C-terminal processing of reaction center protein D1 is essential for the function and assembly of photosystem II in Arabidopsis

Photosystem II (PSII) reaction center protein D1 is synthesized as a precursor (pD1) with a short C-terminal extension. The pD1 is processed to mature D1 by carboxyl-terminal peptidase A to remove the C-terminal extension and form active protein. Here we report functional characterization of the Arabidopsis gene encoding D1 C-terminal processing enzyme (AtCtpA) in the chloroplast thylakoid lumen. Recombinant AtCtpA converted pD1 to mature D1 and a mutant lacking AtCtpA retained all D1 in precursor form, confirming that AtCtpA is solely responsible for processing. As with cyanobacterial ctpa, a knockout Arabidopsis atctpa mutant was lethal under normal growth conditions but was viable with sucrose under low-light conditions. Viable plants, however, showed deficiencies in PSII and thylakoid stacking. Surprisingly, unlike its cyanobacterial counterpart, the Arabidopsis mutant retained both monomer and dimer forms of the PSII complexes that, although nonfunctional, contained both the core and extrinsic subunits. This mutant was also essentially devoid of PSII supercomplexes, providing an unexpected link between D1 maturation and supercomplex assembly. A knock-down mutant expressing about 2% wild-type level of AtCtpA showed normal growth under low light but was stunted and accumulated pD1 under high light, indicative of delayed C-terminal processing. Although demonstrating the functional significance of C-terminal D1 processing in PSII biogenesis, our study reveals an unsuspected link between D1 maturation and PSII supercomplex assembly in land plants, opening an avenue for exploring the mechanism for the association of light-harvesting complexes with the PSII core complexes.Photosystem II (PSII) consists of more than 20 subunits. Assembly of this photosystem is a multistep process that functions in a highly coordinated fashion (1–3). The process starts with PSII initiation complexes (D2, PsbE, PsbF, and PsbI), and then D1 and CP47 are sequentially recruited to form CP47-RC complexes, followed by addition of PsbH, PsbM, PsbTc, and PsbR subunits. Next, CP43 and other subunits are added to generate PSII monomers, which develop into PSII dimers. Finally, light-harvesting complex (LHC) II is attached to form PSII supercomplexes. The D1 protein of PSII is prone to photodamage under excessive light conditions (4). To sustain photosynthesis, damaged D1 protein is degraded and replaced with a newly synthesized copy via PSII repair—a highly complex and critical process whose mechanism remains unclear (3, 4).In most oxygen-evolving photosynthetic organisms, D1 protein is synthesized as a precursor (pD1) with a C-terminal tail. The pD1 protein is integrated into the thylakoid membrane and forms the initial PSII reaction center combined with other PSII subunits. The C-terminal tail of pD1 must be cleaved by an endopeptidase named the carboxyl terminal peptidase (Ctp) to produce mature D1, the functional form (5). In the cyanobacterium Synechocystis PCC 6803, there are three Ctp homologs (CtpA, CtpB, and CtpC), but only one, CtpA, is responsible for cleavage of the pD1 C-terminal extension (5). Disruption of CtpA leads to a loss of PSII activity and oxygen evolution from failure to form the manganese cluster (4, 6). The processing of pD1 is also critical for the association of extrinsic proteins on the luminal side to stabilize the PSII complexes (6, 7).In contrast to cyanobacteria, our knowledge of the significance of Ctp enzymes and D1 C-terminal processing is limited in land plants. Previous researchers reported the purification of CtpA-like protein from pea (8) and spinach (9). The spinach study further showed that the recombinant Ctp protein expressed from Escherichia coli displays activity against pD1 (9). However, because we lack a genetic approach, the functional significance of CtpA and C-terminal processing remains unknown in those and other land plants. In this study, we applied genetics to identify a gene (At4g17740) encoding a CtpA enzyme in Arabidopsis and showed that it is required for PSII function and chloroplast development. We found that Arabidopsis CtpA is essential for assembling functional PSII core complexes, dimers, and PSII supercomplexes. The enzyme is also critical for the PSII damage–repair cycle during the photoinhibition process.     

