A role for PsbZ in the core complex of photosystem II.
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Photosynthesis↗
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Shedding light on clock controlled cab gene transcription in higher plants.
Research from several laboratories has revealed that the circadian clock regulates the transcription of many higher plant genes. Amongst the most well characterized of these genes are those that encode the chlorophyll a/b binding (Cab) proteins of the light harvesting complexes of photosystem II. Analysis of cab gene expression in both etiolated and green plants reveals a complex interaction between phytochrome(s) and the circadian clock, in which phytochrome appears to regulate the level of cab transcription and the circadian clock regulates the phase. The development of a non-invasive assay for cab transcription based on firefly luciferase expression in transgenic plants has not only made it possible to analyze clock regulated transcription in living plants, but also provides a means to screen for clock mutants in Arabidopsis.
Arabidopsis↗
Mutagenesis of basic residues R151 and R161 in manganese-stabilizing protein of photosystem II causes inefficient binding of chloride to the oxygen-evolving complex.
Manganese-stabilizing protein of photosystem II, an intrinsically disordered polypeptide, contains a high ratio of charged to hydrophobic amino acid residues. Arg151 and Arg161 are conserved in all known MSP sequences. To examine the role of these basic residues in MSP structure and function, three mutants of spinach MSP, R151G, R151D, and R161G, were produced. Here, we present evidence that replacement of Arg151 or Arg161 yields proteins that have lower PSII binding affinity, and are functionally deficient even though about 2 mol of mutant MSP/mol PSII can be rebound to MSP depleted PSII membranes. R161G reconstitutes O(2) evolution activity to 40% of the control, while R151G and R151D reconstitute only 20% of the control activity. Spectroscopic and biochemical techniques fail to detect significant changes in solution structure. More extensive O(2) evolution assays revealed that the Mn cluster is stable in samples reconstituted with each mutated MSP, and that all three Arg mutants have the same ability to retain Ca(2+) as the wild-type protein. Activity assays exploring the effect of these mutations on retention of Cl(-), however, showed that the R151G, R151D, and R161G MSPs are defective in Cl(-) binding to the OEC. The mutants have Cl(-) K(M) values that are about four (R161G) or six times (R151G and R151D) higher than the value for the wild-type protein. The results reported here suggest that conserved positive charges on the manganese-stabilizing protein play a role in proper functional assembly of the protein into PSII, and, consequently, in retention of Cl(-) by the O(2)-evolving complex.
Algal Proteins↗
Photosystem II: the engine of life.
Photosystem II (PS II) is a multisubunit membrane protein complex, which uses light energy to oxidize water and reduce plastoquinone. High-resolution electron cryomicroscopy and X-ray crystallography are revealing the structure of this important molecular machine. Both approaches have contributed to our understanding of the organization of the transmembrane helices of higher plant and cyanobacterial PS II and both indicate that PS II normally functions as a dimer. However the high-resolution electron density maps derived from X-ray crystallography currently at 3.7/3.8 A, have allowed assignments to be made to the redox active cofactors involved in the light-driven water-plastoquinone oxidoreductase activity and to the chlorophyll molecules that absorb and transfer energy to the reaction centre. In particular the X-ray work has identified density that can accommodate the four manganese atoms which catalyse the water-oxidation process. The Mn cluster is located at the lumenal surface of the DI protein and approximately 7 A from the redox active tyrosine residue (YZ) which acts an electron/proton transfer link to the primary oxidant P680.+. The lower resolution electron microscopy studies, however, are providing structural models of larger PS II supercomplexes that are ideal frameworks in which to incorporate the X-ray derived structures.
Chlorophyll↗
Localization of the small CAB-like proteins in photosystem II.
