Characterization of O2 evolution by a wheat photosystem II reaction center complex isolated by a simplified method: disjunction of secondary acceptor quinone and enhanced Ca2+ demand

An O2-evolving photosystem II (PSII) reaction center complex was prepared from wheat by a simple method consisting of octylglucoside solubilization of Triton PSII particles followed by one-step sucrose density gradient centrifugation. The complex contained six species of proteins including the 33-kDa extrinsic protein with the same relative abundance as in the original PSII particles, one cytochrome b559, 4 Mn, and about 40 chlorophyll (Chl) per O2-evolving unit, and evolved O2 at a high rate of 1400-1700 mumol O2/mg Chl/h. O2 evolution by the complex was dependent on acceptor species, showing a hierarchy, ferricyanide greater than dichlorobenzoquinone greater than phenylbenzoquinone greater than dimethylbenzoquinone greater than duroquinone, and insensitive to DCMU, indicative of disjunction of the secondary quinone acceptor of PSII from the electron transport pathway. O2 evolution also showed a marked dependence on Cl- and Ca2+: about 10-fold acceleration by Cl- and an additional 2- to 3-fold by Ca2+. Comparison of the dissociation constants for Cl- and Ca2+ between the complex and NaCl-washed PSII particles revealed that octylglucoside treatment gives rise to a new Ca2+-sensitive site by removal of some unknown factor(s) other than the extrinsic 22- and 16-kDa proteins, while it preserves the Cl(-)-sensitive site as native as in NaCl-washed PSII particles. Analysis of the relationship between Cl- demand and Ca2+ demand revealed that Ca2+ absence noncompetitively inhibits the Cl(-)-supported O2 evolution, indicative of the independence of the binding site of these two factors.     

Structural characterization of photosystem II complex from red alga Porphyridium cruentum retaining extrinsic subunits of the oxygen-evolving complex.

The structure of photosystem II (PSII) complex isolated from thylakoid membranes of the red alga Porphyridium cruentum was investigated using electron microscopy followed by single particle image analysis. The dimeric complexes observed contain all major PSII subunits (CP47, CP43, D1 and D2 proteins) as well as the extrinsic proteins (33 kDa, 12 kDa and the cytochrome c(550)) of the oxygen-evolving complex (OEC) of PSII, encoded by the psbO, psbU and psbV genes, respectively. The single particle analysis of the top-view projections revealed the PSII complex to have maximal dimensions of 22 x 15 nm. The analysis of the side-view projections shows a maximal thickness of the PSII complex of about 9 nm including the densities on the lumenal surface that has been attributed to the proteins of the OEC complex. These results clearly demonstrate that the red algal PSII complex is structurally very similar to that of cyanobacteria and to the PSII core complex of higher plants. In addition, the arrangement of the OEC proteins on the lumenal surface of the PSII complex is consistent to that obtained by X-ray crystallography of cyanobacterial PSII.     

Calcium depletion modifies the structure of the photosystem II O(2)-evolving complex.

