Cyt C550 Research Articles

The deep red state of photosystem II in Cyanidioschyzon merolae

We identified and characterised the deep red state (DRS), an optically-absorbing charge transfer state of PSII, which lies at lower energy than P680, in the red algae Cyanidioschyzon merolae by means of low temperature absorption and magnetic circular dichroism spectroscopies. The photoactive DRS has been previously studied in PSII of the higher plant Spinacia oleracea, and in the cyanobacterium Thermosynechococcus vulcanus. We found the DRS in PSII of C. merolae has similar spectral properties. Treatment of PSII with dithionite leads to reduction of cytochrome (cyt) b559 and the PsbV-based cyt c550 as well as the disassembly of the oxygen-evolving complex. Whereas the overall visible absorption spectrum of PSII was little affected, the DRS absorption in the reduced sample was no longer seen. This bleaching of the DRS is discussed in terms of a corresponding lack of a DRS feature in D1D2/cyt b559 reaction centre preparations of PSII.

Optical identification of the long-wavelength (700–1700 nm) electronic excitations of the native reaction centre, Mn4CaO5 cluster and cytochromes of photosystem II in plants and cyanobacteria

Visible/UV absorption in PS II core complexes is dominated by the chl-a absorptions, which extend to ~700nm. A broad 700–730nm PS II core complex absorption in spinach has been assigned [1] to a charge transfer excitation between ChlD1 and ChlD2. Emission from this state, which peaks at 780nm, has been seen [2] for both plant and cyanobacterial samples. We show that Thermosynechococcus vulcanus PS II core complexes have parallel absorbance in the 700–730nm region and similar photochemical behaviour to that seen in spinach. This establishes the low energy charge transfer state as intrinsic to the native PS II reaction centre. High-sensitivity MCD measurements made in the 700–1700nm region reveal additional electronic excitations at ~770nm and ~1550nm. The temperature and field dependence of MCD spectra establish that the system peaking near 1550nm is a heme-to-Fe(III) charge transfer excitation. These transitions have not previously been observed for cyt b559 or cyt c550. The distinctive characteristics of the MCD signals seen at 770nm allow us to assign absorption in this region to a dz2→dx2−y2 transition of Mn(III) in the Ca-Mn4O5 cluster of the oxygen evolving centre. Current measurements were performed in the S1 state. Detailed analyses of this spectral region, especially in higher S states, promise to provide a new window on models of water oxidation.

A High Redox Potential Form of Cytochrome c550 in Photosystem II from Thermosynechococcus elongatus

Cytochrome c(550) (cyt c(550)) is a component of photosystem II (PSII) from cyanobacteria, red algae, and some other eukaryotic algae. Its physiological role remains unclear. In the present work, measurements of the midpoint redox potential (E(m)) were performed using intact PSII core complexes preparations from a histidine-tagged PSII mutant strain of the thermophilic cyanobacterium Thermosynechococcus (T.) elongatus. When redox titrations were done in the absence of redox mediators, an E(m) value of +200 mV was obtained for cyt c(550). This value is ∼300 mV more positive than that previously measured in the presence of mediators (E(m) = -80 mV). The shift from the high potential form (E(m) = +200 mV) to the low potential form (E(m) = -80 mV) of cyt c(550) is attributed to conformational changes, triggered by the reduction of a component of PSII that is sequestered and out of equilibrium with the medium, most likely the Mn(4)Ca cluster. This reduction can occur when reduced low potential redox mediators are present or under highly reducing conditions even in the absence of mediators. Based on these observations, it is suggested that the E(m) of +200 mV obtained without mediators could be the physiological redox potential of the cyt c(550) in PSII. This value opens the possibility of a redox function for cyt c(550) in PSII.