Photoassembly of the manganese cluster and oxygen evolution from monomeric and dimeric CP47 reaction center photosystem II complexes

Isolated subcomplexes of photosystem II from spinach (CP47RC), composed of D1, D2, cytochrome b(559), CP47, and a number of hydrophobic small subunits but devoid of CP43 and the extrinsic proteins of the oxygen-evolving complex, were shown to reconstitute the Mn(4)Ca(1)Cl(x) cluster of the water-splitting system and to evolve oxygen. The photoactivation process in CP47RC dimers proceeds by the same two-step mechanism as observed in PSII membranes and exhibits the same stoichiometry for Mn(2+), but with a 10-fold lower affinity for Ca(2+) and an increased susceptibility to photodamage. After the lower Ca(2+) affinity and the 10-fold smaller absorption cross-section for photons in CP47 dimers is taken into account, the intrinsic rate constant for the rate-limiting calcium-dependent dark step is indistinguishable for the two systems. The monomeric form of CP47RC also showed capacity to photoactivate and catalyze water oxidation, but with lower activity than the dimeric form and increased susceptibility to photodamage. After optimization of the various parameters affecting the photoactivation process in dimeric CP47RC subcores, 18% of the complexes were functionally reconstituted and the quantum efficiency for oxygen production by reactivated centers approached 96% of that observed for reconstituted photosystem II-enriched membranes.     

Potential for hydrogen production with inducible chloroplast gene expression in Chlamydomonas

An inducible chloroplast gene expression system was developed in Chlamydomonas reinhardtii by taking advantage of the properties of the copper-sensitive cytochrome c(6) promoter and of the nucleus-encoded Nac2 chloroplast protein. This protein is specifically required for the stable accumulation of the chloroplast psbD RNA and acts on its 5' UTR. A construct containing the Nac2 coding sequence fused to the cytochrome c(6) promoter was introduced into the nac2-26 mutant strain deficient in Nac2. In this transformant, psbD is expressed in copper-depleted but not in copper-replete medium. Because psbD encodes the D2 reaction center polypeptide of photosystem II (PSII), the repression of psbD leads to the loss of PSII. We have tested this system for hydrogen production. Upon addition of copper to cells pregrown in copper-deficient medium, PSII levels declined to a level at which oxygen consumption by respiration exceeded oxygen evolution by PSII. The resulting anaerobic conditions led to the induction of hydrogenase activity. Because the Cyc6 promoter is also induced under anaerobic conditions, this system opens possibilities for sustained cycling hydrogen production. Moreover, this inducible gene expression system is applicable to any chloroplast gene by replacing its 5' UTR with the psbD 5' UTR in the same genetic background. To make these strains phototrophic, the 5' UTR of the psbD gene was replaced by the petA 5' UTR. As an example, we show that the reporter gene aadA driven by the psbD 5' UTR confers resistance to spectinomycin in the absence of copper and sensitivity in its presence in the culture medium.     

PSB27: A thylakoid protein enabling Arabidopsis to adapt to changing light intensity