The cyanobacterial small CAB-like proteins (SCPs) consist of one-helix proteins that resemble transmembrane regions of the light-harvesting proteins of plants. To determine whether these proteins are associated with protein complexes in the thylakoid membrane, an abundant member of the SCP family, ScpD, was marked with a His tag, and proteins co-isolating with His-tagged ScpD were identified. These proteins included the major Photosystem (PS) II components as well as FtsH, which is involved in degradation of the PSII complex. To ascertain specific interaction between ScpD and the PSII complex, the His-tagged protein fraction was subjected to two-dimensional blue native/SDS-PAGE. Again, PSII components were co-isolated with ScpD-His, and ScpD-His was found to interact most strongly with CP47. ScpD association was most prominent with the monomeric form of PSII, suggesting ScpD association with PSII that is repaired. Using antibodies that recognize both ScpC and ScpD, we found the ScpC protein, which is very similar in primary structure to ScpD, to also co-isolate with the PSII complex. In contrast, ScpE did not co-isolate with a major protein complex in thylakoids. A fourth member of the SCP family, ScpB, could not be immunodetected, but was found by mass spectrometry in samples co-isolating with ScpD-His. Therefore, ScpB may be associated with ScpD as well. No association between SCPs and PSI could be demonstrated. On the basis of these and other data presented, we suggest that members of the SCP family can associate with damaged PSII and can serve as a temporary pigment reservoir while PSII components are being replaced.
Chlorophyll↗
A new photosystem II reaction center component (4.8 kDa protein) encoded by chloroplast genome.
The photosystem II reaction center complex, so-called D1-D2-cytochrome b-559 complex, isolated from higher plants contains a new component of about 4.8 kDa [(1988) Plant Cell Physiol. 29, 1233-1239]. The partial amino acid sequence of this component from spinach was determined after release of N-terminal blockage. The determined sequence matched an open reading frame (ORF36) of the chloroplast genome from tobacco and liverwort, which is located downstream from the psbK gene and forms an operon with psbK. The predicted product consists of 36 amino acid residues and has a single membrane-spanning segment. High homology between the tobacco and liverwort genes, and its presence in the reaction center complex suggest an important role for this component in the photosystem II complex. Since this gene corresponds to a part of the formerly designated psbI gene, we propose to revise the definition of psbI as the gene encoding the 4.8 kDa reaction center component.
Amino Acid Sequence↗
The cab-m7 gene: a light-inducible, mesophyll-specific gene of maize.
Southern blot analysis has revealed the existence in maize of perhaps 12 members of the nuclear cab multigene family encoding the chlorophyll a- and b-binding proteins of the photosystem II light-harvesting complex. Hybridization with 3' probes derived from unsequenced cDNA clones showed that six members of this family differ from one another with respect to expression in mesophyll and/or bundle sheath cells and regulation by light. An additional member of this family, designated cab-m7, that encodes a 28 kDa primary translation product has now been identified. It has been cloned from a maize genomic library and sequenced to begin to define the bases for differences in the expression of these genes. This cab gene is shown to be strongly preferentially expressed in the mesophyll (vs. bundle sheath) cells of maize. Furthermore, the gene is photo-responsive; although small amounts of cab-m7 mRNA are present in etiolated leaves, the mRNA pool is 8-fold larger after six hours of illumination. DNA sequences upstream of the cab-m7 gene resemble those found in the 5'-flanking regions of some other plant genes.
Amino Acid Sequence↗
Tubular crystals of a photosystem II core complex.
An oxygen evolving photosystem II core complex containing all three extrinsic proteins (33, 23, 17 kDa) was isolated from spinach and reconstituted into tubular two-dimensional crystals of 72.9 nm diameter and 1-2 micrometers length. While the 17 and 23 kDa polypeptides were lost during crystallization, the extrinsic 33 kDa protein was retained. The optical spectrum of the crystallized core was characteristic of an intact PSII core complex. Immunoelectron microscopy revealed that the lumenal surface of the PSII complex was exposed at the outside of the cylindrical tubes. The projection of the complex was determined from flattened tubular crystals by negative stain electron microscopy and image analysis to 2.0 nm resolution. Rhombic unit cells (a = 16.2 nm, b = 13.7 nm; gamma = 142.4 degrees) contained one PSII complex.
Crystallization↗
Stepwise transition of the tetra-manganese complex of photosystem II to a binuclear Mn2(micro -O)2 complex in response to a temperature jump: a time-resolved structural investigation employing x-ray absorption spectroscopy.
