Identity of the Photosystem II reaction center polypeptide

A Photosystem-II (PS-II)-enriched chloroplast submembrane fraction has been subjected to non-denaturing gel-electrophoresis. Two chlorophyll a (Chl a)-binding proteins associated with the core complex were isolated and spectrally characterized. The Chl protein with apparent apoprotein mass of 47 kDa (CP47) displayed a 695 nm fluorescence emission maximum (77 K) and light-induced absorption characteristics indicating the presence of the reaction center Chl, P-680, and its primary electron acceptor, pheophytin. A Chl protein of apparent apoprotein mass of 43 kDa (CP43) displayed a fluorescence emission maximum at 685 nm. We conclude that CP43 serves as an antenna Chl protein and the PS II reaction center is located in CP47.     

Isolation and characterization of photosystem II from the filamentous sporophyte of Porphyra yezoensis

Thylakoid membranes were isolated and purified from diploid filamentous sporophytes of Porphyra yezoensis Ueda using sucrose density gradient ultracentrifugation (SDGUC). After thylakoid membranes were solubilized with SDS, the phtosystem II (PSII) particles with high 2, 6-dichloroindophenol (DCIP) photoreduction activity were isolated by SDGUC. The absorption and fluorescence spectra, DCIP photoreduction activity and oxygen evolution activity of the thylakoid membranes and PSII particles were determined. The polypeptide composition of purified PSII particles was distinguished by SDS-PAGE. Results showed that PSII particles of sporophytes differed from the gametophytes in spectral properties and polypeptide composition. Apart from 55 kDa D1-D2 heterodimer, CP47, CP43, 33 kDa protein, D1, D2, cyt b559 and 12 kDa protein were identified from PSII particles from sporophytes; a new 102 kDa protein was also detected. However, cyt c-550, 20 kDa, 14 kDa and 16 kDa proteins found in PSII particles from gametophytes were not detected in the sporophytes.     

Heat stress induces an inhibition of excitation energy transfer from phycobilisomes to photosystem II but not to photosystem I in a cyanobacterium Spirulina platensis.

The effects of high temperature (30-52.5 degrees C) on excitation energy transfer from phycobilisomes (PBS) to photosystem I (PSI) and photosystem II (PSII) in a cyanobacterium Spirulina platensis grown at 30 degrees C were studied by measuring 77 K chlorophyll (Chl) fluorescence emission spectra. Heat stress had a significant effect on 77 K Chl fluorescence emission spectra excited either at 436 or 580 nm. In order to reveal what parts of the photosynthetic apparatus were responsible for the changes in the related Chl fluorescence emission peaks, we fitted the emission spectra by Gaussian components according to the assignments of emission bands to different components of the photosynthetic apparatus. The 643 and 664 nm emissions originate from C-phycocyanin (CPC) and allophycocyanin (APC), respectively. The 685 and 695 nm emissions originate mainly from the core antenna complexes of PSII, CP43 and CP47, respectively. The 725 and 751 nm band is most effectively produced by PSI. There was no significant change in F725 and F751 during heat stress, suggesting that heat stress had no effects on excitation energy transfer from PBS to PSI. On the other hand, heat stress induced an increase in the ratio of Chl fluorescence yield of PBS to PSII, indicating that heat stress inhibits excitation energy transfer from PBS to PSII. However, this inhibition was not associated with an inhibition of excitation energy transfer from CPC to APC since no significant changes in F643 occurred at high temperatures. A dramatic enhancement of F664 occurring at 52.5 degrees C indicates that excitation energy transfer from APC to the PSII core complexes is suppressed at this temperature, possibly due to the structural changes within the PBS core but not to a detachment of PBS from PSII, resulting in an inhibition of excitation energy transfer from APC to PSII core complexes (CP47 + CP43). A decrease in F685 and F695 in heat-stressed cells with excitation at 436 nm seems to suggest that heat stress did not inhibit excitation energy transfer from the Chl a binding proteins CP47 and CP43 to the PSII reaction center and the decreased Chl fluorescence yields from CP43 and CP47 could be explained by the inhibition of the energy transfer from APC to PSII core complexes (CP47 + CP43).     