In earlier studies we have identified FKBP20-2 and CYP38 as soluble proteins of the chloroplast thylakoid lumen that are required for the formation of photosystem II supercomplexes (PSII SCs). Subsequent work has identified another potential candidate functional in SC formation (PSB27). We have followed up on this possibility and isolated mutants defective in the PSB27 gene. In addition to lack of PSII SCs, mutant plants were severely stunted when cultivated with light of variable intensity. The stunted growth was associated with lower PSII efficiency and defective starch accumulation. In response to high light exposure, the mutant plants also displayed enhanced ROS production, leading to decreased biosynthesis of anthocyanin. Unexpectedly, we detected a second defect in the mutant, namely in CP26, an antenna protein known to be required for the formation of PSII SCs that has been linked to state transitions. Lack of PSII SCs was found to be independent of PSB27, but was due to a mutation in the previously described cp26 gene that we found had no effect on light adaptation. The present results suggest that PSII SCs, despite being required for state transitions, are not associated with acclimation to changing light intensity. Our results are consistent with the conclusion that PSB27 plays an essential role in enabling plants to adapt to fluctuating light intensity through a mechanism distinct from photosystem II supercomplexes and state transitions.Photosynthetic light reactions entail the coordinated function of several large membrane complexes: photosystem I (PSI), photosystem II (PSII), cytochrome b6f complex, and CF₀-CF₁ complex. PSII catalyzes the initial step of photosynthesis, the light-dependent oxidation of water that yields molecular oxygen and reduced plastoquinone. The native form of PSII residing in the thylakoid membrane is believed to be organized into several types of supercomplexes (SCs), including the PSII core and the peripheral light harvesting complex II (LHCII), that play a primary role in the harvesting of light, transfer of excitation energy to the reaction center and regulation of light utilization. Several monomeric antenna proteins including CP24, CP26, and CP29 regulate the interaction of the PSII core with LHCII trimers (1–4). Regulation is achieved through photoprotective mechanisms that dissipate absorbed excess energy as heat in response to stress conditions such as high light intensity (5, 6).In natural settings, both the intensity and the spectral quality of light vary extensively, sometimes within very short periods. The changes in light intensity result in imbalanced excitation of PSI and PSII, and with that may lower the efficiency of the photosynthetic light reactions. In acclimating to the changing conditions, plants modify their thylakoid proteins and reorganize their photosynthetic machinery (7, 8). In a rapid response, designated state transitions (9), LHCII associates reversibly with PSII or PSI. Under high light intensity, excessive activation of PSII increases the reduced plastoquinone pool and thereby activates protein kinase STN7 which, in turn, phosphorylates LHCII and prompts the migration of LHCII from PSII (state 1) to PSI (state 2) (10, 11). Oxidation of the plastoquinone pool by the higher activity of PSI then activates protein phosphatase PPH1 that dephosphorylates LHCII and promotes a return to the original association of LHCII with PSII (state 1) (12, 13). In this context, the formation of LHCII-PSII SCs is expected to be a prerequisite for state transitions and, therefore, essential for adaptation to changing light intensity. However, it remains to be rigorously tested if state transitions play a crucial role in plant adaptation to changing light intensity.In the present study, we have identified an Arabidopsis mutant that lacked PSII supercomplexes and grew poorly under changing light intensity. The mutant harbored a T-DNA insertion in the gene encoding the thylakoid lumen protein, PSB27, implicating its function in both the assembly of PSII SCs and adaptation to changing light. A detailed comparative genetic and biochemical analysis confirmed the requirement for PSB27 in enabling plants to adapt to changing light, but, surprisingly, revealed that this adaptation is independent of PSII SC assembly. A second, previously unrecognized defect was localized in CP26, a protein unrelated to PSB27, that is linked to PSII SC assembly. These results prompt the conclusion that PSB27 plays a fundamental role in enabling plants to adapt to changes in light intensity independently of the formation of PSII SCs.     

Identification of the special pair of photosystem II in a chlorophyll d-dominated cyanobacterium

The composition of photosystem II (PSII) in the chlorophyll (Chl) d-dominated cyanobacterium Acaryochloris marina MBIC 11017 was investigated to enhance the general understanding of the energetics of the PSII reaction center. We first purified photochemically active complexes consisting of a 47-kDa Chl protein (CP47), CP43' (PcbC), D1, D2, cytochrome b(559), PsbI, and a small polypeptide. The pigment composition per two pheophytin (Phe) a molecules was 55 +/- 7 Chl d, 3.0 +/- 0.4 Chl a, 17 +/- 3 alpha-carotene, and 1.4 +/- 0.2 plastoquinone-9. The special pair was detected by a reversible absorption change at 713 nm (P713) together with a cation radical band at 842 nm. FTIR difference spectra of the specific bands of a 3-formyl group allowed assignment of the special pair. The combined results indicate that the special pair comprises a Chl d homodimer. The primary electron acceptor was shown by photoaccumulation to be Phe a, and its potential was shifted to a higher value than that in the Chl a/Phe a system. The overall energetics of PSII in the Chl d system are adjusted to changes in the redox potentials, with P713 as the special pair using a lower light energy at 713 nm. Taking into account the reported downward shift in the potential of the special pair of photosystem I (P740) in A. marina, our findings lend support to the idea that changes in photosynthetic pigments combine with a modification of the redox potentials of electron transfer components to give rise to an energetic adjustment of the total reaction system.     