A 5 min exposure of photosystem II to a pH 3 citric acid solution is a simple method for selective removal of Ca(2+) from the O(2)-evolving complex. The resulting preparation retains the 23 and 17 kDa extrinsic polypeptides, but the activity of this material is only 10-20% of that of an untreated control sample. Biochemical characterization of citrate-treated photosystem II reveals that some reaction centers lose the extrinsic proteins during citrate treatment. Furthermore, a comparison of photosystem II preparations treated with citrate, or depleted of 23 and 17 kDa extrinsic polypeptides by high-salt treatment, shows that low concentrations of a small reductant, NH(2)OH, which has little effect on the activity of intact photosystem II, can reduce and inhibit the Mn cluster in both types of preparations. In contrast, a large reductant, hydroquinone, cannot access the majority of O(2)-evolving centers in citrate-treated preparations, while 23 and 17 kDa-depleted material is rapidly inactivated by the reductant. Incubation of the citrate-treated samples in high ( approximately 60 mM) concentrations of CaCl(2) restores 50% of the lost activity; this Ca(2+)-reconstituted activity is chelator-insensitive, indicating that rebinding of Ca(2+) restores the structural integrity of the O(2)-evolving complex. A characterization of Ca(2+) and Cl(-) affinities in steady-state activity assays shows that citrate-treated preparations exhibit a Cl(-) requirement similar to that of polypeptide-depleted photosystem II, while Ca(2+) reactivation of O(2) evolution appears to occur at two structurally distinct sites. One site exhibits a high Ca(2+) affinity, similar to that found in polypeptide-depleted samples, but a second, lower-affinity site also exists, with a K(M) that is approximately 10 times greater than that of the high-affinity site, which is associated with centers that retain the extrinsic polypeptides. These data indicate that citrate-induced Ca(2+) depletion causes release of the 23 and 17 kDa extrinsic polypeptides from some photosystem II reaction centers, and also modifies the structure of the polypeptide-retaining O(2)-evolving centers so that the Mn cluster is exposed to small, but not large, reductants. This change may be due to subtle modifications to the structure of the photosystem II extrinsic proteins that produces a new pathway between the solvent and the Mn cluster or, alternatively, to the opening of an existing channel in the intrinsic lumenal polypeptide domain, between the solvent and the Mn cluster, that is normally occluded by a bound Ca(2+) atom.     

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.     

A novel protein in Photosystem II of a diatom Chaetoceros gracilis is one of the extrinsic proteins located on lumenal side and directly associates with PSII core components

The gene encoding a novel extrinsic protein (Psb31) found in Photosystem II (PSII) of a diatom, Chaetoceros gracilis, was cloned and sequenced. The deduced protein contained three characteristic leader sequences targeted for chloroplast endoplasmic reticulum membrane, chloroplast envelope membrane and thylakoid membrane, indicating that Psb31 is encoded in the nuclear genome and constitutes one of the extrinsic proteins located on the lumenal side. Homologous genes were found in a red alga and chromophytic algae but not in other organisms. Genes encoding the other four extrinsic proteins in C. gracilis PSII were also cloned and sequenced, and their leader sequences were characterized and compared. To search for the nearest neighbor relationship between Psb31 and the other PSII components, we crosslinked the PSII particles with the water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and found that Psb31 directly associates with PSII core components through electrostatic interaction, suggesting that the novel Psb31 protein is one of the extrinsic proteins constituting the functional oxygen-evolving complex of C. gracilis PSII.     

Reactions of hydroxylamine with the electron-donor side of photosystem II

The reaction of hydroxylamine with the O2-evolving center of photosystem II (PSII) in the S1 state delays the advance of the H2O-oxidation cycle by two charge separations. In this paper, we compare and contrast the reactions of hydroxylamine and N-methyl-substituted analogues with the electron-donor side of PSII in both O2-evolving and inactivated [tris(hydroxymethyl)aminomethane- (Tris-) washed] spinach PSII membrane preparations. We have employed low-temperature electron paramagnetic resonance (EPR) spectroscopy in order to follow the oxidation state of the Mn complex in the O2-evolving center and to detect radical oxidation products of hydroxylamine. When the reaction of hydroxylamine with the S1 state in O2-evolving membranes is allowed to proceed to completion, the S2-state multiline EPR signal is suppressed until after three charge separations have occurred. Chemical removal of hydroxylamine from treated PSII membrane samples prior to illumination fails to reverse the effects of the dark reaction, which argues against an equilibrium coordination of hydroxylamine to a site in the O2-evolving center. Instead, the results indicate that the Mn complex is reduced by two electrons by hydroxylamine, forming the S-1 state. An additional two-electron reduction of the Mn complex to a labile "S-3" state probably occurs by a similar mechanism, accounting for the release of Mn(II) ions upon prolonged dark incubation of O2-evolving membranes with high concentrations of hydroxylamine. In N,N-dimethylhydroxylamine-treated, Tris-washed PSII membranes, which lack O2 evolution activity owing to loss of the Mn complex, a large yield of dimethyl nitroxide radical is produced immediately upon illumination at temperatures above 0 degrees C. The dimethyl nitroxide radical is not observed upon illumination under similar conditions in O2-evolving PSII membranes, suggesting that one-electron photooxidations of hydroxylamine do not occur in centers that retain a functional Mn complex. We suggest that the flash-induced N2 evolution observed in hydroxylamine-treated spinach thylakoid membrane preparations arises from recombination of hydroxylamine radicals formed in inactivated O2-evolving centers.     