Comparison of chlorophyll a spectra in wild-type and mutant barley chloroplasts grown under day or intermittent light

The absorption (640–710 nm) and fluorescence emission (670–710 nm) spectra (77 K) of wild-type and Chl b-less, mutant, barley chloroplasts grown under either day or intermittent light were analysed by a RESOL curve-fitting program. The usual four major forms of Chl a at 662, 670, 678 and 684 nm were evident in all of the absorption spectra and three major components at 686, 693 and 704 nm in the emission spectra. A broad Chl a component band at 651 nm most likely exists in all chlorophyll spectra in vivo. The results show that the mutant lacks not only Chl b, but also the Chl a molecules which are bound to the light-harvesting, Chl a/b, protein complex of normal plants. It also appears that the absorption spectrum of this antenna complex is not modified appreciably by its isolation from thylakoid membranes.Abbreviations Chl chlorophyll - DL daylight - ImL intermittent light - WT wildtype - LHC light-harvesting Chl a/b protein complex - S.E. standard error of the mean DBP-CIW No. 763.     

Effect of monogalactosyldiacylglycerol on the interaction between photosystem II core complex and its antenna complexes in liposomes of thylakoid lipids

The non-bilayer lipid monogalactosyldiacylglycerol (MGDG) is the most abundant type of lipid in the thylakoid membrane and plays an important role in regulating the structure and function of photosynthetic membrane proteins. In this study, we have reconstituted the isolated major light-harvesting complexes of photosystem II (PSII) (LHCIIb) and a preparation consisting of PSII core complexes and minor LHCII of PSII (PSIICC) into liposomes that consisted of digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG), with or without MGDG. Transmission electron microscopy and freeze-fracture studies showed unilamellar proteoliposomes, and demonstrated that most of the MGDG is incorporated into bilayer structures. The impact of MGDG on the functional interaction between LHCIIb and PSIICC was investigated by low temperature (77 K) fluorescence emission spectra and the photochemical activity of PSII. The additional incorporation of LHCIIb into liposomes containing PSIICC markedly increased oxygen evolution of PSIICC. Excitation at 480 nm of chlorophyll (Chl) b in LHCIIb stimulated a characteristic fluorescence emission of the Chl a in PSII (684.2 nm), rather than that of the Chl a in LHCIIb (680 nm) in the LHCIIb–PSIICC proteoliposomes, which indicated that the energy was transferred from LHCIIb to PSIICC in liposome membranes. Increasing the percentage of MGDG in the PSIICC–LHCIIb proteoliposomes enhanced the photochemical activity of PSII, due to a more efficient energy transfer from LHCIIb to PSIICC and, thus, an enlarged antenna cross section of PSII.     

Spectral analysis on origination of the bands at 437 nm and 475.5 nm of chlorophyll fluorescence excitation spectrum in Arabidopsis chloroplasts

Chlorophyll fluorescence has been often used as an intrinsic optical molecular probe to study photosynthesis. In this study, the origin of bands at 437 and 475.5 nm in the chlorophyll fluorescence excitation spectrum for emission at 685 nm in Arabidopsis chloroplasts was investigated using various optical analysis methods. The results revealed that this fluorescence excitation spectrum was related to the absorption characteristics of pigment molecules in PSII complexes. Moreover, the excitation band centred at 475.5 nm had a blue shift, but the excitation band at 437 nm changed relatively less due to induction of non‐photochemical quenching (NPQ). Furthermore, fluorescence emission spectra showed that this blue shift occurred when excitation energy transfer from both chlorophyll b (Chl b) and carotenoids (Cars) to chlorophyll a (Chl a) was blocked. These results demonstrate that the excitation band at 437 nm was mainly contributed by Chl a, while the excitation band at 475.5 nm was mainly contributed by Chl b and Cars. The chlorophyll fluorescence excitation spectrum, therefore, could serve as a useful tool to describe specific characteristics of light absorption and energy transfer between light‐harvesting pigments. Copyright © 2015 John Wiley & Sons, Ltd.     