In oxygenic photosynthesis, water is oxidized at a protein-cofactor complex comprising four Mn atoms and, presumably, one calcium. Using multilayers of Photosystem II membrane particles, we investigated the time course of the disassembly of the Mn complex initiated by a temperature jump from 25 degrees C to 47 degrees C and terminated by rapid cooling after distinct heating periods. We monitored polarographically the oxygen-evolution activity, the amount of the Y(D)(ox) radical and of released Mn(2+) by EPR spectroscopy, and the structure of the Mn complex by x-ray absorption spectroscopy (XAS, EXAFS). Using a novel approach to analyze time-resolved EXAFS data, we identify three distinct phases of the disassembly process: (1) Loss of the oxygen-evolution activity and reduction of Y(D)(ox) occur simultaneously (k(1) = 1.0 min(-1)). EXAFS spectra reveal the concomitant loss of an absorber-backscatterer interaction between heavy atoms separated by approximately 3.3 A, possibly related to Ca release. (2) Subsequently, two Mn(III) or Mn(IV) ions seemingly separated by approximately 2.7 A in the native complex are reduced to Mn(II) and released (k(2) = 0.18 min(-1)). The x-ray absorption spectroscopy data is highly suggestive that the two unreleased Mn ions form a di- micro -oxo bridged Mn(III)(2) complex. (3) Finally, the tightly-bound Mn(2)( micro -O)(2) unit is slowly reduced and released (k(3) = 0.014 min(-1)).
Absorptiometry, Photon↗
Isolation and biochemical characterisation of monomeric and dimeric photosystem II complexes from spinach and their relevance to the organisation of photosystem II in vivo.
Membranes enriched in photosystem II were isolated from spinach and further solubilised using n-octyl beta-D-glucopyranoside (OctGlc) and n-dodecyl beta-D-maltoside (DodGlc2). The OctGlc preparation had high rates of oxygen evolution and when subjected to size-exclusion HPLC and sucrose density gradient centrifugation, in the presence of DodGlc2, separated into dimeric (430 kDa), monomeric (236 kDa) photosystem II cores and a fraction containing photosystem II light-harvesting complex (Lhcb) proteins. The dimeric core fraction was more stable, contained higher levels of chlorophyll, beta-carotene and plastoquinone per photosystem II reaction centre and had a higher oxygen-evolving activity than the monomeric cores. Their subunit composition was similar (CP43, CP47, D1, D2, cytochrome b 559 and several lower-molecular-mass components) except that the level of 33-kDa extrinsic protein was lower in the monomeric fraction. Direct solubilisation of photosystem-II-enriched membranes with DodGlc2, followed by sucrose density gradient centrifugation, yielded a super complex (700 kDa) containing the dimeric form of the photosystem II core and Lhcb proteins: Lhcb1, Lhcb2, Lhcb4 (CP29), and Lhcb5 (CP26). Like the dimeric and monomeric photosystem II core complexes, the photosystem II-LHCII complex had lost the 23-kDa and 17-kDa extrinsic proteins, but maintained the 33-kDa protein and the ability to evolve oxygen. It is suggested, with a proposed model, that the isolated photosystem II-LHCII super complex represents an in vivo organisation that can sometimes form a lattice in granal membranes of the type detected by freeze-etch electron microscopy [Seibert, M., DeWit, M. & Staehelin, L. A. (1987) J. Cell Biol. 105, 2257-2265].
Chloroplasts↗
A small chloroplast-encoded protein as a novel architectural component of the light-harvesting antenna.
A small conserved open reading frame in the plastid genome, ycf9, encodes a putative membrane protein of 62 amino acids. To determine the function of this reading frame we have constructed a knockout allele for targeted disruption of ycf9. This allele was introduced into the tobacco plastid genome by biolistic transformation to replace the wild-type ycf9 allele. Homoplasmic ycf9 knockout plants displayed no phenotype under normal growth conditions. However, under low light conditions, their growth rate was significantly reduced as compared with the wild-type, due to a lowered efficiency of the light reaction of photosynthesis. We show that this phenotype is caused by the deficiency in a pigment-protein complex of the light-harvesting antenna of photosystem II and hence by a reduced efficiency of photon capture when light availability is limiting. Our results indicate that, in contrast to the current view, light-harvesting complexes do not only consist of the classical pigment-binding proteins, but may contain small structural subunits in addition. These subunits appear to be crucial architectural factors for the assembly and/or maintenance of stable light-harvesting complexes.