3D map of the plant photosystem II supercomplex obtained by cryoelectron microscopy and single particle analysis

Here we describe the first 3D structure of the photosystem II (PSII) supercomplex of higher plants, constructed by single particle analysis of images obtained by cryoelectron microscopy. This large multisubunit membrane protein complex functions to absorb light energy and catalyze the oxidation of water and reduction of plastoquinone. The resolution of the 3D structure is 24 A and emphasizes the dimeric nature of the supercomplex. The extrinsic proteins of the oxygen-evolving complex (OEC) are readily observed as a tetrameric cluster bound to the lumenal surface. By considering higher resolution data, obtained from electron crystallography, it has been possible to relate the binding sites of the OEC proteins with the underlying intrinsic membrane subunits of the photochemical reaction center core. The model suggests that the 33 kDa OEC protein is located towards the CP47/D2 side of the reaction center but is also positioned over the C-terminal helices of the D1 protein including its CD lumenal loop. In contrast, the model predicts that the 23/17 kDa OEC proteins are positioned at the N-terminus of the D1 protein incorporating the AB lumenal loop of this protein and two other unidentified transmembrane helices. Overall the 3D model represents a significant step forward in revealing the structure of the photosynthetic OEC whose activity is required to sustain the aerobic atmosphere on our planet.     

Changes in Protein Composition and Mn Abundance in Photosystem II Particles on Photoactivation of the Latent O(2)-Evolving System in Flash-Grown Wheat Leaves

Protein composition and Mn abundance were compared between the two photosystem II (PSII) particle preparations obtained before and after photoactivation of the latent O₂-evolving system in intermittently flashed wheat leaves. The following results have been obtained: (a) nonphotoactivated PSII particles were devoid of two extrinsic proteins which corresponded to the 24 and 16 kilodalton proteins in spinach particles, although the particles contained all the intrinsic proteins and the 33 kilodalton extrinsic protein. (b) The two extrinsic proteins absent in nonphotoactivated PSII particles were present in nonphotoactivated thylakoids, but were easily removed by a hypotonic shock followed by brief sonication. Such removal of the proteins did not occur in photoactivated thylakoids. (c) Nonphotoactivated PSII particles contained 1.5 Mn/400 chlorophyll, while photoactivated particles contained 8 Mn/400 chlorophyll. (d) Nonphotoactivated thylakoids contained 6 Mn/400 chlorophyll, but most of them were removed from thylakoids by a hypotonic shock in the presence of ethylenediaminetetraacetate. Such removal of Mn did not occur in photoactivated thylakoids.     

Mutations of arginine 64 within the putative Ca(2+)-binding lumenal interhelical a-b loop of the photosystem II D1 protein disrupt binding of the manganese stabilizing protein and cytochrome c(550) in Synechocystis sp. PCC6803

Mutations D1-R64E, D1-R64Q, and D1-R64V in the putative calcium-binding lumenal interhelical a-b loop of the photosystem II (PSII) D1 protein were characterized in terms of impact on growth, extrinsic protein binding, photoactivation, and properties of the H(2)O-oxidation complex. The D1-R64E charge reversal mutation greatly weakened the binding of the extrinsic manganese-stabilizing protein (MSP) and, to a considerably lesser extent, weakened the binding of cytochrome c(550) (c550). Both D1-R64Q and D1-R64E exhibited an increased requirement for Ca(2+) in the cell growth medium. Bare platinum electrode measurements of O(2)-evolving membranes showed a retarded appearance of O(2) following single turn-over flashes, especially in the case of the D1-R64E mutant. The D1-R64E mutant also had a pronounced tendency to lose O(2) evolution activity in the dark and exhibited an increased relative quantum yield of photoactivation, which are characteristics shared by mutants that lack extrinsic proteins. S(2) and S(3) decay measurements in the isolated membranes indicate that D1-R64E and D1-R64Q have faster decays of these higher S-states as compared to the wild-type. However, fluorescence decay in the presence of DCMU, which monitors primarily Q(A)(-) charge recombination with PSII donors, showed somewhat slower decays. Taken together, the fluorescence and S-state decay indicate that the midpoint of either Q(B)(-) has been modified to be more negative in the mutants or that a recombination path presumably involving either Q(B)(-) or Y(D) has become kinetically more accessible.     