Changes in the photosynthetic characteristics and photosystem stoichiometries in wild-type and Chl <Emphasis Type="Italic">b</Emphasis>-deficient mutant rice seedlings under various irradiances

By using a wild-type rice (Oryza sativa L. cv. Norin No. 8) and the chlorophyll (Chl) b-deficient mutant derived from Norin No. 8 (chlorina 11), the present study monitored the oxygen evolution, contents of Chl a and b, β-carotene, and lutein in leaf and the contents of cytochrome f, and the reaction centres of photosystem I (PSI) and photosystem II (PSII) in thylakoids. The oxygen evolution, maximal quantum yield of PSII (F_v/F_m) and Chl concentration remained constant in both Norin No. 8 and chlorina 11 under 5 and 2% of full sunlight for six days. On the other hand, on the thylakoid level, the PSII reaction centre of chlorina 11 was more stable even under high irradiance, while approximately 40% decrease in levels of the PSII reaction centre occurred under 2% of full sunlight for six days. However, under such conditions, by regulating the stoichiometry of active PSII and PSI centres, the light absorption balance in both rice types was adjusted between the two photosystems. The present study attempted to examine whether the light absorption balance between PSII and PSI is altered to effectively conduct photosynthesis in the wild-type and Chl b-deficient mutant rice seedlings.     

Antenna chlorophyll a complexes in mutant and developing barley

The chlorophyll a antenna of photosystems I and II were each isolated after detergent treatment by gel electrophoresis or sucrose gradient centrifugation from a b-less mutant of barley grown in daylight and from wildtype barley developed in intermittent light. We identified each fraction by both its electrophoretic position and PS I activity (P700 content) in the case of the mutant, and by both PS I and PS II activity (DCIP reduction from DPC) in the light-limited plants. The proportion of Chl a in each photosystem was estimated from the amount in each gel or sucrose gradient band, and from addition of the areas under the absorption spectra (650–710 nm) of each fraction to match the spectrum of the solubilized thylakoids. The latter method was possible because the spectrum (77 K) of each fraction was unique; in the mutant about 70% of chlorophyll is associated with PS I and 30% with PS II. In the light-limited plants, the reverse is true with nearly 70% associated with PS II. RESOL analyses of both absorption and fluorescence emission spectra of all isolated fractions indicated an abnormal arrangement of antenna chlorophyll molecules in the light-limited, developing membranes even though their reaction centers are fully functional.Abbreviations DCIP dichlorophenolindophenol - DOC deoxycholate - DPC diphenylcarbazide - DL daylight - ImL intermittent light - LHC light-harvesting Chl a/b protein complex - PAGE polyacrylamide gel electrophoresis DPB-CIW No. 778     

Chlorophyll-protein composition of the thylakoid membrane from Prochlorothrix hollandica, a prokaryote containing chlorophyll b

The chlorophyll-protein complexes of the thylakoid membrane from Prochlorothrix hollandica were identified following electrophoresis under nondenaturing conditions. Five complexes, CP1-CP5, were resolved and these green bands were analyzed by spectroscopic and immunological methods. CP1 contains the photosystem I (PSI) reaction center, as this complex quenched fluorescence at room temperature, and had a 77 K fluorescence emission peak at 717 nm. CP4 contains the major chlorophyll-a-binding proteins of the photosystem II (PSII) core, because this complex contained polypeptides which cross-reacted to antibodies raised against Chlamydomonas PSII proteins 5 and 6. Furthermore, fluorescence excitation studies at 77 K indicated that only a Chl a is bound to CP4. Complexes CP2, CP3 and CP5 contained functionally bound Chl a and b as judged by absorption spectroscopy at 20 degrees C and fluorescence excitation spectra at 77 K. CP2, CP3 and CP5 all contain polypeptides of 30-33 kDa which are immunologically distinct from the LHC-II complex of higher plant thylakoids.     

Energy transfer of aromatic amino acids in photosystem 2 core antenna complexes CP43 and CP47

Energy transfer of aromatic amino acids in photosystem 2 (PS2) core antenna complexes CP43 and CP47 was studied using absorption spectroscopy, fluorescence spectroscopy, and the 0.35 nm crystal structure of PS2 core complex. The energy of tyrosines (Tyrs) was not effectively transferred to tryptophans (Trps) in CP43 and CP47. The fluorescence emission spectrum of CP43 and CP47 by excitation at 280 nm should be a superposition of the Tyr and Trp fluorescence emission spectra. The aromatic amino acids in CP43 and CP47 could transfer their energy to chlorophyll (Chl) a molecules by the Dexter mechanism and the Föster mechanism, and the energy transfer efficiency in CP47 was much higher than that in CP43. In CP47 the Föster mechanism must be the dominant energy transfer mechanism between aromatic amino acids and Chl a molecules, whereas in CP43 the Dexter mechanism must be the dominant one. Hence solar ultraviolet radiation brings not only damages but also benefits to plants.     

