Cloning, expression of the psbU gene, and functional studies of the recombinant 12-kDa protein of photosystem II from a red alga Cyanidium caldarium.

The encoding extrinsic 12-kDa protein of oxygen-evolving PS II complex from a red alga, Cyanidium caldarium, was cloned and sequenced by means of PCR and a rapid amplification of cDNA ends (RACE) procedure. The gene encodes a putative polypeptide of 154 amino acids with a calculated molecular mass of 16,714 Da. The full sequence of the protein includes two characteristic transit peptides, one for transfer across the chloroplast envelope and another for targeting into the thylakoid lumen. This indicates that the protein is encoded in the nuclear genome. The mature protein consists of 93 amino acids with a calculated molecular mass of 10,513 Da. The cloned gene was successfully expressed in Escherichia coli and the resulting protein was purified, reconstituted to CaCl2-washed PS II complex together with the other extrinsic proteins of 33 and 20 kDa and cyt c-550. The recombinant 12-kDa protein bound completely with the PSII complex, which resulted in a restoration of oxygen evolution equal to the level achieved by binding of the native 12-kDa protein.     

Distribution of the extrinsic proteins as a potential marker for the evolution of photosynthetic oxygen-evolving photosystem II

Distribution of photosystem II (PSII) extrinsic proteins was examined using antibodies raised against various extrinsic proteins from different sources. The results showed that a glaucophyte (Cyanophora paradoxa) having the most primitive plastids contained the cyanobacterial-type extrinsic proteins (PsbO, PsbV, PsbU), and the primitive red algae (Cyanidium caldarium) contained the red algal-type extrinsic proteins (PsO, PsbQ', PsbV, PsbU), whereas a prasinophyte (Pyraminonas parkeae), which is one of the most primitive green algae, contained the green algal-type ones (PsbO, PsbP, PsbQ). These suggest that the extrinsic proteins had been diverged into cyanobacterial-, red algal- and green algal-types during early phases of evolution after a primary endosymbiosis. This study also showed that a haptophyte, diatoms and brown algae, which resulted from red algal secondary endosymbiosis, contained the red algal-type, whereas Euglena gracilis resulted from green algal secondary endosymbiosis contained the green algal-type extrinsic proteins, suggesting that the red algal- and green algal-type extrinsic proteins have been retained unchanged in the different lines of organisms following the secondary endosymbiosis. Based on these immunological analyses, together with the current genome data, the evolution of photosynthetic oxygen-evolving PSII was discussed from a view of distribution of the extrinsic proteins, and a new model for the evolution of the PSII extrinsic proteins was proposed.     

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

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

Cross-reconstitution of various extrinsic proteins and photosystem II complexes from cyanobacteria, red algae and higher plants

Photosystem II (PSII) contains different extrinsic proteins required for oxygen evolution among different organisms. Cyanobacterial PSII contains the 33 kDa, 12 kDa proteins and cytochrome (cyt) c-550; red algal PSII contains a 20 kDa protein in addition to the three homologous cyanobacterial proteins; whereas higher plant PSII contains the 33 kDa, 23 kDa and 17 kDa proteins. In order to understand the binding and functional properties of these proteins, we performed cross-reconstitution experiments with combinations of PSII and extrinsic proteins from three different sources: higher plant (spinach), red alga (Cyanidium caldarium) and cyanobacterium (Synechococcus vulcanus). Among all of the extrinsic proteins, the 33 kDa protein is common to all of the organisms and is totally exchangeable in binding to PSII from any of the three organisms. Oxygen evolution of higher plant and red algal PSII was restored to a more or less similar level by binding of any one of the three 33 kDa proteins, whereas oxygen evolution of cyanobacterial PSII was restored to a larger extent with its own 33 kDa protein than with the 33 kDa protein from other sources. In addition to the 33 kDa protein, the red algal 20 kDa, 12 kDa proteins and cyt c-550 were able to bind to cyanobacterial and higher plant PSII, leading to a partial restoration of oxygen evolution in both organisms. The cyanobacterial 12 kDa protein and cyt c-550 partially bound to the red algal PSII, but this binding did not restore oxygen evolution. The higher plant 23 kDa and 17 kDa proteins bound to the cyanobacterial and red algal PSII only through non-specific interactions. Thus, only the red algal extrinsic proteins are partially functional in both the cyanobacterial and higher plant PSII, which implies a possible intermediate position of the red algal PSII during its evolution from cyanobacteria to higher plants.     