Molecular characterization of PsbW, a nuclear-encoded component of the photosystem II reaction center complex in spinach.

We describe the isolation and characterization of cDNAs encoding the precursor polypeptide of the 6.1-kDa polypeptide associated with the reaction center core of the photosystem II complex from spinach. PsbW, the gene encoding this polypeptide, is present in a single copy per haploid genome. The mature polypeptide with 54 amino acid residues is characterized by a hydrophobic transmembrane segment, and, although an intrinsic membrane protein, it carries a bipartite transit peptide of 83 amino acid residues which directs the N terminus of the mature protein into the chloroplast lumen. Thylakoid integration of this polypeptide does not require a delta pH across the membrane, nor is it azide-sensitive, suggesting that the polypeptide chain inserts spontaneously in an as yet unknown way. The PsbW mRNA levels are light regulated. Similar to cytochrome b559 and PsbS, but different from the chlorophyll-complexing polypeptides D1, D2, CP43, and CP47 of photosystem II, PsbW is present in etiolated spinach seedlings.     

Identification of the mobile light-harvesting complex II polypeptides for state transitions in Chlamydomonas reinhardtii

State transition in photosynthesis is a short-term balancing mechanism of energy distribution between photosystem I (PSI) and photosystem II (PSII). When PSII is preferentially excited (state 2), a pool of mobile light-harvesting complex II (LHCII) antenna proteins is thought to migrate from PSII to PSI, but biochemical evidence for a physical association between LHCII proteins and PSI in state 2 is weak. Here, using the green alga Chlamydomonas reinhardtii, which has a high capacity for state transitions, we report the isolation of PSI-light-harvesting complex I (LHCI) super-complexes from cells locked into state 1 and state 2. We solubilized the thylakoid membranes with a mild detergent, separated the proteins by sucrose density gradient centrifugation, and subjected gradient fractions to gel-filtration chromatography. Three LHCII polypeptides were associated with a PSI-LHCI supercomplex only in state 2; we identified them as two minor monomeric LHCII proteins (CP26 and CP29) and one previously unreported major LHCII protein type II, or LhcbM5. These three LHCII proteins, in addition to the major trimeric LHCII proteins, were phosphorylated upon transition to state 2. The corresponding phylogenetic tree indicates that among the LHCII proteins associated with PSII, these three LHCII proteins are the most similar to the LHC proteins for PSI (LHCI). Our results are important because CP26, CP29, and LhcbM5, which have been viewed as belonging solely to the PSII complex, are now postulated to shuttle between PSI and PSII during state transitions, thereby acting as docking sites for the trimeric LHCII proteins in both PSI and PSII.     

A chloroplast cyclophilin functions in the assembly and maintenance of photosystem II in Arabidopsis thaliana

Photosynthetic light reactions rely on the proper function of large protein complexes (including photosystems I and II) that reside in the thylakoid membrane. Although their composition, structure, and function are known, the repertoire of assembly and maintenance factors is still being determined. Here we show that an immunophilin of the cyclophilin type, CYP38, plays a critical role in the assembly and maintenance of photosystem II (PSII) supercomplexes (SCs) in Arabidopsis. Mutant plants with the CYP38 gene interrupted by T-DNA insertion showed stunted growth and were hypersensitive to high light. Leaf chlorophyll fluorescence analysis and thylakoid membrane composition indicated that cyp38 mutant plants had defects in PSII SCs. Sucrose supplementation enabled the rescue of the mutant phenotype under low-light conditions, but failed to mitigate hypersensitivity to high-light stress. Protein radiolabeling assays showed that, although individual thylakoid proteins were synthesized equally in mutant and wild type, the assembly of the PSII SC was impaired in the mutant. In addition, the D1 and D2 components of the mutant PSII had a short half-life under high-light stress. The results provide evidence that CYP38 is necessary for the assembly and stabilization of PSII.     