Highly purified thermo-stable oxygen-evolving photosystem II core complex from the thermophilic cyanobacterium Synechococcus elongatus having His-tagged CP43

The carboxyl terminus of the CP43 subunit of photosystem II (PSII) in the thermophilic cyanobacterium, Synechococcus elongatus, was genetically tagged with six consecutive histidine residues to create a metal binding site on the PSII supramolecular complex. The histidine-tagging enabled rapid isolation of an intact cyanobacterial PSII core complex from dodecyl maltoside-solubilized thylakoids by a simple one-step Ni(2+)-affinity column chromatography. The isolated core complex was in a dimeric form with a molecular mass of about 580 kDa, consisting of five major intrinsic membrane proteins (CP47, CP43, D1, D2 and cytochrome b-559), three extrinsic proteins (33 kDa, 12 kDa, and cytochrome c-550), and a few low molecular mass membrane proteins, and evolved oxygen at a rate as high as 3,400 mumol (mg Chl)-1 h-1 at 45 degrees C with ferricyanide as an electron acceptor. The core complex emitted thermoluminescence B2-, B1- and Q-bands arising from S2QB-, S3QB- and S2QA- charge recombinations at respective emission temperatures of 45, 38 and 20 degrees C, all of which were higher by about 15 degrees C as compared with those in mesophilic spinach BBY membranes. These results indicated that the isolated core complex well retained the intact properties of thermoluminescence of thermophilic cyanobacterial cells, the deeper stabilization of PSII charge pairs. The isolated complex was extremely stable in terms of both protein composition and function, exhibiting no release of extrinsic proteins, no proteolytic degradation in any of its subunits, accompanied by only a slight (less than 10%) loss in oxygen evolution, after dark-incubation at 20 degrees C for 8 d. These properties of the thermophilic PSII core complex are highly useful for various types of studies on PSII.     

The Psb27 protein facilitates manganese cluster assembly in photosystem II

Photosystem II (PSII) is a large membrane protein complex that uses light energy to convert water to molecular oxygen. This enzyme undergoes an intricate assembly process to ensure accurate and efficient positioning of its many components. It has been proposed that the Psb27 protein, a lumenal extrinsic subunit, serves as a PSII assembly factor. Using a psb27 genetic deletion strain (Deltapsb27) of the cyanobacterium Synechocystis sp. PCC 6803, we have defined the role of the Psb27 protein in PSII biogenesis. While the Psb27 protein was not essential for photosynthetic activity, various PSII assembly assays revealed that the Deltapsb27 mutant was defective in integration of the Mn(4)Ca(1)Cl(x) cluster, the catalytic core of the oxygen-evolving machinery within the PSII complex. The other lumenal extrinsic proteins (PsbO, PsbU, PsbV, and PsbQ) are key components of the fully assembled PSII complex and are important for the water oxidation reaction, but we propose that the Psb27 protein has a distinct function separate from these subunits. We show that the Psb27 protein facilitates Mn(4)Ca(1)Cl(x) cluster assembly in PSII at least in part by preventing the premature association of the other extrinsic proteins. Thus, we propose an exchange of lumenal subunits and cofactors during PSII assembly, in that the Psb27 protein is replaced by the other extrinsic proteins upon assembly of the Mn(4)Ca(1)Cl(x) cluster. Furthermore, we show that the Psb27 protein provides a selective advantage for cyanobacterial cells under conditions such as nutrient deprivation where Mn(4)Ca(1)Cl(x) cluster assembly efficiency is critical for survival.     