Comparison of binding and functional properties of two extrinsic components,Cyt c550 and a 12 kDa protein,in cyanobacterial PSII with those in red algal PSII

Cyt c550 and 12 kDa protein are two extrinsic proteins of photosystem II (PSII) found in cyanobacteria and some eukaryotic algae. The binding patterns of these two extrinsic proteins are different between cyanobacterial (Thermosynechococcus vulcanus) and red algal (Cyanidium caldarium) PSIIs [Shen and Inoue (1993) Biochemistry 32: 1825; Enami et al. (1998) Biochemistry 39: 2787]. In order to elucidate the possible causes responsible for these differences, we first cloned the psbV gene encoding Cyt c550 from a red alga, Cyanidium caldarium, which was compared with the homologous sequences from other organisms. Cross-reconstitution experiments were then performed with different combinations of the extrinsic proteins and the cyanobacterial or red algal PSII. (1). Both the cyanobacterial and red algal Cyt c550 bound directly to the cyanobacterial PSII, whereas none of them bound directly to the red algal PSII, indicating that direct binding of Cyt c550 to PSII principally depends on the structure of PSII intrinsic proteins but not that of Cyt c550 itself. (2). Cyt c550 was functionally exchangeable between the red algal and the cyanobacterial PSII, and the red algal 12 kDa protein functionally bound to the cyanobacterial PSII, whereas the cyanobacterial 12 kDa protein did not bind to the red algal PSII. (3). The antibody against the cyanobacterial or red algal 12 kDa protein reacted with its original one but not with the homologous protein from the other organism, whereas the antibody against the red algal Cyt c550 reacted with both cyanobacterial and red algal Cyt c550. These results imply that the structure and function of Cyt c550 have been largely conserved, whereas those of the 12 kDa protein have been changed, in the two organisms studied here.     

Comparison of the structure of the extrinsic 33 kDa protein from different organisms

The psbO gene encoding the extrinsic 33 kDa protein of oxygen-evolving photosystem II (PSII) complex was cloned and sequenced from a red alga, Cyanidium caldarium. The gene encodes a polypeptide of 333 residues, of which the first 76 residues served as transit peptides for transfer across the chloroplast envelope and thylakoid membrane. The mature protein consists of 257 amino acids with a calculated molecular mass of 28,290 Da. The sequence homology of the mature 33 kDa protein was 42.9-50.8% between the red alga and cyanobacteria, and 44.7-48.6% between the red alga and higher plants. The cloned gene was expressed in Escherichia coli, and the recombinant protein was purified, subjected to protease-treatments. The cleavage sites of the 33 kDa protein by chymotrypsin or V8 protease were determined and compared among a cyanobacterium (Synechococcus elongatus), a euglena (Euglena gracilis), a green alga (Chlamydomonas reinhardtii) and two higher plants (Spinacia oleracea and Oryza sativa). The cleavage sites by chymotrypsin were at 156F and 190F for the cyanobacterium, 159M, 160F and 192L for red alga, 11Y and 151F for euglena, 10Yand 150F for green alga, and 16Y for spinach, respectively. The cleavage sites by V8 protease were at 181E (cyanobacterium), 182E and 195E (red alga), 13E, 67E, 69E, 153D and 181E (euglena), 176E and 180E (green alga), and 18E or 19E (higher plants). Since most of the residues at these cleavage sites were conserved among the six organisms, the results indicate that the structure of the 33 kDa protein, at least the structure based on the accessibility by proteases, is different among these organisms. In terms of the cleavage sites, the structure of the 33 kDa protein can be divided into three major groups: cyanobacterial and red algal-type has cleavage sites at residues around 156-195, higher plant-type at residues 16-19, and euglena and green algal-type at residues of both cyanobacterial and higher plant-types.     