Photosystem II reaction center damage and repair cycle: chloroplast acclimation strategy to irradiance stress

A daily occurrence in the life of a plant is the function of a photosystem II (PSII) damage and repair cycle in chloroplasts. This unique phenomenon involves the frequent turnover of D1, the 32-kDa reaction-center protein of PSII (chloroplast psbA gene product). In the model organism Dunaliella salina (a green alga), growth under low light (100 mol of photons per m2 per sec) entails damage, degradation, and replacement of D1 every 7 hr. Growth under irradiance stress (2200 micromol of photons per m2 per sec) entails damage to D1 every 20 min. The rate of de novo D1 biosynthesis under conditions of both low light and irradiance stress was found to be fairly constant on a per chloroplast or cell basis. The response of D. salina to the enhanced rate of damage entails an accumulation of photodamaged centers (80% of all PSII) and the formation of thylakoid membranes containing a smaller quantity of photosystem I (PSI) centers (about 10% of that in cells grown under low light). These changes contribute to a shift in the PSII/PSI ratio from 1.4:1 under low-light conditions to 15:1 under irradiance stress. The accumulation of photodamaged PSII under irradiance stress reflects a chloroplast inability to match the rate of D1 degradation or turnover with the rate of damage for individual PSII complexes. The altered thylakoid membrane organization ensures that a small fraction of PSII centers remains functional under irradiance stress and sustains electron flow from H2O to ferredoxin with rates sufficient for chloroplast photosynthesis and cell growth.     

Genetically engineered mutant of the cyanobacterium Synechocystis 6803 lacks the photosystem II chlorophyll-binding protein CP-47

CP-47 is absent in a genetically engineered mutant of cyanobacterium Synechocystis 6803, in which the psbB gene [encoding the chlorophyll-binding photosystem II (PSII) protein CP-47] was interrupted. Another chlorophyll-binding PSII protein, CP-43, is present in the mutant, and functionally inactive PSII-enriched particles can be isolated from mutant thylakoids. We interpret these data as indicating that the PSII core complex of the mutant still assembles in the absence of CP-47. The mutant lacks a 77 K fluorescence emission maximum at 695 nm, suggesting that the PSII reaction center is not functional. The absence of primary photochemistry was indicated by EPR and optical measurements: no chlorophyll triplet originating from charge recombination between P680⁺ and Pheo^- was observed in the mutant, and there were no flash-induced absorption changes at 820 nm attributable to chlorophyll P680 oxidation. These observations lead us to conclude that CP-47 plays an essential role in the activity of the PSII reaction center.     

Functional asymmetry of photosystem II D1 and D2 peripheral chlorophyll mutants of Chlamydomonas reinhardtii

The peripheral accessory chlorophylls (Chls) of the photosystem II (PSII) reaction center (RC) are coordinated by a pair of symmetry-related histidine residues (D1-H118 and D2-H117). These Chls participate in energy transfer from the proximal antennae complexes (CP43 and CP47) to the RC core chromophores. In addition, one or both of the peripheral Chls are redox-active and participate in a low-quantum-yield electron transfer cycle around PSII. We demonstrate that conservative mutations of the D2-H117 residue result in decreased Chl fluorescence quenching efficiency attributed to reduced accumulation of the peripheral accessory Chl cation, Chl(Z)(+). In contrast, identical symmetry-related mutations at residue D1-H118 had no effect on Chl fluorescence yield or quenching kinetics. Mutagenesis of the D2-H117 residue also altered the line width of the Chl(Z)(+) EPR signal, but the line shape of the D1-H118Q mutant remained unchanged. The D1-H118 and D2-H117 mutations also altered energy transfer properties in PSII RCs. Unlike wild type or the D1-H118Q mutant, D2-H117N RCs exhibited a reduced CD doublet in the red region of Chl absorbance band, indicative of reduced energetic coupling between P680 and the peripheral accessory Chl. In addition, transient absorption measurements of D2-H117N RCs, excited on the blue side of the Chl absorbance band, exhibited a ( approximately 400 fs) pheophytin Q(X) band bleach lifetime component not seen in wild-type or D1-H118Q RCs. The origin of this component may be related to delayed fast-energy equilibration of the excited state between the core pigments of this mutant.     

