Tight association of CpcL with photosystem I in Anabaena sp. PCC 7120 grown under iron-deficient conditions.

Phycobilisomes (PBSs), which are huge pigment-protein complexes displaying distinctive color variations, bind to photosystem cores for excitation-energy transfer. It is known that isolation of supercomplexes consisting of PBSs and photosystem I (PSI) or PBSs and photosystem II is challenging due to weak interactions between PBSs and the photosystem cores. In this study, we succeeded in purifying PSI-monomer-PBS and PSI-dimer-PBS supercomplexes from the cyanobacterium Anabaena sp. PCC 7120 grown under iron-deficient conditions by anion-exchange chromatography, followed by trehalose density gradient centrifugation. The absorption spectra of the two types of supercomplexes showed apparent bands originating from PBSs, and their fluorescence-emission spectra exhibited characteristic peaks of PBSs. Two-dimensional blue-native (BN)/SDS-PAGE of the two samples showed a band of CpcL, which is a linker protein of PBS, in addition to PsaA/B. Since interactions of PBSs with PSI are easily dissociated during BN-PAGE using thylakoids from this cyanobacterium grown under iron-replete conditions, it is suggested that iron deficiency for Anabaena induces tight association of CpcL with PSI, resulting in the formation of PSI-monomer-PBS and PSI-dimer-PBS supercomplexes. Based on these findings, we discuss interactions of PBSs with PSI in Anabaena.

Isolation and characterization of trimeric and monomeric PSI cores from Acaryochloris marina MBIC11017.

Photosystem I (PSI) catalyzes light-induced electron-transfer reactions and has been observed to exhibit various oligomeric states and different energy levels of chlorophylls (Chls) in response to oligomerization. However, the biochemical and spectroscopic properties of a PSI monomer containing Chls d are not well understood. In this study, we successfully isolated and characterized PSI monomers from the cyanobacterium Acaryochloris marina MBIC11017, and compared their properties with those of the A. marina PSI trimer. The PSI trimers and monomers were prepared using trehalose density gradient centrifugation after anion-exchange and hydrophobic interaction chromatography. The polypeptide composition of the PSI monomer was found to be consistent with that of the PSI trimer. The absorption spectrum of the PSI monomer showed the Qy band of Chl d at 704 nm, which was blue-shifted from the peak at 707 nm observed in the PSI-trimer spectrum. The fluorescence-emission spectrum of the PSI monomer measured at 77 K exhibited a peak at 730 nm without a broad shoulder in the range of 745-780 nm, which was clearly observed in the PSI-trimer spectrum. These spectroscopic properties of the A. marina PSI trimer and monomer suggest different formations of low-energy Chls d between the two types of PSI cores. Based on these findings, we discuss the location of low-energy Chls d in A. marina PSIs.

Development of serial X-ray fluorescence holography for radiation-sensitive protein crystals.

X-ray fluorescence holography (XFH) is a powerful atomic resolution technique capable of directly imaging the local atomic structure around atoms of a target element within a material. Although it is theoretically possible to use XFH to study the local structures of metal clusters in large protein crystals, the experiment has proven difficult to perform, especially on radiation-sensitive proteins. Here, the development of serial X-ray fluorescence holography to allow the direct recording of hologram patterns before the onset of radiation damage is reported. By combining a 2D hybrid detector and the serial data collection used in serial protein crystallography, the X-ray fluorescence hologram can be directly recorded in a fraction of the measurement time needed for conventional XFH measurements. This approach was demonstrated by obtaining the Mn Kα hologram pattern from the protein crystal Photosystem II without any X-ray-induced reduction of the Mn clusters. Furthermore, a method to interpret the fluorescence patterns as real-space projections of the atoms surrounding the Mn emitters has been developed, where the surrounding atoms produce large dark dips along the emitter-scatterer bond directions. This new technique paves the way for future experiments on protein crystals that aim to clarify the local atomic structures of their functional metal clusters, and for other related XFH experiments such as valence-selective XFH or time-resolved XFH.

Biochemical and spectroscopic characterization of PSI-LHCI from the red alga Cyanidium caldarium.