Cross-reconstitution of the extrinsic proteins and Photosystem II complexes from Chlamydomonas reinhardtii and Spinacia oleracea

Cross-reconstitution of the extrinsic proteins and Photosystem II (PS II) from a green alga, Chlamydomonas reinhardtii, and a higher plant,Spinacia oleracea, was performed to clarify the differences of binding properties of the extrinsic proteins between these two species of organisms. (1) Chlamydomonas PsbP and PsbQ directly bound to Chlamydomonas PS II independent of the other extrinsic proteins but not to spinach PS II. (2) Chlamydomonas PsbP and PsbQ directly bound to the functional sites of Chlamydomonas PS II independent of the origins of PsbO, while spinach PsbP and PsbQ only bound to non-functional sites on Chlamydomonas PS II. (3) Both Chlamydomonas PsbP and spinach PsbP functionally bound to spinach PS II in the presence of spinach PsbO. (4) While Chlamydomonas PsbP functionally bound to spinach PS II in the presence of Chlamydomonas PsbO, spinach PsbP bound loosely to spinach PS II in the presence of Chlamydomonas PsbO with no concomitant restoration of oxygen evolution. (5) Chlamydomonas PsbQ bound to spinach PS II in the presence of Chlamydomonas PsbP and PsbO or spinach PsbO but not to spinach PS II in the presence of spinach PsbP and Chlamydomonas PsbO or spinach PsbO. (6) Spinach PsbQ did not bind to spinach PS II in the presence of Chlamydomonas PsbO and PsbP. On the basis of these results, we showed a simplified scheme for binding patterns of the green algal and higher plant extrinsic proteins with respective PS II.     

Organisation of Photosystem I and Photosystem II in red alga Cyanidium caldarium: encounter of cyanobacterial and higher plant concepts

Structure and organisation of Photosystem I and Photosystem II isolated from red alga Cyanidium caldarium was determined by electron microscopy and single particle image analysis. The overall structure of Photosystem II was found to be similar to that known from cyanobacteria. The location of additional 20 kDa (PsbQ') extrinsic protein that forms part of the oxygen evolving complex was suggested to be in the vicinity of cytochrome c-550 (PsbV) and the 12 kDa (PsbU) protein. Photosystem I was determined as a monomeric unit consisting of PsaA/B core complex with varying amounts of antenna subunits attached. The number of these subunits was seen to be dependent on the light conditions used during cell cultivation. The role of PsaH and PsaG proteins of Photosystem I in trimerisation and antennae complexes binding is discussed.     

Structures and functions of the extrinsic proteins of photosystem II from different species

This minireview presents a summary of information available on the variety and binding properties of extrinsic proteins that form the oxygen-evolving complex of photosystem II (PSII) of cyanobacteria, red alga, diatom, green alga, euglena, and higher plants. In addition, the structure and function of extrinsic PsbO, PsbV, and PsbU proteins are summarized based on the crystal structure of thermophilic cyanobacterial PSII together with biochemical and genetic studies from various organisms.     

Kojic Acid Oxidase

Photosystem II (PSII), which catalyzes photosynthetic water oxidation, is composed of more than 20 subunits, including membrane-intrinsic and -extrinsic proteins. The extrinsic proteins of PSII shield the catalytic Mn₄CaO₅ cluster from exogenous reductants and serve to optimize oxygen evolution at physiological ionic conditions. These proteins include PsbO, found in all oxygenic organisms, PsbP and PsbQ, specific to higher plants and green algae, and PsbU, PsbV, CyanoQ, and CyanoP in cyanobacteria. Furthermore, red algal PSII has PsbQ′ in addition to PsbO, PsbV, and PsbU, and diatoms have Psb31 in supplement to red algal-type extrinsic proteins, exemplifying the functional divergence of these proteins during evolution. This review provides an updated summary of recent findings on PSII extrinsic proteins and discusses their binding, function, and evolution within various photosynthetic organisms.     