Amino Acid Sequence↗
Physiologically active chloroplasts contain pools of unassembled extrinsic proteins of the photosynthetic oxygen-evolving enzyme complex in the thylakoid lumen.
The oxygen-evolving complex (OEC) of photosystem II (PS II) consists of at least three extrinsic membrane-associated protein subunits, OE33, OE23, and OE17, with associated Mn2+, Ca2+, and Cl- ions. These subunits are bound to the lumen side of PS II core proteins embedded in the thylakoid membrane. Our experiments reveal that a significant fraction of each subunit is normally present in unassembled pools within the thylakoid lumen. This conclusion was supported by immunological detection of free subunits after freshly isolated pea thylakoids were fractionated with low levels of Triton X-100. Plastocyanin, a soluble lumen protein, was completely released from the lumen by 0.04% Triton X-100. This gentle detergent treatment also caused the release from the thylakoids of between 10 and 20%, 40 and 60%, and 15 and 50% of OE33, OE23, and OE17, respectively. Measurements of the rates of oxygen evolution from Triton-treated thylakoids, both in the presence and absence of Ca2+, and before and after incubation with hydroquinone, demonstrated that the OEC was not dissociated by the detergent treatment. Thylakoids isolated from spinach released similar amounts of extrinsic proteins after Triton treatment. These data demonstrate that physiologically active chloroplasts contain significant pools of unassembled extrinsic OEC polypeptide subunits free in the lumen of the thylakoids.
Calcium↗
Chromophore organization in the higher-plant photosystem II antenna protein CP26.
The chlorophyll a/b-xanthophyll-protein CP26 complex belongs to the Lhc protein family. It binds nine chlorophylls and two xanthophylls per 26.6 kDa polypeptide. Determination of the characteristics of each binding site is needed for the understanding of functional organization of individual proteins belonging to the photosystem II supramolecular complex. The biochemical and spectroscopic features of native CP26 are presented here together with identification of pigment binding and energy transitions in different sites. The analysis has been performed via a new approach using recombinant CP26 complexes in which the chromophore content has been experimentally modified. Data were interpreted on the basis of homology with CP29 and LHCII complexes, for which detailed knowledge is available from mutation analysis. We propose that one additional Chl b is present in CP26 as compared to CP29 and that it is located in site B2. We also found that in CP26 three chlorophyll binding sites are selective for Chl a, one of them being essential for the folding of the pigment-protein complex. Two xanthophyll binding sites were identified, one of which (L1) is essential for protein folding and specifically binds lutein. The second site (L2) has lower selectivity and can bind any of the xanthophyll species present in thylakoids.
Binding Sites↗
Deletion mutagenesis in Synechocystis sp. PCC6803 indicates that the Mn-stabilizing protein of photosystem II is not essential for O2 evolution.
The photosystem II (PSII) reaction center complex coordinates a cluster of Mn atoms that are involved in the accumulation of oxidizing equivalents generated by light-induced charge separations within the intrinsic portion of the PSII complex. A 33-kDa extrinsic protein, termed the Mn-stabilizing protein (MSP), has been implicated in the stabilization of two of the four Mn atoms of the cluster, yet the precise role of this protein in O2 evolution remains to be elucidated. Here we describe the construction of a mutant of the cyanobacterium Synechocystis sp. PCC6803 in which the entire gene encoding MSP has been deleted. Northern and immunoblot analyses indicate that other PSII proteins are expressed and accumulated, despite the absence of MSP. Fluorescence emission spectra at 77 K indicate PSII assembles in the mutant, but that the binding of MSP is required for the normal fluorescence characteristics of the PSII complex, and suggest a specific interaction between MSP and CP47. Fluorescence induction measurements indicate a reduced rate of forward electron transport to the primary electron donor, P680, in the mutant. It is concluded that in contrast to previous reports, MSP is not required for the assembly of active PSII complexes nor is it essential for H2O-splitting activity in vivo.
Blotting, Northern↗
Revealing the structure of the oxygen-evolving core dimer of photosystem II by cryoelectron crystallography.
