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The water-oxidizing complex (WOC), also known as the oxygen-evolving complex (OEC), of photosystem II in oxygenic photosynthetic organisms efficiently catalyzes water oxidation. It is, therefore, responsible for the presence of oxygen in the Earth's atmosphere. The WOC is a manganese–calcium (Mn4CaO5(H2O)4) cluster housed in a protein complex. In this review, we focus on water exchange chemistry of metal hydrates and discuss the mechanisms and factors affecting this chemical process. Further, water exchange rates for both the biological cofactor and synthetic manganese water splitting are discussed. The importance of fully unveiling the water exchange mechanism to understand the chemistry of water oxidation is also emphasized here. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.

Photosystem II (PSII) photochemical efficiency (chlorophyll fluorescence ratio Fv/Fm) was recorded in vivo in Synechocystis 6803 during high light illumination and during a subsequent shift of the cells to darkness. A continuing decrease in the Fv/Fm ratio was observed even after the cells were transferred to darkness, provided the temperature was high enough. The decrease in the PSII efficiency after the shifting of the cells to darkness correlated directly with the loss of the D1 protein under different temperatures, suggesting that temperature-dependent proteolysis of the D1 protein in darkness induces the loss of PSII photochemical efficiency under these conditions. Furthermore, the amount of FtsH protease was found to increase during the high light treatment. This observation suggests that the synthesis of the FtsH protein is a light-regulated process and that this protease most probably has a key role in an efficient degradation of the D1 protein even under post-illuminative conditions, provided the temperature is high enough to prevent the initial reversible steps of photoinhibition.

This minireview is about the path that led me to the identification of the Photosystem II reaction center in oxygenic photosynthesis. It is based mostly on my own experiences and viewpoints. Thus, the article is essentially a personal account, and does not include all contributions that led to the identification of this functional unit of Photosystem II.

We report the results of persistent spectral hole burning (PSHB) at 4 K in the isolated photosystem II reaction center complex (PSII RC) in the presence and absence of externally added dibromothymoquinone DBMIB (2,6-dibromo-3-methyl-5-isopropyl-p-benzoquinone). The purified PSII complex (D1–D2–cytochrome b559 reaction center complex) has six molecules of chlorophyll a and lacks intrinsic electron acceptor plastoquinone (designated QA and QB, respectively). DBMIB replaces the function of QA. The efficiency for the hole formation at a long wavelength (680 nm) was higher than that at a shorter wavelength (671 nm). Addition of DBMIB lowered the efficiency at shorter wavelengths (671–674 nm) with little effect at longer wavelengths (677–680 nm). Temperature excursion experiments at 4–30 K indicated the higher temperature stability of the hole at the longer wavelength suggesting that the extent of thermally induced backward reaction of hole formation, i.e., the structural relaxation behavior of the matrix around the pigments was smaller at the longer wavelength. The hole burned at 671 nm can be attributed to the pheophytin in D2 subunit, while the hole at 680 nm to the electron-acceptor pheophytin in D1 subunit. The possibility remains that the peripheral Chl 667 is responsible for the hole at 671 nm. The temperature dependence of Debye–Waller factor also suggested that the PSII RC complex has stronger electron–phonon interaction than usual amorphous polymer systems.

More than one hundred mutants of Synechocystis sp. PCC 6803 impaired in photoautotrophic growth were generated by in vitro random PCR mutagenesis targeted to a region of the psbAII gene corresponding to a 210 amino acid (Ser148–Ala357) segment of the D1 protein. The 90 random mutants that could translate the full-length D1 protein carried 1–9 (on average 3.0) amino acid substitutions in the targeted region. Mutations were often found in the obligate photoheterotrophic strains at specific residues that have been reported or speculated to be important in the function of PSII, such as Y161, H198, H272, E333 and H337. This verifies the usefulness of the present method to identify functionally important residues in PSII. Other residues that were often mutated in the strains with impaired photoautotrophy included non-charged residues around the lumenal edges of transmembrane helices C, D and E, such as I192 and N296. Eleven mutants carried a single-point mutation in residues, such as Q165, Q187, W278, A294 and N298, and these identified the functional importance of these residues, most of which were on the donor side of PSII. A preliminary characterization of some of the mutants obtained in this study is provided.