Light-harvesting complexes (LHCs) have been diversified in oxygenic photosynthetic organisms, and play an essential role in capturing light energy which is transferred to two types of photosystem cores to promote charge-separation reactions. Red algae are one of the groups of photosynthetic eukaryotes, and their chlorophyll (Chl) a-binding LHCs are specifically associated with photosystem I (PSI). In this study, we purified three types of preparations, PSI-LHCI supercomplexes, PSI cores, and isolated LHCIs, from the red alga Cyanidium caldarium, and examined their properties. The polypeptide bands of PSI-LHCI showed characteristic PSI and LHCI components without contamination by other proteins. The carotenoid composition of LHCI displayed zeaxanthins, ß-cryptoxanthins, and ß-carotenes. Among the carotenoids, zeaxanthins were enriched in LHCI. On the contrary, both zeaxanthins and ß-cryptoxanthins could not be detected from PSI, suggesting that zeaxanthins and ß-cryptoxanthins are bound to LHCI but not PSI. A Qy peak of Chl a in the absorption spectrum of LHCI was shifted to a shorter wavelength than those in PSI and PSI-LHCI. This tendency is in line with the result of fluorescence-emission spectra, in which the emission maxima of PSI-LHCI, PSI, and LHCI appeared at 727, 719, and 677 nm, respectively. Time-resolved fluorescence spectra of LHCI represented no 719 and 727-nm fluorescence bands from picoseconds to nanoseconds. These results indicate that energy levels of Chls around/within LHCIs and within PSI are changed by binding LHCIs to PSI. Based on these findings, we discuss the expression, function, and structure of red algal PSI-LHCI supercomplexes.

Structure of a monomeric photosystem I core associated with iron-stress-induced-A proteins from Anabaena sp. PCC 7120.

Iron-stress-induced-A proteins (IsiAs) are expressed in cyanobacteria under iron-deficient conditions. The cyanobacterium Anabaena sp. PCC 7120 has four isiA genes; however, their binding property and functional roles in PSI are still missing. We analyzed a cryo-electron microscopy structure of a PSI-IsiA supercomplex isolated from Anabaena grown under an iron-deficient condition. The PSI-IsiA structure contains six IsiA subunits associated with the PsaA side of a PSI core monomer. Three of the six IsiA subunits were identified as IsiA1 and IsiA2. The PSI-IsiA structure lacks a PsaL subunit; instead, a C-terminal domain of IsiA2 occupies the position of PsaL, which inhibits the oligomerization of PSI, leading to the formation of a PSI monomer. Furthermore, excitation-energy transfer from IsiAs to PSI appeared with a time constant of 55 ps. These findings provide insights into both the molecular assembly of the Anabaena IsiA family and the functional roles of IsiAs.

Cryo-electron microscopy structure of the intact photosynthetic light-harvesting antenna-reaction center complex from a green sulfur bacterium.

The photosynthetic reaction center complex (RCC) of green sulfur bacteria (GSB) consists of the membrane-imbedded RC core and the peripheric energy transmitting proteins called Fenna-Matthews-Olson (FMO). Functionally, FMO transfers the absorbed energy from a huge peripheral light-harvesting antenna named chlorosome to the RC core where charge separation occurs. In vivo, one RC was found to bind two FMOs, however, the intact structure of RCC as well as the energy transfer mechanism within RCC remain to be clarified. Here we report a structure of intact RCC which contains a RC core and two FMO trimers from a thermophilic green sulfur bacterium Chlorobaculum tepidum at 2.9 Å resolution by cryo-electron microscopy. The second FMO trimer is attached at the cytoplasmic side asymmetrically relative to the first FMO trimer reported previously. We also observed two new subunits (PscE and PscF) and the N-terminal transmembrane domain of a cytochrome-containing subunit (PscC) in the structure. These two novel subunits possibly function to facilitate the binding of FMOs to RC core and to stabilize the whole complex. A new bacteriochlorophyll (numbered as 816) was identified at the interspace between PscF and PscA-1, causing an asymmetrical energy transfer from the two FMO trimers to RC core. Based on the structure, we propose an energy transfer network within this photosynthetic apparatus.

Structural elucidation of vascular plant photosystem I and its functional implications.

In vascular plants, bryophytes and algae, the photosynthetic light reaction takes place in the thylakoid membrane where two transmembrane supercomplexes PSII and PSI work together with cytochrome b 6 f and ATP synthase to harvest the light energy and produce ATP and NADPH. Vascular plant PSI is a 600-kDa protein-pigment supercomplex, the core complex of which is partly surrounded by peripheral light-harvesting complex I (LHCI) that captures sunlight and transfers the excitation energy to the core to be used for charge separation. PSI is unique mainly in absorption of longer-wavelengths than PSII, fast excitation energy transfer including uphill energy transfer, and an extremely high quantum efficiency. From the early 1980s, a lot of effort has been dedicated to structural and functional studies of PSI-LHCI, leading to the current understanding of how more than 200 cofactors are kept at the correct distance and geometry to facilitate fast energy transfer in this supercomplex at an atomic level. In this review, we review the history of studies on vascular plant PSI-LHCI, summarise the present research progress on its structure, and present some new and further questions to be answered in future studies.