Differences in the Interactions between the Subunits of Photosystem II Dependent on D1 Protein Variants in the Thermophilic Cyanobacterium Thermosynechococcus elongatus

The main cofactors involved in the oxygen evolution activity of Photosystem II (PSII) are located in two proteins, D1 (PsbA) and D2 (PsbD). In Thermosynechococcus elongatus, a thermophilic cyanobacterium, the D1 protein is encoded by either the psbA(1) or the psbA(3) gene, the expression of which is dependent on environmental conditions. It has been shown that the energetic properties of the PsbA1-PSII and those of the PsbA3-PSII differ significantly (Sugiura, M., Kato, Y., Takahashi, R., Suzuki, H., Watanabe, T., Noguchi, T., Rappaport, F., and Boussac, A. (2010) Biochim. Biophys. Acta 1797, 1491-1499). In this work the structural stability of PSII upon a PsbA1/PsbA3 exchange was investigated. Two deletion mutants lacking another PSII subunit, PsbJ, were constructed in strains expressing either PsbA1 or PsbA3. The PsbJ subunit is a 4-kDa transmembrane polypeptide that is surrounded by D1 (i.e. PsbA1), PsbK, and cytochrome b(559) (Cyt b(559)) in existing three-dimensional models. It is shown that the structural properties of the PsbA3/ΔPsbJ-PSII are not significantly affected. The polypeptide contents, the Cyt b(559) properties, and the proportion of PSII dimer were similar to those found for PsbA3-PSII. In contrast, in PsbA1/ΔPsbJ-PSII the stability of the dimer is greatly diminished, the EPR properties of the Cyt b(559) likely indicates a decrease in its redox potential, and many other PSII subunits are lacking. These results shows that the 21-amino acid substitutions between PsbA1 and PsbA3, which appear to be mainly conservative, must include side chains that are involved in a network of interactions between PsbA and the other PSII subunits.

Isolation and characterization of the oxygen-evolving photosystem II complex from the economical red alga Bangia fusco-purpurea

The highly pure and active photosystem II (PSII) complex was isolated from Bangia fusco-purpurea (Dillw) Lyngb., an important economic red alga in China, through two steps of sucrose density gradient ultracentrifugation and characterized by the room absorption and fluorescence emission spectra, DCIP (2,6-dichloroindophenol) reduction, and oxygen evolution rates. The PSII complex from B. fusco-purpurea had the characteristic absorption peaks of chlorophyll (Chl) a (436 and 676 nm) and typical fluorescence emission peak at 685 nm (Ex = 436 nm). Moreover, the acquired PSII complex displayed high oxygen evolution (139 μmol O2/(mg Chl h) in the presence of 2.5 mM 2,6-dimethybenzoqinone as an artificial acceptor and was active in photoreduction of DCIP (2,6-dichloroindophenol) by DPC (1,5-diphenylcarbazide) at 163 U/(mg Chl a h). SDS-PAGE also suggested that the purified PSII complex contained four intrinsic proteins (D1, D2, CP43, and CP47) and four extrinsic proteins (33-kD protein, 20-kD protein, cyt c-550, and 14-kD protein).

Spectroscopic and Functional Characterizations of Cyanobacterium Synechocystis PCC 6803 Mutants on and near the Heme Axial Ligand of Cytochrome b559 in Photosystem II

The functional role of cytochrome (cyt) b(559) in photosystem II (PSII) was investigated in H22K alpha and Y18S alpha cyt b(559) mutants of the cyanobacterium Synechocystis sp. PCC6803. H22K alpha and Y18S alpha cyt b(559) mutant carries one amino acid substitution on and near one of heme axial ligands of cyt b(559) in PSII, respectively. Both mutants grew photoautotrophically, assembled stable PSII, and exhibited the normal period-four oscillation in oxygen yield. However, both mutants showed several distinct chlorophyll a fluorescence properties and were more susceptible to photoinhibition than wild type. EPR results indicated the displacement of one of the two axial ligands to the heme of cyt b(559) in H22K alpha mutant reaction centers, at least in isolated reaction centers. The maximum absorption of cyt b(559) in Y18S alpha mutant PSII core complexes was shifted to 561 nm. Y18S alpha and H22K alpha mutant PSII core complexes contained predominately the low potential form of cyt b(559). The findings lend support to the concept that the redox properties of cyt b(559) are strongly influenced by the hydrophobicity and ligation environment of the heme. When the cyt b(559) mutations placed in a D1-D170A genetic background that prevents assembly of the manganese cluster, accumulation of PSII is almost completely abolished. Overall, our data support a functional role of cyt b(559) in protection of PSII under photoinhibition conditions in vivo.