Absence of the PsbQ protein results in destabilization of the PsbV protein and decreased oxygen evolution activity in cyanobacterial photosystem II

We have previously reported that cyanobacterial photosystem II (PS II) contains a protein homologous to PsbQ, the extrinsic 17-kDa protein found in higher plant and green algal PS II (Kashino, Y., Lauber, W. M., Carroll, J. A., Wang, Q., Whitmarsh, J., Satoh, K., and Pakrasi, H. B. (2002) Biochemistry 41, 8004-8012) and that it has regulatory role(s) on the water oxidation machinery (Thornton, L. E., Ohkawa, H., Roose, J. L., Kashino, Y., Keren, N., and Pakrasi, H. B. (2004) Plant Cell 16, 2164-2175). In this work, the localization and the function of PsbQ were assessed using the cyanobacterium Synechocystis sp. PCC 6803. From the predicted sequence, cyanobacterial PsbQ is expected to be a lipoprotein on the luminal side of the thylakoid membrane. Indeed, experiments in this work show that upon Triton X-114 fractionation of thylakoid membranes, PsbQ partitioned in the hydrophobic phase, and trypsin digestion revealed that PsbQ was highly exposed to the luminal space of thylakoid membranes. Detailed functional assays were conducted on the psbQ deletion mutant (DeltapsbQ) to analyze its water oxidation machinery. PS II complexes purified from DeltapsbQ mutant cells had impaired oxygen evolution activity and were remarkably sensitive to NH(2)OH, which indicates destabilization of the water oxidation machinery. Additionally, the cytochrome c(550) (PsbV) protein partially dissociated from purified DeltapsbQ PS II complexes, suggesting that PsbQ contributes to the stability of PsbV in cyanobacterial PS II. Therefore, we conclude that the major function of PsbQ is to stabilize the PsbV protein, thereby contributing to the protection of the catalytic Mn(4)-Ca(1)-Cl(x) cluster of the water oxidation machinery.     

Oxygen-evolving extrinsic proteins (PsbO,P,Q,R): bioinformatic and functional analysis

The water-splitting and oxygen-evolving (OE) reaction is carried out by a large multisubunit protein complex, Photosystem II (PSII), that has two distinct regions: a membrane intrinsic-region that includes most of the PSII subunits and a lumenal extrinsic-region that is in close association to the manganese catalytic center. The recently determined PSII 3D structures from cyanobacteria provide a considerable amount of new knowledge about the OE architecture (K.N. Ferreira, T.M. Iverson, K. Maghlaoui, J. Barber, S. Iwata, Architecture of the photosynthetic oxygen-evolving center, Science 303 (2004) 1831-1838; B. Loll, J. Kern, W. Saenger, A. Zouni, J. Biesiadka, Towards complete cofactor arrangement in the 3.0 A resolution structure of photosystem II, Nature 438 (2005) 1040-1044). Most of the intrinsic core PSII polypeptides have been well conserved through evolution from ancient cyanobacteria to modern plants, keeping the essence of PSII light driven reactions from prokaryotes to eukaryotes; but what is striking is the large number of changes that have occurred in the oxygen-evolving extrinsic proteins (OEEp) associated to PSII lumenal side. For unknown reasons plant PSII has required the "invention" of three OEEps: PsbP (23 kDa), PsbQ (16 kDa) and PsbR (10 kDa); associated to the ubiquitous OEEp PsbO (33 kDa). This set of proteins seems to be required in plants for the full activity and stability of the OE center in vivo, but their specific function is not clear. In this paper, bioinformatics and functional data show that the OEEps present in plants and green algae are very distinct from their prokaryotic counterparts. Moreover, clear differences are found for PsbQ from higher plants and green algae; and a relationship has been found between PsbR and the Mn cluster.     