The D1 protein of photosystem II is synthesized in precursor with a carboxyl-terminal extension. Interestingly, there is quite a range in chain length of the extension, which roughly depends upon the class of organisms. In cyanobacteria, e.g. in Synechocystis sp. PCC6803, the extension consists of 16 amino acid residues, seven residues longer than its counterpart in higher plants. In this study, we examined the D1 processing in Synechocystis sp. PCC6803 by pulse-chase experiments and detected a proteolytic intermediate of this process. This finding suggests that the elongated extension in this organism is excised with a sequential two-step proteolysis, which differs markedly from the manner observed in higher plants.

CtpA, which is classified as a novel type of serine protease with a Ser/Lys catalytic dyad, is responsible for the C-terminal processing of precursor D1 protein (pD1) of the photosystem II reaction center, a process that is indispensable for the integration of water-splitting machinery in photosynthesis. In this study, overexpression in Escherichia coli and one-step purification of spinach CtpA were carried out to analyze the characteristics of this new type of protease and to elucidate the molecular interactions in the C-terminal processing of pD1 on the thylakoid membrane. The successful accumulation of functional CtpA inE. coli may argue against the possibility, based on homology to E. coli Tsp, that the enzyme is involved in the degradation of incomplete proteins in chloroplasts, e.g. by utilizing the ssrA-tagging system. Analysis using a synthetic pD1 oligopeptide demonstrated that the enzymatic properties (including substrate recognition) of overexpressed CtpA with an extra sequence of GSHMLE at the N terminus were indistinguishable from those of the native enzyme. CtpA was insensitive to penem, which has been shown to inhibit some Ser/Lys-type proteases, suggesting that the catalytic center of CtpA is quite unique. By using the substrate in different molecular environments (i.e. synthetic pD1 oligopeptide in solution and pD1 in photosystem II-enriched thylakoid membrane), we observed a dramatic difference in the pH profile and affinity for the substrate, suggesting the presence of a specific interaction of CtpA with a factor(s) that modulates the pH dependence of proteolytic action in response to physiological conditions.

A cDNA encoding a putative RNA and/or DNA helicase has been isolated from Arabidopsis thaliana cDNA libraries. The cloned cDNA is 5166 bases long, and its largest open reading frame encodes 1538 amino acids. The central region of the predicted protein is homologous to a group of nucleic acid helicases from the DEAD/H family. However, the N- and C-terminal regions of the Arabidopsis cDNA product are distinct from these animal DEIH proteins. We have found that the C-terminal region contains three characteristic sequences: (i) two DNA-binding segments that form a probe helix (PH) involved in DNA recognition; (ii) an SV40-type nuclear localization signal; and (iii) 11 novel tandem-repeat sequences each consisting of about 28 amino acids. We have designated this cDNA as NIH (nuclear DEIH-boxhelicase). Functional character-ization of a recombinant fusion product containing the repeated region indicates that NIH may form homodimers, and that this is the active form in solution. Based on this information and the observation that the sequence homology is limited to the DEAH regions, we conclude that the biological roles of the plant helicase NIH differ from those of the animal DEIH family.

The D1 protein of the photosystem II reaction center is thought to be the most light-sensitive component of the photosynthetic machinery. To understand the mechanisms underlying the light sensitivity of D1, we performed in vitro random mutagenesis of the psbA gene that codes for D1, transformed the unicellular cyanobacterium Synechocystis sp. PCC 6803 with mutated psbA, and selected phototolerant transformants that did not bleach in high intensity light. A region of psbA2 coding for 178 amino acids of the carboxyl-terminal portion of the peptide was subjected to random mutagenesis by low fidelity polymerase chain reaction amplification or by hydroxylamine treatment. This region contains the binding sites for Q(B), D2 (through Fe), and P680. Eighteen phototolerant mutants with single and multiple amino acid substitutions were selected from a half million transformants exposed to white light at 320 micromol m(-2) s(-1). A strain transformed with non-mutagenized psbA2 became bleached under the same conditions. Site-directed mutagenesis has confirmed that one or more substitutions of amino acids at residues 234, 254, 260, 267, 322, 326, and 328 confers phototolerance. The rate of degradation of D1 protein was not appreciably affected by the mutations. Reduced bleaching of mutant cyanobacterial cells may result from continued buildup of photosynthetic pigment systems caused by changes in redox signals originating from D1.