Protein Unfolding:1H-NMR Studies of Paramagnetic Ferricytochrome c-550 from Horse Heart

Electronic transfer protein cytochrome c-550 from horse heart is studied in the unfolded state by means of paramagnetic 1H NMR. The protein contains 104 aminoacid residues and a heme group with low spin FeIII ion in the oxidized form of protein. The global secondary structure is of the α-helix type as occurs in the case of very other cytochromes c investigated such as cyt c-550 from Thiobacillus versutus or cyt c-551 from Pseudomonas aeruginosa. We have studied the coordination characteristic and electronic properties of heme iron horse heart ferricytochrome c-550 at increasing denaturing conditions (up to 3.1 M GuHCl and 288-323 K). The 1H T1 values of the signals were measured and some resonance assignments made based on EXSY experiments. The electronic structure of the iron(III) is discussed on the basis of the temperature dependence of the isotropic shifts and relaxation times. These results show that it is produced a change of spin, from low-spin iron(III) (2T2, S=1/2) in the folded state to high-spin iron(III) (6A1, S=5/2) in the unfolded state. It seems to be possible that in the opened structure the ferricyt c-550 loses one axial ligand (His/-) appearing the spin transition.

Extinction coefficients of cytochromes b559 and c550 of Thermosynechococcus elongatus and Cyt b559/PS II stoichiometry of higher plants

“Reduced minus oxidized” difference extinction coefficients Δ ɛ in the α-bands of Cyt b559 and Cyt c550 were determined by using functionally and structurally well-characterized PS II core complexes from the thermophilic cyanobacterium Thermosynechococcus elongatus. Values of 25.1 ± 1.0 mM −1 cm −1 and 27.0 ± 1.0 mM −1 cm −1 were obtained for Cyt b559 and Cyt c550, respectively. Anaerobic redox titrations covering the wide range from −250 up to +450 mV revealed that the heme groups of both Cyt b559 and Cyt c550 exhibit homogenous redox properties in the sample preparation used, with E m values at pH 6.5 of 244 ± 11 mV and −94 ± 21 mV, respectively. No HP form of Cyt b559 could be detected. Experiments performed on PS II membrane fragments of higher plants where the content of the high potential form of Cyt b559 was varied by special treatments (pH, heat) have shown that the α-band extinction of Cyt b559 does not depend on the redox form of the heme group. Based on the results of this study the Cyt b559/PSII stoichiometry is inferred to be 1:1 not only in thermophilic cyanobacteria as known from the crystal structure but also in PSII of plants. Possible interrelationships between the structure of the Q B site and the microenvironment of the heme group of Cyt b559 are discussed.

Redox potential of cytochrome c550 in the cyanobacterium Thermosynechococcus elongates

Cytochrome c550 (cyt c550) from photosystem II (PSII) exists in the PSII-bound form but can be released from PSII by treatment with divalent cations or Tris, yielding the isolated form. We calculated heme redox potentials ( E m) based on the crystal structures of cyt c550 by solving the Poisson–Boltzmann equation. In the isolated form, the calculated E m are −240 mV at pH 6.0 and −352 mV at pH 9.0. This pH-dependence is predominantly due to deprotonation of the heme-propionic group near Asn-49. In the PSII-bound form, the calculated E m was up-shifted by 160 mV versus the isolated form due to a conformational change of protein backbone, yielding E m = −84 mV.