Structural-functional organization of the main light harvesting complex and photosystem 2 of higher plants

The interrelation between spectral and structural–functional properties of LhcIIb was studied. The dipole strength of the main Q_y bands of chlorophylls (Chl a 30.8 D²; Chl b 18.5 D²) and chlorophyll a/b ratio (Chl a/Chl b = 7 : 6) were determined for LhcIIb. The Chl a/Chl b value shows that the subunit of this complex contains seven Chl a and six Chl b molecules. Individual bands of chlorophylls (bands in stokes and anti-Stokes parts at 77 K were Lorentzian and Gaussian, respectively) were resolved using synchronized deconvolution of absorption, CD, and LD bands of chlorophylls. Seven of these bands belonged to Chl a. Parameters of absorption bands of Chl a indicate that seven molecules represent a united cluster (heptamer) with exciton interactions, determining the spectrum of LhcIIb in the Chl a absorption region. Parameters of absorption bands of Chl b show the existence of three clusters: monomer (639.6 nm), dimer (645.2 and 647.4 nm), and trimer (649.8 and 654.1 nm). These clusters and their properties agree with the well-known structure of porphyrin groups of the LhcIIb subunit (Kuhlbrandt, 1994). A distorted ring of seven porphyrins in the stromal range of the subunit corresponds to Chl a heptamer; a separately located molecule near the N-terminal domain on the stromal side of the subunit corresponds to Chl b monomer; a dimer and a trimer of porphyrins in the lumenal range of the subunit correspond to the dimer and trimer of Chl b, respectively. The calculated lifetimes of the excitation energy (exciton) transfer in subunit and trimer of LhcIIb confirm this location of pigments. The geometry of the Chl a heptamer (mutual orientation of transition dipole moments) was determined by the steady-state Kasha–Tinoco approximation using parameters of individual bands of exciton splitting. The calculated parameters of mutual orientation of Chl a dipoles agree with the topography of the stromal porphyrins found by electron crystallography (Kuhlbrandt, 1994). A structural model of the granal multicentral macrocomplex of PSII (MPSII) is suggested. The lifetimes of the exciton migration between the main pigment–protein compartments of MPSII were calculated. The results of calculation are consistent with the structural model of the photosystem. The location of pigments provides for fast exciton hopping between Chl a clusters of neighboring proteins in the MPSII along the stromal surface within the membrane (5-25 psec) and between stacked membranes (40 psec) of chloroplast grana.     

Energy dissipation efficiency in the CP43 assembly intermediate complex of photosystem II

Photosystem II in oxygenic organisms is a large membrane bound rapidly turning over pigment protein complex. During its biogenesis, multiple assembly intermediates are formed, including the CP43-preassembly complex (pCP43). To understand the energy transfer dynamics in pCP43, we first engineered a His-tagged version of the CP43 in a CP47-less strain of the cyanobacterium Synechocystis 6803. Isolated pCP43 from this engineered strain was subjected to advanced spectroscopic analysis to evaluate its excitation energy dissipation characteristics. These included measurements of steady-state absorption and fluorescence emission spectra for which correlation was tested with Stepanov relation. Comparison of fluorescence excitation and absorptance spectra determined that efficiency of energy transfer from β-carotene to chlorophyll a is 39 %. Time-resolved fluorescence images of pCP43-bound Chl a were recorded on streak camera, and fluorescence decay dynamics were evaluated with global fitting. These demonstrated that the decay kinetics strongly depends on temperature and buffer used to disperse the protein sample and fluorescence decay lifetime was estimated in 3.2–5.7 ns time range, depending on conditions. The pCP43 complex was also investigated with femtosecond and nanosecond time-resolved absorption spectroscopy upon excitation of Chl a and β-carotene to reveal pathways of singlet excitation relaxation/decay, Chl a triplet dynamics and Chl a → β-carotene triplet state sensitization process. The latter demonstrated that Chl a triplet in the pCP43 complex is not efficiently quenched by carotenoids. Finally, detailed kinetic analysis of the rise of the population of β-carotene triplets determined that the time constant of the carotenoid triplet sensitization is 40 ns.     