Oxygen-evolving extrinsic proteins (PsbO,P,Q,R): Bioinformatic and functional analysis

The water-splitting and oxygen-evolving (OE) reaction is carried out by a large multisubunit protein complex, Photosystem II (PSII), that has two distinct regions: a membrane intrinsic-region that includes most of the PSII subunits and a lumenal extrinsic-region that is in close association to the manganese catalytic center. The recently determined PSII 3D structures from cyanobacteria provide a considerable amount of new knowledge about the OE architecture (K.N. Ferreira, T.M. Iverson, K. Maghlaoui, J. Barber, S. Iwata, Architecture of the photosynthetic oxygen-evolving center, Science 303 (2004) 1831-1838; B. Loll, J. Kern, W. Saenger, A. Zouni, J. Biesiadka, Towards complete cofactor arrangement in the 3.0 A resolution structure of photosystem II, Nature 438 (2005) 1040-1044). Most of the intrinsic core PSII polypeptides have been well conserved through evolution from ancient cyanobacteria to modern plants, keeping the essence of PSII light driven reactions from prokaryotes to eukaryotes; but what is striking is the large number of changes that have occurred in the oxygen-evolving extrinsic proteins (OEEp) associated to PSII lumenal side. For unknown reasons plant PSII has required the “invention” of three OEEps: PsbP (23 kDa), PsbQ (16 kDa) and PsbR (10 kDa); associated to the ubiquitous OEEp PsbO (33 kDa). This set of proteins seems to be required in plants for the full activity and stability of the OE center in vivo, but their specific function is not clear. In this paper, bioinformatics and functional data show that the OEEps present in plants and green algae are very distinct from their prokaryotic counterparts. Moreover, clear differences are found for PsbQ from higher plants and green algae; and a relationship has been found between PsbR and the Mn cluster.     

PsbU provides a stable architecture for the oxygen-evolving system in cyanobacterial photosystem II

PsbU is a lumenal peripheral protein in the photosystem II (PS II) complex of cyanobacteria and red algae. It is thought that PsbU is replaced functionally by PsbP or PsbQ in plant chloroplasts. After the discovery of PsbP and PsbQ homologues in cyanobacterial PS II [Thornton et al. (2004) Plant Cell 16, 2164-2175], we investigated the function of PsbU using a psbU deletion mutant (DeltaPsbU) of Synechocystis 6803. In contrast to the wild type, DeltaPsbU did not grow when both Ca2+ and Cl- were eliminated from the growth medium. When only Ca2+ was eliminated, DeltaPsbU grew well, whereas when Cl- was eliminated, the growth rate was highly suppressed. Although DeltaPsbU grew normally in the presence of both ions under moderate light, PS II-related disorders were observed as follows. (1) The mutant cells were highly susceptible to photoinhibition. (2) Both the efficiency of light utilization under low irradiance and the chlorophyll-specific maximum rate of oxygen evolution in DeltaPsbU cells were 60% lower than those of the wild type. (3) The decay of the S2 state in DeltaPsbU cells was decelerated. (4) In isolated PS II complexes from DeltaPsbU cells, the amounts of the other three lumenal extrinsic proteins and the electron donation rate were drastically decreased, indicating that the water oxidation system became significantly labile without PsbU. Furthermore, oxygen-evolving activity in DeltaPsbU thylakoid membranes was highly suppressed in the absence of Cl-, and 60% of the activity was restored by NO3- but not by SO4(2-), indicating that PsbU had functions other than stabilizing Cl-. On the basis of these results, we conclude that PsbU is crucial for the stable architecture of the water-splitting system to optimize the efficiency of the oxygen evolution process.     

The PsbQ′ protein affects the redox potential of the Q<Subscript>A</Subscript> in photosystem II

Red alga contains four extrinsic proteins in photosystem II (PSII), which are PsbO, PsbV, PsbU, and PsbQ′. Except for the PsbQ′, the composition is the same in cyanobacterial PSII. Reconstitution analysis of cyanobacterial PSII has shown that oxygen-evolving activity does not depend on the presence of PsbQ′. Recently, the structure of red algal PSII was elucidated. However, the role of PsbQ′ remains unknown. In this study, the function of the acceptor side of PSII was analyzed in PsbQ′-reconstituted PSII by redox titration of Q_A and thermoluminescence. The redox potential of Q_A was positively shifted when PsbQ′ was attached to the PSII. The positive shift of Q_A is thought to cause a decrease in the amount of triplet chlorophyll in PSII. On the basis of these results, we propose that PsbQ′ has a photoprotective function when irradiated with strong light.     

