membranes. However, the importance of those interactions needs to be examined at the cellular level. To this end, we developed a system expressing exogenous PsbP in the background of the Chlamydomonas BF25 mutant lacking native PsbP. Expression of His-tagged PsbP successfully restored the oxygen-evolving activity and photoautotrophic growth of the mutant, while PsbP-?15 lacking the N-terminal 15 residues, which are crucial for the oxygen-evolving activity of spinach PSII in vitro, only partially did. This demonstrated the importance of N-terminal sequence of PsbP for the photosynthetic activity in vivo. Furthermore, the PSII–LHCII supercomplex can be specifically purified from the Chlamydomonas cells having His-tagged PsbP using a metal affinity chromatography. This study provides a platform not only for the functional analysis of PsbP in vivo but also for structural analysis of the PSII–LHCII supercomplex from green algae.     

Three-dimensional structure of Chlamydomonas reinhardtii and Synechococcus elongatus photosystem II complexes allows for comparison of their oxygen-evolving complex organization

Electron microscopy and single-particle analyses have been carried out on negatively stained photosystem II (PSII) complexes isolated from the green alga Chlamydomonas reinhardtii and the thermophilic cyanobacterium Synechococcus elongatus. The analyses have yielded three-dimensional structures at 30-A resolution. Biochemical analysis of the C. reinhardtii particle suggested it to be very similar to the light-harvesting complex II (LHCII).PSII supercomplex of spinach, a conclusion borne out by its three-dimensional structure. Not only was the C. reinhardtii LHCII.PSII supercomplex dimeric and of comparable size and shape to that of spinach, but the structural features for the extrinsic OEC subunits bound to the lumenal surface were also similar thus allowing identification of the PsbO, PsbP, and PsbQ OEC proteins. The particle isolated from S. elongatus was also dimeric and retained its OEC proteins, PsbO, PsbU, and PsbV (cytochrome c(550)), which were again visualized as protrusions on the lumenal surface of the complex. The overall size and shape of the cyanobacterial particle was similar to that of a PSII dimeric core complex isolated from spinach for which higher resolution structural data are known from electron crystallography. By building the higher resolution structural model into the projection maps it has been possible to relate the positioning of the OEC proteins of C. reinhardtii and S. elongatus with the underlying transmembrane helices of other major intrinsic subunits of the core complex, D1, D2, CP47, and CP43 proteins. It is concluded that the PsbO protein is located over the CP47 and D2 side of the reaction center core complex, whereas the PsbP/PsbQ and PsbV/PsbU are positioned over the lumenal surface of the N-terminal region of the D1 protein. However, the mass attributed to PsbV/PsbU seems to bridge across to the PsbO, whereas the PsbP/PsbQ proteins protrude out more from the lumenal surface. Nevertheless, within the resolution and quality of the data, the relative positions of the center of masses for OEC proteins of C. reinhardtii and S. elongatus are similar and consistent with those determined previously for the OEC proteins of spinach.     

Effects of the lack of phosphatidylglycerol on the donor side of photosystem II

Our previous studies with the pgsA mutant of the cyanobacterium Synechocystis sp. PCC6803 (hereafter termed pgsA mutant), which is defective for the biosynthesis of phosphatidylglycerol (PG), revealed an important role for PG in the electron acceptor side of photosystem II (PSII), especially in the electron transport between plastoquinones Q(A) and Q(B). This study now shows that PG also plays an important role in the electron donor side of PSII, namely, the oxygen-evolving system. Analyses of purified PSII complexes indicated that PSII from PG-depleted pgsA mutant cells sustained only approximately 50% of the oxygen-evolving activity compared to wild-type cells. Dissociation of the extrinsic proteins PsbO, PsbV, and PsbU, which are required for stabilization of the manganese (Mn) cluster, followed by the release of a Mn atom, was observed in PSII of the PG-depleted mutant cells. The released PsbO rebound to PSII when PG was added back to the PG-depleted mutant cells, even when de novo protein synthesis was inhibited. Changes in photosynthetic activity of the PG-depleted pgsA mutant cells induced by heat treatment or dark incubation resembled those of DeltapsbO, DeltapsbV, and DeltapsbU mutant cells. These results suggest that PG plays an important role in binding extrinsic proteins required for sustaining a functional Mn cluster on the donor side of PSII.