Random mutations were introduced into the DNA fragment of the psbA2 gene of Synechocystis sp. PCC 6803, which encodes the carboxyl-terminal 178 amino acid region of the D1 protein of the PSII reaction center, by in vitro random mutagenesis to obtain D1 species resistant to herbicides and to understand the protein-herbicide interactions. The mutants were screened on the criterion of resistance to either 1 microM DCMU or 10 microM atrazine. In these mutants, amino acid substitutions were distributed throughout the entire area of the targeted region in the D1 protein. However, in every mutant, except for one case, the substitution was present in the region described as the "herbicide-binding niche", i.e., between Phe211 and Leu275, although some amino acid substitutions which were not previously described were found at residues known to be involved with herbicide affinity. Thus, the result of random mutagenesis basically supports the validity of the proposed structural model for the D1 protein, as well as of the herbicide-binding niche. Preliminary characterization of the herbicide-resistant mutants obtained in this study has also been conducted.

Recent advances in the studies on chlorophyll-protein complexes of higher plants are summarized in this article. Special emphasis is laid on the isolation, pigment composition and the absorption and fluorescence properties of the complexes.

Genes for σ-like factors of bacterial-type RNA polymerase have not been characterized from any multicellular eukaryotes, although they probably play a crucial role in the expression of plastid photosynthesis genes. We have cloned three distinct cDNAs, designated SIG1 , SIG2 , and SIG3 , for polypeptides possessing amino acid sequences for domains conserved in σ 70 factors of bacterial RNA polymerases from the higher plant Arabidopsis thaliana . Each gene is present as one copy per haploid genome without any additional sequences hybridized in the genome. Transient expression assays using green fluorescent protein demonstrated that N-terminal regions of the SIG2 and SIG3 ORFs could function as transit peptides for import into chloroplasts. Transcripts for all three SIG genes were detected in leaves but not in roots, and were induced in leaves of dark-adapted plants in rapid response to light illumination. Together with results of our previous analysis of tissue-specific regulation of transcription of plastid photosynthesis genes, these results indicate that expressed levels of the genes may influence transcription by regulating RNA polymerase activity in a green tissue-specific manner.

Mechanisms underlying suppressed levels of transcripts for plastid photosynthesis genes in nongreen tissues such as roots and calli were analyzed in Arabidopsis thaliana, a plant suitable for further genetic dissection. A region encoding promoters of rbcL, the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, and the atpB/E operon for the [beta] and [epsilon] subunits of coupling factor one were cloned and sequenced. Transcripts for rbcL, atpB/E, and psbA, the gene for the D1 protein in the photosystem II reaction center, were barely detectable in roots of A. thaliana, whereas 16S rRNA was detected at a low level. The run-on transcription experiment revealed that expression of rbcL, atpB/E, and psbA was regulated at transcription. The copy number of plastid DNA in roots was one-fifth that in green leaves on the basis of total cellular DNA, suggesting that in the latter the DNA copy-number regulation also exists in plastid gene expression. Digestion of DNA with methyl-sensitive and -insensitive isoschizomeric endonucleases and subsequent polymerase chain reaction, as well as in vitro transcription of plastid DNAs with Escherichia coli RNA polymerase, resulted in no evidence of regulation by DNA modification. In spite of predominant suppression of expression of rbcL, atpB/E, and psbA at transcription in roots and calli, 16S rRNA levels were decreased because of low RNA stability.

Transient absorbance changes of the primary electron donor chlorophyll a (P680) and acceptor pheophytin a (H) were measured at 77 K by nanosecond laser spectroscopy in the Dl-D2-cytochrome bSS9 photosystem II reaction center complex containing dibromomethylisopropyl benzoquinone (DBMIB). After the laser excitation of the reaction center in the presence of DBMIB, only the P680+(DBMIB) state was detected. P680+ mainly decayed with a t 1/e of 11 ms. In the absence of DBMIB, the excitation produced the P680+H~ radical pair. The radical pair produced the triplet state (P680T) with a t 1/e of 50 ns, and P680T then decayed with a tVt of 2.1 ms. It was concluded that H~ was oxidized by DBMIB in a time range faster than the detecting time resolution (3.5 ns) even at 77 K. The rapid oxidation of H~ by DBMIB was also confirmed by the suppression of delayed fluorescence with a decay t1/e of 50 ns. The P680+(DBMIB")/P680(DBMIB) difference spectrum exhibited a Q, band with a peak at 682 nm with a shoulder at 673 nm. The spectral shape was almost temperature insensitive between 77 and 265 K. The feature of this spectrum in the wavelength range between 330 and 720 nm was compared with that of P680 T/P680 or H"/H at 77 K.