A unique photosystem I reaction center from a chlorophyll d-containing cyanobacterium Acaryochloris marina.

Photosystem I (PSI) is a large protein supercomplex that catalyzes the light-dependent oxidation of plastocyanin (or cytochrome c6 ) and the reduction of ferredoxin. This catalytic reaction is realized by a transmembrane electron transfer chain consisting of primary electron donor (a special chlorophyll (Chl) pair) and electron acceptors A0 , A1 , and three Fe4 S4 clusters, FX , FA , and FB . Here we report the PSI structure from a Chl d-dominated cyanobacterium Acaryochloris marina at 3.3 Å resolution obtained by single-particle cryo-electron microscopy. The A. marina PSI exists as a trimer with three identical monomers. Surprisingly, the structure reveals a unique composition of electron transfer chain in which the primary electron acceptor A0 is composed of two pheophytin a rather than Chl a found in any other well-known PSI structures. A novel subunit Psa27 is observed in the A. marina PSI structure. In addition, 77 Chls, 13 α-carotenes, two phylloquinones, three Fe-S clusters, two phosphatidyl glycerols, and one monogalactosyl-diglyceride were identified in each PSI monomer. Our results provide a structural basis for deciphering the mechanism of photosynthesis in a PSI complex with Chl d as the dominating pigments and absorbing far-red light.

Molecular organizations and function of iron-stress-induced-A protein family in Anabaena sp. PCC 7120.

Iron-stress-induced-A proteins (IsiAs) are expressed in cyanobacteria under iron-deficient conditions, and surround photosystem I (PSI) trimer with a ring formation. A cyanobacterium Anabaena sp. PCC 7120 has four isiA genes; however, it is unknown how the IsiAs are associated with PSI. Here we report on molecular organizations and function of the IsiAs in this cyanobacterium. A deletion mutant of the isiA1 gene was constructed, and the four types of thylakoids were prepared from the wild-type (WT) and ΔisiA1 cells under iron-replete (+Fe) and iron-deficient (-Fe) conditions. Immunoblotting analysis exhibits a clear expression of the IsiA1 in the WT-Fe. The PSI-IsiA1 supercomplex is found in the WT-Fe, and excitation-energy transfer from IsiA1 to PSI is verified by time-resolved fluorescence analyses. Instead of the IsiA1, both IsiA2 and IsiA3 are bound to PSI monomer in the ΔisiA1-Fe. These findings provide insights into multiple-expression system of the IsiA family in this cyanobacterium.

Time-resolved studies of metalloproteins using X-ray free electron laser radiation at SACLA.

BACKGROUND: The invention of the X-ray free-electron laser (XFEL) has provided unprecedented new opportunities for structural biology. The advantage of XFEL is an intense pulse of X-rays and a very short pulse duration (<10 fs) promising a damage-free and time-resolved crystallography approach. SCOPE OF REVIEW: Recent time-resolved crystallographic analyses in XFEL facility SACLA are reviewed. Specifically, metalloproteins involved in the essential reactions of bioenergy conversion including photosystem II, cytochrome c oxidase and nitric oxide reductase are described. MAJOR CONCLUSIONS: XFEL with pump-probe techniques successfully visualized the process of the reaction and the dynamics of a protein. Since the active center of metalloproteins is very sensitive to the X-ray radiation, damage-free structures obtained by XFEL are essential to draw mechanistic conclusions. Methods and tools for sample delivery and reaction initiation are key for successful measurement of the time-resolved data. GENERAL SIGNIFICANCE: XFEL is at the center of approaches to gain insight into complex mechanism of structural dynamics and the reactions catalyzed by biological macromolecules. Further development has been carried out to expand the application of time-resolved X-ray crystallography. This article is part of a Special Issue entitled Novel measurement techniques for visualizing 'live' protein molecules.

Probing structure-function relationships in early events in photosynthesis using a chimeric photocomplex.