Crystal structure of oxygen-evolving photosystem II from Thermosynechococcus vulcanus at 3.7-A resolution

Photosystem II (PSII) is a multisubunit membrane protein complex performing light-induced electron transfer and water-splitting reactions, leading to the formation of molecular oxygen. The first crystal structure of PSII from a thermophilic cyanobacterium Thermosynechococcus elongatus was reported recently [Zouni, A., Witt, H. T., Kern, J., Fromme, P., Krauss, N., Saenger, W. & Orth, P. (2001) Nature 409, 739-743)] at 3.8-A resolution. To analyze the PSII structure in more detail, we have obtained the crystal structure of PSII from another thermophilic cyanobacterium, Thermosynechococcus vulcanus, at 3.7-A resolution. The present structure was built on the basis of the sequences of PSII large subunits D1, D2, CP47, and CP43; extrinsic 33- and 12-kDa proteins and cytochrome c550; and several low molecular mass subunits, among which the structure of the 12-kDa protein was not reported previously. This yielded much information concerning the molecular interactions within this large protein complex. We also show the arrangement of chlorophylls and cofactors, including two beta-carotenes recently identified in a region close to the reaction center, which provided important clues to the secondary electron transfer pathways around the reaction center. Furthermore, possible ligands for the Mn-cluster were determined. In particular, the C terminus of D1 polypeptide was shown to be connected to the Mn cluster directly. The structural information obtained here provides important insights into the mechanism of PSII reactions.     

Mechanism of photosystem II photoinactivation and D1 protein degradation at low light: the role of back electron flow

Light intensities that limit electron flow induce rapid degradation of the photosystem II (PSII) reaction center D1 protein. The mechanism of this phenomenon is not known. We propose that at low excitation rates back electron flow and charge recombination between the QB*- or QA*- semiquinone acceptors and the oxidized S(2,3) states of the PSII donor side may cause oxidative damage via generation of active oxygen species. Therefore, damage per photochemical event should increase with decreasing rates of PSII excitation. To test this hypothesis, the effect of the dark interval between single turnover flashes on the inactivation of water oxidation, charge separation and recombination, and the degradation of D1 protein were determined in spinach thylakoids. PSII inactivation per flash increases as the dark interval between the flashes increases, and a plateau is reached at dark intervals, allowing complete charge recombination of the QB*-/S2,3 or QA*-/S2 states (about 200 and 40 s, respectively). At these excitation rates: (i) 0.7% and 0.4% of PSII is inactivated and 0.4% and 0.2% of the D1 protein is degraded per flash, respectively, and (ii) the damage per flash is about 2 orders of magnitude higher than that induced by equal amount of energy delivered by excess continuous light. No PSII damage occurs if flashes are given in anaerobic conditions. These results demonstrate that charge recombination in active PSII is promoted by low rates of excitation and may account for a the high quantum efficiency of the rapid turnover of the D1 protein induced by limiting light.     

Psb27, a transiently associated protein, binds to the chlorophyll binding protein CP43 in photosystem II assembly intermediates