Here we present cryoelectron crystallographic analysis of an isolated dimeric oxygen-evolving complex of photosystem II (at a resolution of approximately 0.9 nm), revealing that the D1-D2 reaction center (RC) proteins are centrally located between the chlorophyll-binding proteins, CP43 and CP47. This conclusion supports the hypothesis that photosystems I and II have similar structural features and share a common evolutionary origin. Additional density connecting the two halves of the dimer, which was not observed in a recently described CP47-RC complex that did not include CP43, may be attributed to the small subunits that are involved in regulating secondary electron transfer, such as PsbH. These subunits are possibly also required for stabilization of the dimeric photosystem II complex. This complex, containing at least 29 transmembrane helices in its asymmetric unit, represents one of the largest membrane protein complexes studied at this resolution.
Cryoelectron Microscopy↗
Synthesis and turnover of photosystem II reaction center polypeptides in cyanobacterial D2 mutants.
Several mutations in psbD (coding for the photosystem II reaction protein D2) lead to a loss of the entire photosystem II complex from the thylakoid membrane. For two site-directed D2 mutants, E69V (Glu-69 mutated to Val) and ET-9 (introducing a premature stop codon in psbD), and two psbD deletion mutants, the synthesis and degradation of photosystem II reaction center components were analyzed. In E69V, no D2 or psbC-encoded CP43 was detected upon 5-min pulse labeling or Western blotting, whereas a psbDI/C transcript was present. Either D2 and CP43 are very unstable in this mutant, or the mutation causes an inhibition of translation of the psbDC operon. In E69V, the predominant lifetime of D1 is several minutes, but a small fraction of D1 remains stable in the thylakoid for several hours. In E69V, also a 43-kDa band is detected by D1 antisera. We attribute this band to a D1 cross-linking product possibly involved in D1 degradation. We identified a D1 precursor in E69V and psbDI/C/DII-, two mutants with particularly short D1 lifetimes. A unique feature of the E69V mutant is that destabilization of the photosystem II complex is triggered by a single-site mutation leading to extremely rapid turnover or lack of synthesis of D2.
Blotting, Northern↗
Activation of the silent psbA1 gene in the cyanobacterium Synechocystis sp strain 6803 produces a novel and functional D1 protein.
The photosystem II reaction center protein D1 in Synechocystis sp strain 6803 is encoded by the psbA2 and psbA3 genes of the three-membered psbA gene family. The silent and divergent psbA1 copy of the psbA gene family was activated by exchanging part of its upstream region with a corresponding fragment of the psbA2 copy. The light-regulated expression of the activated psbA1 gene showed that the inserted psbA2 segment contains the information necessary for light-dependent as well as high-light-stimulated transcription. The activated psbA1 gene expressed a novel D1 protein, D1'. A mutant strain containing psbA1 as the only active psbA gene grew photoautotrophically at a rate comparable to that of the wild type. This finding demonstrates that despite its unusual amino acid sequence, D1 is exchangeable for D1 in the photosystem II complex, at least under normal laboratory conditions. The D1' protein was found to have a degradation rate similar to that of the D1 protein under low- or high-light conditions. Another mutant containing the activated psbA1 gene together with the psbA2 and psbA3 genes produced both the D1 and D1' proteins.
Amino Acid Sequence↗
Thylakoid protein phosphorylation and the thiol redox state.
Illumination of thylakoid membranes leads to the phosphorylation of a number of photosystem II-related proteins, including the reaction center proteins D1 and D2 as well as the light-harvesting complex (LHCII). Regulation of light-activated thylakoid protein phosphorylation has mainly been ascribed to the redox state of the electron carrier plastoquinone. In this work, we show that this phosphorylation in vitro is also strongly influenced by the thiol disulfide redox state. Phosphorylation of the light-harvesting complex of photosystem II was found to be favored by thiol-oxidizing conditions and strongly downregulated at moderately thiol-reducing conditions. In contrast, phosphorylation of the photosystem II reaction center proteins D1 and D2 as well as that of other photosystem II subunits was found to be stimulated up to 2-fold by moderately thiol-reducing conditions and kept at a high level also at highly reducing conditions. These responses of the level of thylakoid protein phosphorylation to changes in the thiol disulfide redox state are reminiscent of those observed in vivo in response to changes in the light intensity and point to the possibility of a second loop of redox regulation of thylakoid protein phosphorylation via the ferredoxin-thioredoxin system.
Adenosine Triphosphatases↗