The native core light-harvesting complex (LH1) from the thermophilic purple phototrophic bacterium Thermochromatium tepidum requires Ca2+ for its thermal stability and characteristic absorption maximum at 915 nm. To explore the role of specific amino acid residues of the LH1 polypeptides in Ca-binding behavior, we constructed a genetic system for heterologously expressing the Tch. tepidum LH1 complex in an engineered Rhodobacter sphaeroides mutant strain. This system contained a chimeric pufBALM gene cluster (pufBA from Tch. tepidum and pufLM from Rba. sphaeroides) and was subsequently deployed for introducing site-directed mutations on the LH1 polypeptides. All mutant strains were capable of phototrophic (anoxic/light) growth. The heterologously expressed Tch. tepidum wild-type LH1 complex was isolated in a reaction center (RC)-associated form and displayed the characteristic absorption properties of this thermophilic phototroph. Spheroidene (the major carotenoid in Rba. sphaeroides) was incorporated into the Tch. tepidum LH1 complex in place of its native spirilloxanthins with one carotenoid molecule present per αß-subunit. The hybrid LH1-RC complexes expressed in Rba. sphaeroides were characterized using absorption, fluorescence excitation, and resonance Raman spectroscopy. Site-specific mutagenesis combined with spectroscopic measurements revealed that α-D49, ß-L46, and a deletion at position 43 of the α-polypeptide play critical roles in Ca binding in the Tch. tepidum LH1 complex; in contrast, α-N50 does not participate in Ca2+ coordination. These findings build on recent structural data obtained from a high-resolution crystallographic structure of the membrane integrated Tch. tepidum LH1-RC complex and have unambiguously identified the location of Ca2+ within this key antenna complex.

Isolation and characterization of PSI-LHCI super-complex and their sub-complexes from a red alga Cyanidioschyzon merolae.

Photosystem I (PSI)-light-harvesting complex I (LHCI) super-complex and its sub-complexes PSI core and LHCI, were purified from a unicellular red alga Cyanidioschyzon merolae and characterized. PSI-LHCI of C. merolae existed as a monomer with a molecular mass of 580 kDa. Mass spectrometry analysis identified 11 subunits (PsaA, B, C, D, E, F, I, J, K, L, O) in the core complex and three LHCI subunits, CMQ142C, CMN234C, and CMN235C in LHCI, indicating that at least three Lhcr subunits associate with the red algal PSI core. PsaG was not found in the red algae PSI-LHCI, and we suggest that the position corresponding to Lhca1 in higher plant PSI-LHCI is empty in the red algal PSI-LHCI. The PSI-LHCI complex was separated into two bands on native PAGE, suggesting that two different complexes may be present with slightly different protein compositions probably with respective to the numbers of Lhcr subunits. Based on the results obtained, a structural model was proposed for the red algal PSI-LHCI. Furthermore, pigment analysis revealed that the C. merolae PSI-LHCI contained a large amount of zeaxanthin, which is mainly associated with the LHCI complex whereas little zeaxanthin was found in the PSI core. This indicates a unique feature of the carotenoid composition of the Lhcr proteins and may suggest an important role of Zea in the light-harvesting and photoprotection of the red algal PSI-LHCI complex.

Biochemical and structural study of Arabidopsis hexokinase 1.

Hexokinase 1 from Arabidopsis thaliana (AtHXK1) plays a dual role in glycolysis and sugar sensing for vital metabolic and physiological processes. The uncoupling of glucose signalling from glucose metabolism was demonstrated by the analysis of two mutants (AtHXK1(G104D) and AtHXK1(S177A)) that are catalytically inactive but still functional in signalling. In this study, substrate-binding experiments indicate that the two catalytically inactive mutants have a high affinity for glucose, and an ordered substrate-binding mechanism has been observed for wild-type AtHXK1. The structure of AtHXK1 was determined both in its inactive unliganded form and in its active glucose-bound form at resolutions of 1.8 and 2.0 Å, respectively. These structures reveal a domain rearrangement of AtHXK1 upon glucose binding. The 2.1 Šresolution structure of AtHXK1(S177A) in the glucose-bound form shows similar glucose-binding interactions as the wild type. A glucose-sensing network has been proposed based on these structures. Taken together, the results provide a structural explanation for the dual functions of AtHXK1.

Application of peptide gemini surfactants as novel solubilization surfactants for photosystems I and II of cyanobacteria.

We designed novel peptide gemini surfactants (PG-surfactants), DKDKC12K and DKDKC12D, which can solubilize Photosystem I (PSI) of Thermosynecoccus elongatus and Photosystem II (PSII) of Thermosynecoccus vulcanus in an aqueous buffer solution. To assess the detailed effects of PG-surfactants on the original supramolecular membrane protein complexes and functions of PSI and PSII, we applied the surfactant exchange method to the isolated PSI and PSII. Spectroscopic properties, light-induced electron transfer activity, and dynamic light scattering measurements showed that PSI and PSII could be solubilized not only with retention of the original supramolecular protein complexes and functions but also without forming aggregates. Furthermore, measurement of the lifetime of light-induced charge-separation state in PSI revealed that both surfactants, especially DKDKC12D, displayed slight improvement against thermal denaturation below 60 °C compared with that using ß-DDM. This degree of improvement in thermal resistance still seems low, implying that the peptide moieties did not interact directly with membrane protein surfaces. By conjugating an electron mediator such as methyl viologen (MV(2+)) to DKDKC12K (denoted MV-DKDKC12K), we obtained derivatives that can trap the generated reductive electrons from the light-irradiated PSI. After immobilization onto an indium tin oxide electrode, a cathodic photocurrent from the electrode to the PSI/MV-DKDKC12K conjugate was observed in response to the interval of light irradiation. These findings indicate that the PG-surfactants DKDKC12K and DKDKC12D provide not only a new class of solubilization surfactants but also insights into designing other derivatives that confer new functions on PSI and PSII.