Photosystem II (PSII), a large multisubunit pigment–protein complex localized in the thylakoid membrane of cyanobacteria and chloroplasts, mediates light-driven evolution of oxygen from water. Recently, a high-resolution X-ray structure of the mature PSII complex has become available. Two PSII polypeptides, D1 and CP43, provide many of the ligands to an inorganic Mn₄Ca center that is essential for water oxidation. Because of its unusual redox chemistry, PSII often undergoes degradation followed by stepwise assembly. Psb27, a small luminal polypeptide, functions as an important accessory factor in this elaborate assembly pathway. However, the structural location of Psb27 within PSII assembly intermediates has remained elusive. Here we report that Psb27 binds to CP43 in such assembly intermediates. We treated purified genetically tagged PSII assembly intermediate complexes from the cyanobacterium Synechocystis 6803 with chemical cross-linkers to examine intermolecular interactions between Psb27 and various PSII proteins. First, the water-soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was used to cross-link proteins with complementary charged groups in close association to one another. In the His27△ctpAPSII preparation, a 58-kDa cross-linked species containing Psb27 and CP43 was identified. This species was not formed in the HT3△ctpA△psb27PSII complex in which Psb27 was absent. Second, the homobifunctional thiol-cleavable cross-linker 3,3′-dithiobis(sulfosuccinimidylpropionate) (DTSSP) was used to reversibly cross-link Psb27 to CP43 in His27△ctpAPSII preparations, which allowed the use of liquid chromatography/tandem MS to map the cross-linking sites as Psb27K⁶³↔CP43D³²¹ (trypsin) and CP43K²¹⁵↔Psb27D⁵⁸AGGLK⁶³↔CP43D³²¹ (chymotrypsin), respectively. Our data suggest that Psb27 acts as an important regulatory protein during PSII assembly through specific interactions with the luminal domain of CP43.     

Site-directed mutagenesis in photosystem II of the cyanobacterium Synechocystis sp. PCC 6803: Donor D is a tyrosine residue in the D2 protein

The chemical nature of electron donor(s) in photosystem II in photosynthetic membranes was analyzed by site-directed mutagenesis of the gene encoding the protein D2 of the photosystem II reaction center. Mutation of the Tyr-160 residue of the D2 protein into phenylalanine results in the disappearance of the electron paramagnetic resonance signal II_S originating from D⁺, the oxidized form of the slow photosystem II electron donor D. Signal II_S is still present if a neighboring residue in D2, Met-159, is mutated into arginine. Both mutants have normal rereduction kinetics of the oxidized primary electron donor, P680⁺, in octyl glucoside-extracted thylakoids, indicating that D is not directly involved in P680⁺ reduction. However, overall photosystem II activity appears to be impaired in the Tyr-160-Phe mutant: photosystem II-dependent growth of this mutant is slowed down by a factor of 3-4, whereas photoheterotrophic growth rates in wild type and mutant are essentially identical. Binding studies of diuron, a photosystem II herbicide, show that there is no appreciable decrease in the number of photosystem II centers in the Tyr-160-Phe mutant. The decrease in photosystem II activity in this mutant may be interpreted to indicate a role of D in photoactivation, rather than one as an important redox intermediate in the photosynthetic electron-transport chain.     

Site-directed mutagenesis identifies a tyrosine radical involved in the photosynthetic oxygen-evolving system.

Photosynthetic oxygen evolution takes place in the thylakoid protein complex known as photosystem II. The reaction center core of this photosystem, where photochemistry occurs, is a heterodimer of homologous polypeptides called D1 and D2. Besides chlorophyll and quinone, photosystem II contains other organic cofactors, including two known as Z and D. Z transfers electrons from the site of water oxidation to the oxidized reaction center primary donor, P+.680, while D+. gives rise to the dark-stable EPR spectrum known as signal II. D+. has recently been shown to be a tyrosine radical. Z is probably a second tyrosine located in a similar environment. Indirect evidence indicates that Z and D are associated with the D1 and D2 polypeptides, respectively. To identify the specific tyrosine residue corresponding to D, we have changed Tyr-160 of the D2 polypeptide to phenylalanine by site-directed mutagenesis of a psbD gene in the cyanobacterium Synechocystis 6803. The resulting mutant grows photosynthetically, but it lacks the EPR signal of D+.. We conclude that D is Tyr-160 of the D2 polypeptide. We suggest that the C2 symmetry in photosystem II extends beyond P680 to its immediate electron donor and conclude that Z is Tyr-161 of the D1 polypeptide.     