A preliminary characterization has been carried out for a mutant strain of Synechocystis sp. PCC 6803 obtained by in vitro random mutagenesis applied to the region covering the nucleotides 538–1071 in the coding frame of the psbA2 gene encoding the D1 protein of the photosystem II reaction center. The mutant strain (G-3) was shown to have five substitutions in the amino acid in regions other than so-called ‘PEST-like sequence’ in the D1 protein, i.e. Phe-260 and Phe-273 to Ser, Gln-304 and Gln-310 to Arg, and Val-351 to Ala. The mutant cells were tolerant to high irradiance both in terms of pigmentation and viability. The rate of degradation of D1 protein in this mutant, however, was only slightly different from that of the control strain.

A photochemically inactive pheophytin (Pheo) a molecule in the spinach D1-D2-cyt b559 complex, which contains approximately 6 chlorophyll (Chl) a molecules and two β-carotene molecules for every two Pheo a molecules, was identified by fluorescence excitation spectroscopy at 77K, and confirmed by absorption, linear dichroism (LD), and magnetic circular dichroism spectra. When the emission was monitored at 740 nm, photochemically active Pheo a was detected as peaks at 418, 513, 543, and 681 nm, as described in previous reports. However, when the emission was monitored at 665 nm, two bands were observed at 414 and 537 nm; these bands were insensitive to photochemical reduction of the primary acceptor and thus appear to be due to photochemically inactive Pheo a. The Qy band of this Pheo a molecule at 77K was estimated to be at 671 ± 1 nm, based on the magnitude of the band-shift in photochemically active Pheo a. Two β-carotene molecules in the complex were also discriminated by fluorescence excitation and LD spectra. These observations were discussed in the light of current understanding of the molecular organization of pigments and the relationship of pigment arrangement to optical properties in the reaction center of photosystem II.

Magnetic circular dichroism (MCD) of chlorophylls (Chl) in P700-enriched (12 Chl/P700) photosystem (PS) I particles and D1-D2-cyt b 559 (PSII reaction center) particles isolated from spinach are measured in the wavelength region between 450 and 750 nm. The relative magnitude of MCD to absorption intensity ([MCD]/[Abs]) is found to be a good measure to estimate the interaction of chromophores (dimer or monomer). The difference spectrum between reduced and oxidized state for the 12 Chl/P700 particles can be interpreted to be composed of a positive MCD from P700 and a negative MCD for P700 + . MCD signals for the primary acceptor Chl a monomer (A 0 ) and the reduced cytochrome f are also observed. The MCD signal from pheophytin a in the PSII reaction center is remarkably in the Q x , region as strong as the signal from the reaction center chlorophylls (P680 and accessory) and other attached chlorophylls. Based on the data, the MCD bands, as well as the absorption bands in the Q y region, are deconvoluted into the contribution from reaction center chlorophylls and the other chlorophylls. The [MCD]/[Abs] ratio of P680 or P700 is small and similar to that of special pair bacteriochlorophylls in the reaction center of purple bacteria, indicating the specific feature of MCD signals that originate from a dimer-type interaction.

Environmental ionizing radiations were surveyed in Okayama city during December of 1992, using a NaI (Tl) scintillation survey meter (TCS-161; Aloka, Japan) and a NaI (Tl) γ spectrometer (JSM-102; Aloka, Japan) ; measerments were carried out in fine days.Equivalent dose rate of environmental ionizing radiations was in the range of 0.048-0.171μSv/h, and the average was 0.082±0.019μSv/h. The dose rate was higher in hills than in fields. Therefore, it appears as if rocks have more radioactive materials than soils do.The distribution of the dose rates was analyzed from geological points of view. Mesozoic and Paleozoic layer was in the range of 0.067-0.095μSv/h, and the average was 0.080±0.008μSv/h. Rhyolitic layer was in the range of 0.076-0.105μSv/h, and the average was 0.088±0.010μSv/h. Granitic layer was in the range of 0.057-0.171μSv/h, and the average was 0.098±0.022μSv/h. Neogene Period layer was in the range of 0.057-0.114μSv/h, and the average was 0.076±0.015μSv/h. Quaternary Period layer was in the range of 0.048-0.114μSv/h, and the average was 0.076±0.013μSv/h.By γ spectrometry, 40K, 208Tl, 212Bi, 214Bi, and 228Ac were detected at Mt. Kaigara (0.171μSv/h), Tsushima (0.076μSv/h) and Fujita (0.048μSv/h), especially, 226Ra was detected at Mt. Kaigara. It seems that 40K was distributed uniformly in Okayama city area. Thas, the difference in the dose range is attributable to nuclides other than 40K.Now, we conclude that there has been no change of the dose rates of environmental radiation in Okayama city these 10 years.