Effects of phospholipase and lipase treatments on photosystem II core dimer from a thermophilic cyanobacterium.

Lipids are important components of transmembrane protein complexes. In order to study the roles of lipids in photosystem II (PSII), we treated the PSII core dimer complex from a thermophilic cyanobacterium Thermosynechococcus vulcanus with phospholipase A(2) (PLA(2)) and lipase, and examined their effects on PSII structure and function. PLA(2)-treatment decreased the content of phospholipid, phosphatidylglycerol (PG) by 59%, leading to a decrease of oxygen evolution by 40%. On the other hand, although treatment with lipase specifically decreased the content of monogalactosyldiacylglycerol (MGDG) by 52%, it decreased oxygen evolution only by 16%. This indicates that PG plays a more important role in PSII than MGDG. Both PLA(2)- and lipase-treatments induced neither the dissociation of PSII dimer, nor any loss of polypeptides. The degradation of PG resulted in a damage to the Q(B)-binding site as demonstrated from photoreduction activity of 2,6-dichlorophenolindophenol and chlorophyll fluorescence yields in the absence or presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and the dependencies of oxygen evolution on various electron acceptors before and after PLA(2)- or lipase-treatments. However, there were approximately three and five molecules of PG and MGDG per PSII reaction center left in the PSII dimeric complex after the PLA(2)- and lipase-treatments. These lipids are therefore bound to the interior of the protein matrix and resistant to the lipase treatments. The resistance of these lipids against PLA(2)- and lipase-treatments may be a specific feature of PSII from the thermophilic cyanobacterium, suggesting a possible correlation between binding of lipids and thermostability of PSII.

Self-assembling peptide inspired by a barnacle underwater adhesive protein.

An underwater bioadhesive generally comprises a multiprotein complex that provides a molecular basis for self-assembly. We report here a new class of self-assembling peptide inspired by a 20 kDa barnacle cement protein. Studies on the chemically synthesized 24-residue peptide have revealed that (1) it underwent irreversible self-assembly upon the addition of salt, (2) the self-assembly was started at a salt concentration close to that of seawater with noncovalent intermolecular interactions, (3) the self-assembled material resembled a macroscopic membrane of interwoven nanofilaments, (4) incubation in an alkaline pH range formed the intramolecular disulfide bond of a peptide molecule, thus triggering a conformation change of the molecule, and (5) conformational change of the building block promoted the formation of a nanofiber, resulting in the display of a three-dimensional meshlike mesoscopic structure with defined pores having a diameter of approximately 200 nm. The peptide is likely to provide a suitable basis for further development of peptide-based materials.

ATP-induced hexameric ring structure of the cyanobacterial circadian clock protein KaiC.

BACKGROUND: KaiA, KaiB and KaiC are cyanobacterial circadian clock proteins. KaiC contains two ATP/GTP-binding Walker's motif As, and mutations in these regions affect the clock oscillations. RESULTS: ATP induced the hexamerization of KaiC. The Km value for the ATP for the hexamerization was 1.9 micro m. Triphosphate nucleotides bound to the two Walker's motif As, and their binding functioned cooperatively for the hexamerization. An unhydrolysable substrate, 5'-adenylylimidodiphosphate (AMPPNP), also induced the hexamerization, indicating that nucleotide binding, but not its hydrolysis, is essential for the hexamerization. Mutations in each of the two Walker's motif As that affect the clock phenotype increased the Km value for ATP and inhibited the hexamerization. Thus, the KaiC hexamerization seems to be necessary for its clock function. The KaiC hexamer has the shape of a hexagonal pot with a diameter and height of approximately 100 A and with a relatively large cavity (73 A deep and 18-34 A wide) inside. This pot-shaped structure suggests that KaiC functions in a similar manner to F1-ATPase, helicase or ATP-dependent protease/chaperon, all of which have dynamic activities inside the central cavity of their hexameric rings. CONCLUSION: ATP-induced KaiC hexamerization is necessary for the clock function of KaiC.

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

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

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.