Isolation and Characterization of Photosystem Ⅱ of Porphyra yezoensis Ueda

The thylakoid membranes were isolated and purified from gametophyte of Porphyra yezoensis Ueda (P. yezoensis) by sucrose density gradient ultracentrifugation. After P. yezoensis gametophyte thylakoid membranes were solubilized with SDS, the photosystem Ⅱ (PSⅡ) particles were isolated and purified. The activity of PSⅡ     

Differential changes in degradation of chlorophyll-protein complexes of photosystem I and photosystem II during flag leaf senescence of rice.

The stability of chlorophyll-protein complexes of photosystem I (PSI) and photosystem II (PSII) was investigated by chlorophyll (Chl) fluorescence spectroscopy, absorption spectra and native green gel separation system during flag leaf senescence of two rice varieties (IIyou 129 and Shanyou 63) grown under outdoor conditions. During leaf senescence, photosynthetic CO(2) assimilation rate, carboxylase activity of Rubisco, chlorophyll and carotenoids contents, and the chlorophyll a/b ratio decreased significantly. The 77 K Chl fluorescence emission spectra of thylakoid membranes from mature leaves had two peaks at around 685 and 735 nm emitting mainly from PSII and PSI, respectively. The total Chl fluorescence yields of PSI and PSII decreased significantly with senescence progressing. However, the decrease in the Chl fluorescence yield of PSI was greater than in the yield of PSII, suggesting that the rate of degradation in chlorophyll-protein complexes of PSI was greater than in chlorophyll-protein complexes of PSII. The fluorescence yields for all chlorophyll-protein complexes decreased significantly with leaf senescence in two rice varieties but the extents of their decrease were significantly different. The greatest decrease in the Chl fluorescence yield was in PSI core, followed by LHCI, CP47, CP43, and LHCII. These results indicate that the rate of degradation for each chlorophyll-protein complex was different and the order for the stability of chlorophyll-protein complexes during leaf senescence was: LHCII>CP43>CP47>LHCI>PSI core, which was partly supported by the green gel electrophoresis of the chlorophyll-protein complexes.     

Prolonged incubation with low concentrations of mercury alters energy transfer and chlorophyll (Chl) a protein complexes in Synechococcus 6301: changes in Chl a absorption and emission characteristics and loss of the F695 emission band

Synechococcus PCC 6301 cells grown in the presence of low sublethal levels of (about 2 m) mercury induced alterations in chlorophyll (Chl) a absorption without significant alterations in phycocyanin. Chl a fluorescence emission in Hg²⁺ -raised cells showed a large (about 18 nm) blue shift in the peak emission. No major spectral changes in phycobilisome (PBsome) emission characteristic were noticed, indicating major structural alterations in Chl-protein complexes by incubation with Hg²⁺ ions. Low temperature (77 K) emission spectra of cells grown in the presence of Hg²⁺ showed a loss of the characteristic Chl a emission band at 695 nm (F695), which is known to be linked to photosystem II photochemistry and to originate from the Chl a of core antenna polypeptide CP 47 of photosystem II. The SDS-PAGE polypeptide profile of thylakoids indicates a loss of a polypeptide(s) with a molecular mass between 40 and 60 k Da by Hg²⁺ incubation of cells. Our results suggest that prolonged incubation of Synechococcus 6301 cells with low concentrations of Hg²⁺ affects the Chl a spectral properties and the structure of Chl-protein complexes.     

Isolation and Characterization of an Oxygen Evolving Photosystem 2 Core Complex from the Thermophilic Cyanobacterium Mastigocladus laminosus

A novel purification procedure was developed for the isolation of oxygen evolving photosystem 2 (PS2) from Mastigocladus laminosus. The isolation procedure involves dodecyl maltoside extraction followed by column chromatography using anion exchange resins. The isolated PS2 reaction center (RC) was analyzed for its biochemical and biophysical characteristics. Analysis by SDS polyacrylamide gel electrophoresis revealed that the complex contained five intrinsic membrane proteins (CP 47, CP 43, D1, D2, and cyt b ₅₅₉) and at least three low molecular mass proteins. The complex exhibited high rates of oxygen evolution [333 mmol(O₂) kg^–1(Chl) s^–1] in the presence of 2.5 mM 2,6-dimethylbenzoquinone (DMBQ) as an artificial electron acceptor. The red chlorophyll a absorption peak of this complex was observed at 673.5±0.2 nm. The isolated PS2 core complex was free of photosystem 1 as inferred from its SDS-PAGE and fluorescence spectrum. The electron transfer properties of the Mastigocladus cells and the purified PS2 core complex were further probed by measuring thermoluminescence signals, which indicated the presence of a primary quinone electron acceptor (Q_A) in the purified PS2 core complex.     