Cross-linking Evidence for Multiple Interactions of the PsbP and PsbQ Proteins in a Higher Plant Photosystem II Supercomplex

The extrinsic subunits of membrane-bound photosystem II (PSII) maintain an essential role in optimizing the water-splitting reaction of the oxygen-evolving complex (OEC), even though they have undergone drastic change during the evolution of oxyphototrophs from symbiotic cyanobacteria to chloroplasts. Two specific extrinsic proteins, PsbP and PsbQ, bind to the lumenal surface of PSII in green plants and maintain OEC conformation and stabilize overall enzymatic function; however, their precise location has not been fully resolved. In this study, PSII-enriched membranes, isolated from spinach, were subjected to chemical cross-linking combined with release-reconstitution experiments. We observed direct interactions between PsbP and PsbE, as well as with PsbR. Intriguingly, PsbP and PsbQ were further linked to the CP26 and CP43 light-harvesting proteins. In addition, two cross-linked sites, between PsbP and PsbR, and that of PsbP and CP26, were identified by tandem mass spectrometry. These data were used to estimate the binding topology and location of PsbP, and the putative positioning of PsbQ and PsbR on the lumenal surface of the PSII. Our model gives new insights into the organization of PSII extrinsic subunits in higher plants and their function in stabilizing the OEC of the PSII supercomplex.     

Isolation and characterization of oxygen-evolving thylakoid membranes and photosystem II particles from a marine diatom Chaetoceros gracilis

Thylakoid membranes retaining high oxygen-evolving activity (about 250 micromol O(2)/mg Chl/h) were prepared from a marine centric diatom, Chaetoceros gracilis, after disruption of the cells by freeze-thawing. We also succeeded in purification of Photosystem II (PSII) particles by differential centrifugation of the thylakoid membranes after treatment with 1% Triton X-100. The diatom PSII particles showed an oxygen-evolving activity of 850 and 1045 micromol O(2)/mg Chl/h in the absence and presence of CaCl(2), respectively. The PSII particles contained fucoxanthin chlorophyll a/c-binding proteins in addition to main intrinsic proteins of CP47, CP43, D2, D1, cytochrome b559, and the antenna size was estimated to be 229 Chl a per 2 molecules of pheophytin. Five extrinsic proteins were stoichiometrically released from the diatom PSII particles by alkaline Tris-treatment. Among these five extrinsic proteins, four proteins were red algal-type extrinsic proteins, namely, PsbO, PsbQ', PsbV and PsbU, whereas the other one was a novel, hypothetical protein. This is the first report on isolation and characterization of diatom PSII particles that are highly active in oxygen evolution and retain the full set of extrinsic proteins including an unknown protein.     

Binding and functional properties of the extrinsic proteins in oxygen-evolving photosystem II particle from a green alga,Chlamydomonas reinhardtii having his-tagged CP47

Oxygen-evolving photosystem II (PSII) particles were purified from Chlamydomonas reinhardtii having His-tag extension at the C terminus of the CP47 protein, by a single-step Ni(2+)-affinity column chromatography after solubilization of thylakoid membranes with sucrose monolaurate. The PSII particles consisted of, in addition to intrinsic proteins, three extrinsic proteins of 33, 23 and 17 kDa. The preparation showed a high oxygen-evolving activity of 2,300-2,500 micro mol O(2) (mg Chl)(-1) h(-1) in the presence of Ca(2+) using ferricyanide as the electron acceptor, while its activity was 680-720 micro mol O(2) (mg Chl)(-1) h(-1) in the absence of Ca(2+) and Cl(-) ions. The activity was 710-820 micro mol O(2) (mg Chl)(-1) h(-1) independent of the presence or absence of Ca(2+) and Cl(-) when 2,6-dichloro-p-benzoquinone was used as the acceptor. These activities were scarcely inhibited by DCMU. The kinetics of flash-induced fluorescence decay revealed that the electron transfer from Q(A)(-) to Q(B) was significantly inhibited, and the electron transfer from Q(A)(-) to ferricyanide was largely stimulated in the presence of Ca(2+). These results indicate that the acceptor side, Q(B) site, was altered in the PSII particles but its donor side remained intact. Release-reconstitution experiments revealed that the extrinsic 23 and 17 kDa proteins were released only partially by NaCl-wash, while most of the three extrinsic proteins were removed when treated with urea/NaCl, alkaline Tris or CaCl(2). The 23 and 17 kDa proteins directly bound to PSII independent of the other extrinsic proteins, and the 33 kDa protein functionally re-bound to CaCl(2)-treated PSII which had been reconstituted with the 23 and 17 kDa proteins. These binding properties were largely different from those of the extrinsic proteins in higher plant PSII, and suggest that each of the three extrinsic proteins has their own binding sites independent of the others in the green algal PSII.     