Photosynthetic characteristics along with phototolerance and photoinhibition of photosystem II (PS II) were monitored in Synechocystis sp. PCC 6803 wild type (KC) and its psbAII mutants viz., I6 (N322I, I326F, and F328S), G6 (N267Y), and H7 (Y254C and I314V) that have up to three point mutations, localized in the D-E loop of the D1 polypeptide of PSII reaction centre. These strains exhibited entirely different growth trends upon shifting from 30 micormol m(-2)s(-1) to high irradiance (500 micromol m(-2)s(-1) , 30 degrees C). The I6 and H7 cells grew well, whereas KC and G6 cells showed inability for cell multiplication. The photosynthetic efficiency demonstrated about 50% loss in chlorophyll fluorescence of variable yield (Fv/Fm) within 20-30 min in all mutants, whereas the wild type (KC) cells could reach the same level of loss in 2 hr. I6 and H7 cells showed continuous cell growth and maintenance under long-term exposure of high light compared to G6 mutant and wild type cells. The wild type cells showed slow decrease in their photochemical activity and Fv/Fm values, compared to mutant cells. The recovery seemed to be almost identical, and also stimulated by growth light, inspite of differential photoinhibitory behaviours. Darkness and translational inhibitor lincomycin both were found to be unassociated with the restoration of photoinhibited process of PS II.

A cross-linked product between the D1 and D2 subunits was generated by treating isolated spinach PS II reaction center with a bifunctional cross-linker, 1,6-hexamethylene diisocyanate. To obtain information on the contact site(s) between D1 and D2 proteins, the cross-linked product was cleaved by CNBr, the resultant fragments were separated using a reverse-phase HPLC, and then the partial sequence of the cross-linked polypeptide fragments were determined. By comparing the results with the deduced amino acid sequence of the D1 and D2 proteins from spinach, it is concluded that the C-terminal domains of the D1 subunit (D308-A334) and that of the D2 subunit (Y297-L353) are in close proximity.

Synthetic oligopeptides of different chain lengths of 11 to 38 amino acids, corresponding to the carboxyl-terminal sequence of D1 precursor protein of the photosystem II reaction center, were subjected to a proteolytic cleavage by a processing enzyme isolated from spinach, in order to analyze the recognition signal. Practically the same K m and V max values were obtained for the oligopeptides consisting of more than 19 amino acids; a decrease in affinity, without affecting the V max value, was observed for the peptide consisting of 16 amino acids; no detectable activity was found for the peptide with 11 amino acids. When Asp-342 (12th residue from C-terminus) was replaced with Asn, for the peptide consisting of 16 amino acids, the enzymatic activity was completely abolished. In contrast, replacing Asp-342 with Glu had little effect. The efficiency of these oligopeptides as a substrate mentioned above, together with their effectiveness as an inhibitor, clearly demonstrated that the negative charge on Asp-342 plays a crucial role in the recognition, i.e. binding and cleavage, of the substrate by the processing enzyme, and suggested that the carboxyl-terminal extension consisting of 9 amino acids, by itself is not important in the binding.

Two distinct cross-linked products containing psbI gene product were generated in the photosystem II reaction center complex from spinach by treatment with bifunctional reagents directed to amino groups. The first product, which was generated by a 3,3'-dithiobis(succinimidyl propionate) treatment, is deduced to be formed between the epsilon-amino group of Lys3 of the psbI gene product, and a side-chain amino group present on the stromal extension of the D2 protein. The CNBr cleavage analysis of the cross-linked product predicted that the amino group of the D2 protein engaged in the cross-linking is either one of the three lysine residues on the N-terminal fragment (from N-terminus to Met19) or the Lys268 on the 6th fragment (from Val248 to Met275). The second product, which was also generated on the stromal side by a 1,6-hexamethylene diisocyanate treatment preferentially in alkaline conditions, is predicted to be formed between the epsilon-amino group of Lys3 of the psbI gene product and the N-terminal alpha-amino group of the alpha-subunit of cytochrome b559.