Effects of lanthanum and calcium on photoelectron transport activity and the related protein complexes in chloroplast of cucumber leaves

The effects of lanthanum and calcium ions on electron transport, dichlorephenol indophenol (DCIP) photoreduction, and oxygen evolution activities in chloroplast from cucumber (Cucumis satives L.) were determined. The lanthanum inhibited the whole electron chain-transport activity of chloroplast. DCIP photoreduction and oxygen evolution activities of the photosystem I (PSII) also decrease after treatment with La³⁺. But the diminished activities of PSII and chloroplast caused by La³⁺ could be reversed by Ca²⁺ and even became higher than the control level. The concentration analysis of related protein complexes to photoelectron transport in chloroplast included that La³⁺ induced the concentration of chlorophyll protein complexes increasing but caused some nonchlorophyll protein complexes to decompose partially. This increasing effect of La³⁺ on chlorophyll protein complexes results in the improvement of chlorophyll content, which will improve the absorption of photoelectron and energy transport in the process of photosynthesis.     

A study of molecular interactions in light-harvesting complexes LHCIIb, CP29, CP26 and CP24 by Stark effect spectroscopy

Electric field-induced absorption changes (electrochromism or Stark effect) of the light-harvesting PSII pigment-protein complexes LHCIIb, CP29, CP26 and CP24 were investigated. The results indicate the lack of strong intermolecular interactions in the chlorophyll a (Chl a) pools of all complexes. Characteristic features occur in the electronic spectrum of Chl b, which reflect the increased values of dipole moment and polarizability differences between the ground and excited states of interacting pigment systems. The strong Stark signal recorded for LHCIIb at 650-655 nm is much weaker in CP29, where it is replaced by a unique Stark band at 639 nm. Electrochromism of Chl b in CP26 and CP24 is significantly weaker but increased electrochromic parameters were also noticed for the Chl b transition at 650 nm. The spectra in the blue region are dominated by xanthophylls. The differences in Stark spectra of Chl b are linked to differences in pigment content and organization in individual complexes and point to the possibility of electron exchange interactions between energetically similar and closely spaced Chl b molecules.     

The chlorophyll-protein complexes of Acetabularia. A novel chlorophyll ab complex which forms oligomers

(1) Five minor chlorophyll-protein complexes were isolated from thylakoid membranes of the green alga Acetabularia by SDS-polyacrylamide gel electrophoresis, after SDS or octylglucoside solubilization. None of them were related to CP I (Photosystem I reaction center core) or CP II (chlorophyll $\text{a}\text{b}$ light-harvesting complex). (2) Two complexes (CPa-1 and CPa-2) contained only chlorophyll (Chl) a, with absorption maxima of 673 and 671 nm, and fluorescence emission maxima of 683 nm compared to 676 nm for CP II. The complexes had apparent molecular masses of 43–47 and 38–40 kDa, and contained a single polypeptide of 41 and 37 kDa, respectively. They each account for about 3% of the total chlorophyll. (3) Three complexes had identical spectra, with Chl $\text{a}\text{b}$ ratios of 3–4 compared to 2 for thylakoid membranes, and a pronounced shoulder around 485 nm indicating enrichment in carotenoids. One of them was the complex ‘CP 29’ (Camm, E.L. and Green, B.R. (1980) Plant Physiol. 66, 428–432) and the other two were slightly different oligomeric forms of CP 29. They could be formed from CP 29 during reelectrophoresis; but about half the complex was isolated originally in an oligomeric form. Together they account for at least 7% of the total chlorophyll. Their function is unknown.     

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 μmol O₂/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 μmol O₂/mg Chl/h in the absence and presence of CaCl₂, 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.