A genetically tagged Psb27 protein allows purification of two consecutive photosystem II (PSII) assembly intermediates in Synechocystis 6803, a cyanobacterium

Photosystem II (PSII) is a large membrane bound molecular machine that catalyzes light-driven oxygen evolution from water. PSII constantly undergoes assembly and disassembly because of the unavoidable damage that results from its normal photochemistry. Thus, under physiological conditions, in addition to the active PSII complexes, there are always PSII subpopulations incompetent of oxygen evolution, but are in the process of undergoing elaborate biogenesis and repair. These transient complexes are difficult to characterize because of their low abundance, structural heterogeneity, and thermodynamic instability. In this study, we show that a genetically tagged Psb27 protein allows for the biochemical purification of two monomeric PSII assembly intermediates, one with an unprocessed form of D1 (His27ΔctpAPSII) and a second one with a mature form of D1 (His27PSII). Both forms were capable of light-induced charge separation, but unable to photooxidize water, largely because of the absence of a functional tetramanganese cluster. Unexpectedly, there was a significant amount of the extrinsic lumenal PsbO protein in the His27PSII, but not in the His27ΔctpAPSII complex. In contrast, two other lumenal proteins, PsbU and PsbV, were absent in both of these PSII intermediate complexes. Additionally, the only cytoplasmic extrinsic protein, Psb28 was detected in His27PSII complex. Based on these data, we have presented a refined model of PSII biogenesis, illustrating an important role of Psb27 as a gate-keeper during the complex assembly process of the oxygen-evolving centers in PSII.     

Localization of cyanobacterial photosystem II donor-side subunits by electron microscopy and the supramolecular organization ofphotosystem II in the thylakoid membrane.

A large set of electron microscopy projections of photosystem II (PSII) dimers isolated from the cyanobacterium Synechococcus elongatus was characterized by single particle image analysis. In addition to previously published maps at lower resolution [Boekema, E.J., Hankamer, B., Bald, D., Kruip, J., Nield, J., Boonstra, A.F., Barber, J. & R?gner, M. (1995) Proc. Natl Acad. Sci. USA 92, 175-179], the new side-view projections show densities of all three lumenal extrinsic proteins, i.e. the 33-kDa, 12-kDa and the cytochrome c-550 subunit encoded by psbO, psbU and psbV, respectively. Analysis of the size and shape of the top-view projections revealed a small number of photosystem II particles of about double the size of the usual dimers. Size and quantity of these 'double dimers' correlates with a small fraction of 1000-kDa particles found with HPLC-size-exclusion chromatographic analysis. Because many cyanobacteria contain dimeric photosystem II complexes arranged in rows within the membrane, the double dimers can be considered as the breakdown fragments of these rows. Their analysis enabled the detection of the arrangement of photosystem II within the rows, in which the dimers interact with other dimers mostly with their tips, leaving a rather open center at the interfaces of two dimers. The dimers have a repeating distance of only 11.7 nm. As a consequence, the phycobilisomes, located on top of PSII and functioning in light-harvesting, must be closely packed or almost touch each other, in a manner similar to a recently suggested model [Bald, D., Kruip, J. & R?gner, M. (1996) Photosynthesis Res. 49, 103-118].     