In the presence of externally added ATP, in the dark, isolated pea chloroplasts accumulate two proteins of molecular masses of about 22 and 24 kDa which precipitate with specific antibodies raised against the D1 protein. By chasing in the light, these proteins disappeared on the fluorogram concomitant with the appearance of the precursor- and mature-sized D1 proteins. Polysome analysis indicated that the 22-kDa component is associated with membrane-bound ribosomes and is thus ascribed to a translation intermediate of the D1 protein. On the other hand, the 24-kDa component could not be found in the polysome fraction under the experimental condition used, suggesting the possibility that this component is a degradation product of the D1 protein. The conclusion from this analysis is that the synthesis and/or stable accumulation of the D1 protein requires factor(s) caused by illumination, in addition to ATP, in isolated pea chloroplasts.

Changes in composition of membrane proteins in Synechocystis PCC 6803 induced by the shift of light regime for photosynthetic growth were studied in relation to the regulation of PS I/PS II stoichiometry. Special attention was paid to the changes in abundance of proteins of PS I and PS II complexes. Composition was examined using a LDS-PAGE and a quantitative enzyme immunoassay. Abundance of PsaA/B polypeptides and the PsaC polypeptide of the PS I complex, on a per cell basis, increased under the light regime exciting preferentially PS II and decreased under the light regime exciting mainly PS I. Similar changes were observed with polypeptides of 18.5, 10 and 8.5 kDa. The abundance of other proteins associated with membranes, including PsbA polypeptide of the PS II complex, was fairly constant irrespective of light regime. These results are consistent with our previous observations with other strains of cyanophytes (Anabaena variabilis M2 and Synechocystis PCC 6714) that PS I is the variable component in changes in PS I/PS II stoichiometry in response to changing light regimes for photosynthesis.

The orientation properties of the reaction centre triplet in Photosystem II (PS II) were determined. A high triplet yield was generated in PS II membranes by double reduction of the primary quinone electron acceptor Q A . It is deduced that the triplet state is localised on a chlorophyll, the tetrapyrrolic plane of which is tilted at 30° to the membrane. A similar orientation was found in D1 / D2 / cyt b -559 particles demonstrating that the triplet is confined to the reaction centre. Optical work in the literature has been interpreted as indicating that the triplet is localised on a monomeric chlorophyll and that, in the singlet state, P680 consists of this molecule weakly coupled to a second chlorophyll. The weakness of the coupling, compared to the coupling in the special pair of purple bacteria, allows P680 to be considered as a monomer. Taking the optical data into account, we propose that P680 is a chlorophyll molecule oriented at 30° to the membrane. This result is discussed in terms of the structural analogy between PS II and the reaction centre of purple bacteria. A model is favored in which P680 is a chlorophyll, structurally analogous to one of the monomeric bacteriochlorophylls of the bacterial reaction centre. In addition, the orientation data indicate that this chlorophyll is rotated by 45° in its ring plane compared to the monomeric bacteriochlorophylls in the reaction centre of Rhodopseudomonas viridis .

The cells of Synechocystis sp. PCC 6803 were subjected under photoinhibitory irradiation (600 micromolm(-2)s(-1)) at various temperatures (20-40 degrees C) to study in vivo quality control of photosystem II (PSII). The protease biogenesis and its consequences on photosynthetic efficiency (chlorophyll fluorescence ratio Fv/Fm) of the PSII, D1 degradation and repair were monitored during illumination and darkness. The loss in Fv/Fm value and degradation of D1 protein occurred not only under high light exposure, but also continued when the cells were subjected under dark restoration process after high light exposure. No loss in Fv/Fm value or D1 degradation occurred during recovery under growth/low light (30 micromol m(-2) s(-1)). Further, it helped the resynthesis of new D1 protein, essential to sustain quality control of PSII. In vivo triggering of D1 protein required high light exposure to switch-on the protease biosynthesis to maintain protease pool which induced temperature-dependent enzymatic proteolysis of photodamaged D1 protein during photoinhition and dark incubation. Our findings suggested the involvement and overexpression of a membrane-bound FtsH protease during high light exposure which caused degradation of D1 protein, strictly regulated by high temperature (30-40 degrees C). However, lower temperature (20 degrees C) prevented further loss of photoinhibited PSII efficiency in vivo and also retarded temperature-dependent proteolytic process of D1 degradation.