Isolation and Characterization of Photosystem II of <italic>Porphyra yezoensis</italic> 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 particles was determined with DCIP (2,6-dichloroindophenol) photoreduction reaction. The composition of purified PSII particles was detected by SDS-PAGE. As a result, seven proteins including 55 kD protein, 47 kD protein, 43 kD protein, 33 kD protein, 31 kD protein, 29 kD protein, and 18 kD protein were found. Compared with PSII particles of higher plants and other algae, they were identified as D1/D2 complex, CP47, CP43, 33 kD protein, D1, D2 and cyt c-550 respectively. Besides, other three new proteins of 20 kD, 16 kD and 14 kD respectively were found. Among these extrinsic proteins, the 16 kD and 14 kD proteins had not been reported previously, and the 20 kD protein was found for the first time in multicellular red algae.

Extrinsic proteins of photosystem II: an intermediate member of PsbQ protein family in red algal PS II.

The oxygen-evolving photosystem II (PS II) complex of red algae contains four extrinsic proteins of 12 kDa, 20 kDa, 33 kDa and cyt c-550, among which the 20 kDa protein is unique in that it is not found in other organisms. We cloned the gene for the 20-kDa protein from a red alga Cyanidium caldarium. The gene consists of a leader sequence which can be divided into two parts: one for transfer across the plastid envelope and the other for transfer into thylakoid lumen, indicating that the gene is encoded by the nuclear genome. The sequence of the mature 20-kDa protein has low but significant homology with the extrinsic 17-kDa (PsbQ) protein of PS II from green algae Volvox Carteri and Chlamydomonas reinhardtii, as well as the PsbQ protein of higher plants and PsbQ-like protein from cyanobacteria. Cross-reconstitution experiments with combinations of the extrinsic proteins and PS IIs from the red alga Cy. caldarium and green alga Ch. reinhardtii showed that the extrinsic 20-kDa protein was functional in place of the green algal 17-kDa protein on binding to the green algal PS II and restoration of oxygen evolution. From these results, we conclude that the 20-kDa protein is the ancestral form of the extrinsic 17-kDa protein in green algal and higher plant PS IIs. This provides an important clue to the evolution of the oxygen-evolving complex from prokaryotic cyanobacteria to eukaryotic higher plants. The gene coding for the extrinsic 20-kDa protein was named psbQ' (prime).

Redox properties of the photosystem II cytochromes b559 and c550 in the cyanobacterium Thermosynechococcus elongatus.

Redox properties of cytochrome b559 (Cyt b559) and cytochrome c550 (Cyt c550) have been studied by using highly stable photosystem II (PSII) core complex preparations from a mutant strain of the thermophilic cyanobacterium Thermosynechococcus elongatus with a histidine tag on the CP43 protein of PSII. Two different redox potential forms for Cyt b559 are found in these preparations, with a midpoint redox potential ( E'(m)) of +390 mV in about half of the centers and +275 mV in the other half. The high-potential form, whose E'(m)is pH independent, can be converted into the lower potential form by Tris washing, mild heating or alkaline pH incubation. The E'(m) of the low-potential form is significantly higher than that found in other photosynthetic organisms and is not affected by pH. The possibility that the heme of Cyt b559 in T. elongatus is in a more hydrophobic environment is discussed. Cyt c550 has a higher E'(m)when bound to the PSII core (-80 mV at pH 6.0) than after its extraction from the complex (-240 mV at pH 6.0). The E'(m) of Cyt c550 bound to PSII is pH independent, while in the purified state an increase of about 58 mV/pH unit is observed when the pH decreases below pH 9.0. Thus, Cyt c550 seems to have a single protonateable group which influences the redox properties of the heme. From these electrochemical measurements and from EPR controls it is proposed that important changes in the solvent accessibility to the heme and in the acid-base properties of that protonateable group could occur upon the release of Cyt c550 from PSII.

Factors that determine the unusually low reduction potential of cytochrome c550 in cyanobacterial photosystem II.