Reconstitution of the photosystem II Ca2+ binding site

The roles of Ca(2+) in H(2)O oxidation may be as a site of substrate binding, and as a structural component of the photosystem II O(2)-evolving complex. One indication of this dual role of the metal is revealed by probing the Mn cluster in the Ca(2+) depleted O(2) evolving complex that retains extrinsic 23- and 17-kDa polypeptides with reductants (NH(2)OH and hydroquinone) [Biochemistry 41 (2002) 958]. Calcium appears to bind to photosystem II at a site where it could bind substrate H(2)O. Equilibration of Ca(2+) with this binding site is facilitated by increased ionic strength, and incubation of Ca(2+) reconstitution mixtures at 22 degrees C accelerates equilibration of Ca(2+) with the site. The Ca(2+) reconstituted enzyme system regains properties of unperturbed photosystem II: Sensitivity to NH(2)OH inhibition is decreased, and Cl(-) binding with increased affinity can be detected. The ability of ionic strength and temperature to facilitate rebinding of Ca(2+) to the intact O(2) evolving complex suggests that the structural environment of the oxidizing side of photosystem II may be flexible, rather than rigid.     

Targeted disruption of psbX and biochemical characterization of photosystem II complex in the thermophilic cyanobacterium Synechococcus elongatus

PSII-X is a small hydrophobic protein, which is universally present in photosystem II (PSII) core complex among cyanobacteria and plants. The role of PSII-X was studied by directed mutagenesis and biochemical analysis in the thermophilic cyanobacterium Synechococcus elongatus. The psbX-disrupted mutant could grow photoautotrophically indicative of non-essential function, while it showed growth defect under low CO(2) conditions. An active O(2)-evolving PSII complex was successfully isolated from the mutant and wild type. Protein composition of the isolated PSII complex was the same as wild type except for the absence of PSII-X. O(2) evolution supported by artificial quinones was affected in the psbX-disrupted mutant. At high concentration of 2,6-dichlorobenzoquinone or 2,6-dimethylbenzoquinone, the mutant showed much lower activity than wild type, while not much difference was found at low concentration. These results imply that binding or turnover of quinones at the Q(B) site depends, at least in part, on PSII-X protein in the PSII complex. Gel filtration chromatography of the PSII complex revealed that the dimeric structure of the complex was not greatly affected in the psbX-disrupted mutant.     

具放氧活性的光系统Ⅱ核心复合物的光谱特性和叶绿素蛋白质成分的研究

我们从菠菜叶片分离出一种具有高放氧活性的光系统Ⅱ(PSII)核心复台物.我们对这种PSII核心复合物进行了PSII光化学活性、吸收、荧光光谱特性和叶绿素蛋白质成分的研究.这种PSII核心复合物是一种不含PSI成分,又除去了LHC-Ⅱ的较纯,较小的具有高放氧活性(?)的单位.在文中对PSII反应中心叶绿素的吸收成分和CPa带进行了一些讨论.     

Low-molecular-mass proteins in cyanobacterial photosystem II: identification of psbH and psbK gene products by N-terminal sequencing

The O2-evolving photosystem II core complex was isolated from a thermophilic cyanobacterium, Synechococcus vulcanus Copeland. Analysis by SDS-polyacrylamide gel electrophoresis revealed that the complex contained at least seven low-molecular-mass proteins in addition to the well characterized CP47 apoprotein, CP43 apoprotein, 33 kDa extrinsic protein, D1 protein, D2 protein and large subunit of cytochrome b-559. The separation of these low-molecular-mass proteins were very similar between cyanobacterial and higher plant PS II. N-terminal sequences of the 6.5 kDa and 3.9 kDa proteins of cyanobacterial core complex were determined after blotting to a polyvinylidene difluoride membrane. The sequence of the 6.5 kDa protein showed high homology with an internal sequence of plant psbH gene product, so-called 10 kDa phosphoprotein, but did not conserve the Thr residue which is specifically phosphorylated in plants. The sequence of the 3.9 kDa protein corresponded to the K protein of higher plants (mature form of psbK gene product). These results indicate that the products of both psbH and psbK genes are present in cyanobacterial PS II as well as being associated with the O2-evolving core complex.