This article provides a glimpse into the dawning of research on chlorophyll-protein complexes and a brief recollection of the path that led us to the identification of the photosystem II reaction center, i.e., the polypeptides that carry the site of primary charge separation in oxygenic photosynthesis. A preliminary version of the personal review on the latter topic has already appeared in this journal (Satoh Photosynth Res 76:233-240, 2003).

Flash-induced absorbance changes in the nanosecond to millisecond time ranges have been measured in the Photosystem II reaction center complex consisting of D1 and D2 subunits and cytochrome b -559, in the presence of 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB). The results indicate that DBMIB largely quenches the primary radical pair (P-680 + Pheo − ) and the formation of the triplet ( 3 P-680). Long-lived absorption signals in the red and near-infrared (bleaching at 680 nm and broad increase at 740–830 nm) and in the green (peak at 560 nm) can be attributed to the oxidation of P-680 and to the reduction of cytochrome b -559. These data show that addition of DBMIB induces stabilization of P-680 + and a rapid (perhaps submicrosecond) reduction of cytochrome b -559. The signals attributed to P-680 + and to reduced cytochrome decay in parallel ( t 1 2 = 2 ms ), showing that the cytochrome reduces P-680 + . The stabilization occurred also in the presence of plastoquinone-3 and (with DBMIB) at −29°C in a viscous solution containing 60% glycerol at a low concentration of quinone, suggesting that the quinone reconstitutes the function of Q A and thus mediates electron transport from the reduced pheophytin a to the intrinsic cytochrome.

The D1 protein, a key subunit of photosystem II reaction center, is synthesized as a precursor form with a carboxyl-terminal extension, in oxygenic photosynthetic organisms with some exceptions. This part of the protein is removed by the action of an endopeptidase, and the proteolytic processing is indispensable for the manifestation of oxygen-evolving activity in photosynthesis. The carboxyl-terminus of mature D1 protein, which appears upon the cleavage, has recently been demonstrated to be a ligand for a manganese atom in the Mn(4)Ca-cluster, which is responsible for the water oxidation chemistry in photosystem II, based on the isotope-edited Fourier transform infrared spectroscopy and the X-ray crystallography. On the other hand, the structure of a peptidase involved in the cleavage of precursor D1 protein has been resolved at a higher resolution, and the enzyme-substrate interactions have extensively been analyzed both in vivo and in vitro. The present article briefly summarizes the history of research and the present state of our knowledge on the carboxyl-terminal processing of precursor D1 protein in the photosystem II reaction center.

This minireview is about the path that led me to the identification of the Photosystem II reaction center in oxygenic photosynthesis. It is based mostly on my own experiences and viewpoints. Thus, the article is essentially a personal account, and does not include all contributions that led to the identification of this functional unit of Photosystem II.

This chapter briefly traces some of the early studies and key findings which have led to our current perception and understanding of Photosystem II, the water:plastoquinone oxidoreductase in oxygenic photosynthesis. Starting with the discovery of oxygen and the idea of two photosystems, the progressive identification of the unique structural and functional aspects of Photosystem II are outlined and related to the corresponding chapters in the book. The aim is to integrate the detailed descriptions in the various chapters in the context of the structure and function of Photosystem II as a whole. The chapter ends with a brief perspective for the future study and application of Photosystem II research.

The Dl protein of the photosystem II reaction center turns over the most rapidly of all the proteins of the thylakoid membrane under illumination in vivo. In vitro, the Dl protein sustained cleavage in a surface-exposed loop (DE loop) or cross-linking with another reaction center protein, the D2 protein or cytochrome b 5 5 9 , under illumination. We found that the Dl protein was damaged in essentially the same way in vivo, although the resultant fragments and cross-linked adducts barely accumulated due to digestion by proteases. In vitro studies detected a novel stromal protease(s) that digested the adducts but not the monomeric Dl protein. These observations suggest that, in addition to cleavage, the cross-linking reactions themselves are processes involved in complete degradation of the Dl protein in vivo. Peptide mapping experiments located the cross-linking sites with the D2 protein among residues 226-244, which includes the cross-linking site with cytochrome b 5 5 9 [Barbato, R., et al. (1995) J. Biol. Chem. 270, 24032-24037], in the N-terminal part of the DE loop, while N-terminal amino acid sequencing of the fragment located the cleavage site around residue 260 in the C-terminal part of the loop. We propose a model explaining the occurrence of simultaneous cleavage and cross-linking and discuss the mechanisms of complete degradation of the Dl protein in vivo.