A new purification protocol for cytochrome c550 (cyt c550) from His-tagged SYnechocYstis PCC 6803 photosystem II (PSII) was developed which allows the protein to be isolated in high yield and purity. Electron paramagnetic resonance spectroscopy of cyt c550, both free in solution and in intact PSII preparations, yields identical spectra with g values at 1.50, 2.23, and 2.87, which are characteristic for a ferric low-spin bis-histidine coordinated heme. The resonance Raman spectrum of the isolated protein exhibits features characteristic of bis-histidine axial ligation of the iron and a slight ruffling of the heme macrocycle. Together, these results indicate that the heme structure is not very different from most c-type cytochromes, and thus the structure of the heme does not account for its unusually low reduction potential. A direct electrochemical measurement of the reduction potential was performed using square wave voltammetry on a pyrolytic graphite edge electrode, yielding E1.2=-108 mV (vs. NHE) with a peak separation of 5 mV. This value is 150 mV more positive than that previously measured by redox titrations. Because the behavior of the protein in the electrochemistry experiments is indicative of adsorption to the electrode surface, we surmise that binding of the protein to the electrode excludes solvent water from the heme-binding site. We conclude that the degree of solvent exposure makes a significant contribution to the heme reduction potential. Similarly, the binding of cyt c550 to PSII may also reduce the solvent exposure of the heme, and so the direct electrochemical value of the reduction potential may be relevant to the protein in its native state.

Bioelectrocatalytic Detection of Histamine Using Quinohemoprotein Amine Dehydrogenase and the Native Electron Acceptor Cytochrome c-550

Bioelectrocatalytic sensor of histamine has been constructed using quinohemoprotein amine dehydrogenase (QH-AmDH) as an enzyme and its native electron acceptor cytochrome c-550 (Cyt c-550) as a mediator. Highly reversible electron transfer of Cyt c-550 is achieved at bis(4-pyridyl)disulfide-modified Au electrodes. The electron transfer from substrate-reduced QH-AmDH to oxidized Cyt c-550 is in the diffusion-controlled region, in spite of relatively small redox potential difference. QH-AmDH and Cyt c-550 can be co-entrapped on the electrode surface with a suitable dialysis membrane. As a result, Cyt c-550 is found to be an excellent mediator compared with other artificial ones. The sensor allows the histamine detection down to 500 nM (S/N=3) and the linearity is retained up to 1.5 mM with a relative standard deviation of 4.8 % at 10 µM histamine (n=6). The performance of the histamine biosensor is discussed in view of clinical chemistry.

Structural comparison of oxygen-evolving photosystem II core complexes fromSynechococcus elongatus and Spinacia oleracea

The molecular composition of oxygen evolving s-n-dodecylmaltoside-solubilised PS II core complexes from Synechococcus elongatus and Spinacia oleracea have been structurally analysed by SDS/urea/PAGE, immunoblotting using monospecific antibodies directed against PS II polypeptides, MALDI-TOF-MS and absorbance difference spectroscopy. A detailed cofactor analysis was likewise carried out. According to our investigations PS II core complexes from the cyanobacterium consist of at least 17 different proteins and that of spinach of s 15 subunits. One copy of cytb559 and cyt c550 were identified per cyanobacterial PS II complex. In contrast to spinach PS II core complexes no phosphorylated protein could be identified in the cyanobacterial PS II by phosphoamino acid specific antibodies (a-P-Thr, -P-Ser and -P-Tyr). In addition we addressed the question under which experimental conditions PS II core complexes of both sources occur as monomers or dimers by gel permeation HPLC, static/ dynamic light scattering and analytical ultracentrifugation. The results will be discussed in the light of recent structural models for PS II.