Structure of a photosystem II supercomplex isolated from Prochloron didemni retaining its chlorophyll a/b light-harvesting system

Prochlorophytes are a class of cyanobacteria that do not use phycobiliproteins as light-harvesting systems, but contain chlorophyll (Chl) a/b-binding Pcb proteins. Recently it was shown that Pcb proteins form an 18-subunit light-harvesting antenna ring around the photosystem I (PSI) trimeric reaction center complex of the prochlorophyte Prochlorococcus marinus SS120. Here we have investigated whether the symbiotic prochlorophyte Prochloron didemni also contains the same supermolecular complex. Using cells isolated directly from its ascidian host, we found no evidence for the presence of the Pcb-PSI supercomplex. Instead we have identified and characterized a supercomplex composed of photosystem II (PSII) and Pcb proteins. We show that 10-Pcb subunits associate with the PSII dimeric reaction center core to form a giant complex having an estimated Mr of 1,500 kDa with dimensions of 210 x 290 A. Five-Pcb subunits flank each long side of the dimer and assuming each binds 13 Chl molecules, increase the antenna size of PSII by approximately 200%. Fluorescence emission studies indicate that energy transfer occurs efficiently from the Pcb antenna. Modeling using the x-ray structure of cyanobacterial PSII suggests that energy transfer to the PSII reaction center is via the Chls bound to the CP47 and CP43 proteins.     

Engineering of an alternative electron transfer path in photosystem II

The initial steps of oxygenic photosynthetic electron transfer occur within photosystem II, an intricate pigment/protein transmembrane complex. Light-driven electron transfer occurs within a multistep pathway that is efficiently insulated from competing electron transfer pathways. The heart of the electron transfer system, composed of six linearly coupled redox active cofactors that enable electron transfer from water to the secondary quinone acceptor Q_B, is mainly embedded within two proteins called D1 and D2. We have identified a site in silico, poised in the vicinity of the Q_A intermediate quinone acceptor, which could serve as a potential binding site for redox active proteins. Here we show that modification of Lysine 238 of the D1 protein to glutamic acid (Glu) in the cyanobacterium Synechocystis sp. PCC 6803, results in a strain that grows photautotrophically. The Glu thylakoid membranes are able to perform light-dependent reduction of exogenous cytochrome c with water as the electron donor. Cytochrome c photoreduction by the Glu mutant was also shown to significantly protect the D1 protein from photodamage when isolated thylakoid membranes were illuminated. We have therefore engineered a novel electron transfer pathway from water to a soluble protein electron carrier without harming the normal function of photosystem II.     

Both N-terminal and C-terminal regions of steroid sulfatase are important for enzyme activity

Steroid sulfatase (STS) is localized in the endoplasmic reticulum and catalyzes desulfation of 3beta-hydroxysteroid sulfates. X-linked ichthyosis (XLI) is an inherited skin disorder caused by deficiency of STS enzyme activity. We previously reported a case in which XLI with a one-base change in the STS gene and variation in amino acid Q560P developed. In this study, we performed molecular analysis to determine the importance of terminal regions of STS and the effect of mutant STS on STS enzyme activity. To examine the effect of terminal truncated STS on the enzyme activity, N- and C-terminal truncated STS expression vectors were transfected into COS-1 cells. The activity of truncated STS lacking the N-terminal regions declined, and the activity of C-terminal-truncated STS declined with extension of the truncated C-terminal region. Although the results of pulse-chase experiments showed that a one-base mutant STS (Q560P) and C-terminal-truncated STS (deltaC2 (1-559)) had no effects on protein synthesis and degradation, the mutant STS and C-terminal-truncated STS have dominant negative effect on STS enzyme activity when the STS mutant or truncated STS protein and a wild-type STS protein coexist in cells. Results of coprecipitation of the truncated STS with an STS-FLAG fusion protein showed that STS formed a dimer conformation in cells. In this study, we have shown that both the N-terminal region and C-terminal region are important for STS enzyme activity. The C-terminal mutant has a dominant negative effect on wild-type STS.