Homologs of plant PsbP and PsbQ proteins are necessary for regulation of photosystem ii activity in the cyanobacterium Synechocystis 6803

The mechanism of oxygen evolution by photosystem II (PSII) has remained highly conserved during the course of evolution from ancestral cyanobacteria to green plants. A cluster of manganese, calcium, and chloride ions, whose binding environment is optimized by PSII extrinsic proteins, catalyzes this water-splitting reaction. The accepted view is that in plants and green algae, the three extrinsic proteins are PsbO, PsbP, and PsbQ, whereas in cyanobacteria, they are PsbO, PsbV, and PsbU. Our previous proteomic analysis established the presence of a PsbQ homolog in the cyanobacterium Synechocystis 6803. The current study additionally demonstrates the presence of a PsbP homolog in cyanobacterial PSII. Both psbP and psbQ inactivation mutants exhibited reduced photoautotrophic growth as well as decreased water oxidation activity under CaCl(2)-depleted conditions. Moreover, purified PSII complexes from each mutant had significantly reduced activity. In cyanobacteria, one PsbQ is present per PSII complex, whereas PsbP is significantly substoichiometric. These findings indicate that both PsbP and PsbQ proteins are regulators that are necessary for the biogenesis of optimally active PSII in Synechocystis 6803. The new picture emerging from these data is that five extrinsic PSII proteins, PsbO, PsbP, PsbQ, PsbU, and PsbV, are present in cyanobacteria, two of which, PsbU and PsbV, have been lost during the evolution of green plants.     

Isolation and characterization of oxygen-evolving thylakoid membranes and photosystem II particles from a glaucocystophyte, Cyanophora paradoxa

Conditions for preparing oxygen-evolving thylakoid membranes and PSII complexes, and those for observing the PSII activity were investigated in a glaucocystophyte, Cyanophora paradoxa. The active thylakoid membranes were isolated either with a medium containing glycerol or with that containing high concentrations of sucrose, phosphate, and citrate. Active PSII particles were solubilized by octyl-beta-D-glucoside from thylakoid membranes and were separated by sucrose density gradient centrifugation. The thylakoid membranes and PSII particles showed an oxygen-evolving activity only in high-ionic-strength media. The extrinsic 33 kDa protein (PsbO) and the cytochrome c(550) (PsbV) were found to be present in the PSII particles as in cyanobacteria or red algae, but no 12 kDa protein (PsbU) was detected. The PsbO protein was classified as a land-plant type by its N-terminal amino acid sequence.     

Novel Features of Eukaryotic Photosystem II Revealed by Its Crystal Structure Analysis from a Red Alga

Photosystem II (PSII) catalyzes light-induced water splitting, leading to the evolution of molecular oxygen indispensible for life on the earth. The crystal structure of PSII from cyanobacteria has been solved at an atomic level, but the structure of eukaryotic PSII has not been analyzed. Because eukaryotic PSII possesses additional subunits not found in cyanobacterial PSII, it is important to solve the structure of eukaryotic PSII to elucidate their detailed functions, as well as evolutionary relationships. Here we report the structure of PSII from a red alga Cyanidium caldarium at 2.76 Å resolution, which revealed the structure and interaction sites of PsbQ′, a unique, fourth extrinsic protein required for stabilizing the oxygen-evolving complex in the lumenal surface of PSII. The PsbQ′ subunit was found to be located underneath CP43 in the vicinity of PsbV, and its structure is characterized by a bundle of four up-down helices arranged in a similar way to those of cyanobacterial and higher plant PsbQ, although helices I and II of PsbQ′ were kinked relative to its higher plant counterpart because of its interactions with CP43. Furthermore, two novel transmembrane helices were found in the red algal PSII that are not present in cyanobacterial PSII; one of these helices may correspond to PsbW found only in eukaryotic PSII. The present results represent the first crystal structure of PSII from eukaryotic oxygenic organisms, which were discussed in comparison with the structure of cyanobacterial PSII.