Cloning and expression of a cDNA encoding the extrinsic 20 kDa protein of photosystem II from a red alga Cyanidium caldarium

Oxygen-evolving PS II complex purified from a red alga, Cyanidium caldarium, contains four extrinsic proteins; they are 12 kDa, 20 kDa, 33 kDa proteins and cytochrome (cyt) c-550 [Enami et al. (1995) B.B.A. 1232, 208 - 216]. In this study, the 20 kDa protein was cloned and expressed in E. coli. The 20 kDa protein was purified from CaCl2-treated extracts of purified PS II complex and its N-terminal amino acid sequence was determined. Oligonucleotides synthesized on the basis of its N-amino acid sequence were used as primers for degenerated PCR, and complete sequence of the gene was determined using modified 5'- and 3'- RACE methods. The resulted open reading frame encodes a protein of 218 amino acid residues of which, the first 62 residues were assigned as leader sequences according to the N-terminal sequence of the mature protein. The resulted mature protein contains 146 amino acid residues with a molecular mass of 16386 Da. A search with the Swiss-Prot database revealed that the Cyanidium caldarium 20 kDa protein is 36.3% and 30.3% identical to the extrinsic 17 kDa protein from Volvox and Chlamydomonas, respectively, suggesting that the red algal 20 kDa protein is possibly an ancestor of the 17 kDa protein in green algae. An expression vector containing the 20 kDa gene was constructed in pCAL-n-EK plasmid, and the successful expression of the protein has been confirmed. We are currently performing reconstitution experiments with the recombinant 20 kDa protein to CaCl2-washed red algal PS II to study the binding and functional properties of the protein.

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.

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.

An Independent Role of Cytochrome c-550 in Cyanobacterial Photosystem II As Revealed by Double-Deletion Mutagenesis of the psbO and psbV Genes in Synechocystis sp. PCC 6803

Cytochrome (cyt) c-550 and the 33 kDa protein are two extrinsic components that function in maintaining oxygen evolution in cyanobacterial cells. Deletion of either of the two components has been shown to result in cyanobacterial phenotypes that are still capable of photoautotrophic growth albeit with a reduced rate. In order to study the function of cyt c-550 in cyanobacterial photosystem II (PSII) and its possible interaction with the 33 kDa extrinsic protein, we constructed a mutant lacking both cyt c-550 and the 33 kDa protein by inactivating the psbV and psbO genes simultaneously in a cyanobacterium, Synechocystis sp. PCC 6803. The resultant double-deletion mutant was unable to grow photoautotrophically and showed almost no oxygen-evolving activity (less than 10% of the wild type). This residual activity was also lost rapidly upon illumination, suggesting an increased sensitivity of the mutant cells toward photoinhibition. Thermoluminescence measurements indicated that the mutant virtually cannot undergo normal charge accumulation (S-state transitions) leading to oxygen evolution. Herbicide-binding and Western blot analyses showed that the mutant accumulates the PSII complex to an extent of only 20% of that in wild-type cells. Combined with previous results, the present results indicated that cyt c-550 supported oxygen evolution in the single-deletion mutant lacking the 33 kDa protein alone and vice versa. Thus, both cyt c-550 and the 33 kDa protein function independently in maintaining cyanobacterial oxygen-evolving activity in vivo, and both of them are required for the optimal activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical crosslinking studies of extrinsic proteins in cyanobacterial photosystem II

Chemical crosslinking with a zero-length crosslinker, 1-ethyl-3-(3-dimethylaminopropyl) carbodimide hydrochloride, was applied to a cyanobacterial photosystem II complex retaining three extrinsic proteins, the 33 kDa manganese-stabilizing protein, cytochrome (cyt) c-550 and the 12 kDa protein. Three major crosslinked products were obtained in addition to the crosslinked product between the extrinsic 33 kDa and the intrinsic CP47 proteins. They were identified to be: cyt c-550-12 kDa; cyt c-550-12 kDa-33 kDa; D2-cyt c-550-12 kDa. These results indicate that the three extrinsic proteins are closely located with each other in cyanobacterial PSII, supporting the previous proposal that, like the 33 kDa protein, cyt c-550 and the 12 kDa protein are associated with PSII at the lumenal side of thylakoids. The results also suggested that the D2 reaction center protein provides a direct binding site for the extrinsic cyt c-550.