CtpA, a carboxyl-terminal processing protease, is a member of a novel family of endoproteases that includes a tail-specific protease from Escherichia coli. In oxygenic photosynthetic organisms, CtpA catalyzes C-terminal processing of the D1 protein of photosystem II, an essential event for the assembly of a manganese cluster and consequent light-mediated water oxidation. We introduced site-specific mutations at 14 conserved residues of CtpA in the cyanobacterium Synechocystis sp. PCC 6803 to examine their functional roles. Analysis of the photoautotrophic growth capabilities of these mutants, their ability to process precursor D1 protein and hence evolve oxygen, along with an estimation of the protease content in the mutants revealed that five of these residues are critical for in vivo activity of CtpA. Recent x-ray crystal structure analysis of CtpA from the eukaryotic alga Scenedesmus obliquus (Liao, D.-I., Qian, J., Chisholm, D. A., Jordan, D. B. and Diner, B. A. (2000) Nat. Struct. Biol. 7, 749-753) has shown that the residues equivalent to Ser-313 and Lys-338, two of the five residues mentioned above, form the catalytic center of this enzyme. Our in vivo analysis demonstrates that the three other residues, Asp-253, Arg-255, and Glu-316, are also important determinants of the catalytic activity of CtpA.

A method based on the susceptibility of photosynthetic organisms to nitrofurantoin under illumination was established to screen mutants of Synechocystis sp. PCC 6803 deficient in the function of photosystem II, which were created by random PCR mutagenesis targeted to the psbAII gene coding for the D1 protein of the photosystem II reaction center. In this method, cyanobacterial colonies on a nitrocellulose membrane on a BG11 agar plate were treated with nitrofurantoin at 1.0 mM under white light at 40 microE x m(-2 ) x s(-1) for 2 h, and then kept under normal conditions without nitrofurantoin so that surviving cells could grow. This method was also shown to be useful for screening mutants deficient in the function of photosystem I.

We have isolated a cDNA from Arabidopsis thaliana for a protein consisting of 323 amino acids with similarity to an extracellular nuclease from Staphylococcus . Nuclease assay using toluidine blue-DNA plates has demonstrated that the gene product has nuclease activity dependent on Ca 2+ and inhibited by Zn 2+ , designated CAN ( Ca 2+ -dependent n uclease). Differing from the staphylococcal nuclease, CAN has neither a signal peptide nor any long hydrophobic regions, suggesting that it is not a secreted protein.

Several random mutations have been generated in the psbA2 gene of Synechocystis sp. PCC 6803 [Narusaka, Y., Murakami, A., Saeki, J., Kobayashi, H., and Satoh, K. (1996) Plant Sci. 115, 261-266]. The phototolerant mutant (I6) carrying all the amino acid substitutions in the lumenal side of D1 protein (S322I, I326F, and F328S) and a site-directed mutant of the same phenotype (NDFS) substituted in the stromal side of the protein (N234D and F260S) were characterized by thermoluminescence measurements. We observed (1) no significant differences in their growth rates at either low or high light irradiance, (2) a downshifted B-band in the NDFS mutant, (3) an upshifted Q-band in the I6 mutant, and (4) a damped period four oscillation of thermoluminescence in the B-band of both mutants. By examining the possible implications of these results on the redox properties of the PS II components in the mutants, we concluded that equilibrium constants for sharing an electron between the primary (QA) and secondary acceptor plastoquinones (QB) are decreased in both mutants.

The major proteins involved in the primary processes of photosynthesis are encoded by genes located in the chloroplast genome, and the expression of this gene information is regulated by nuclear-encoded factors often at the later stages, e.g., the translational and post-translational levels, in response to the development and the varying environmental conditions. The carboxyl-terminal (C-terminal) processing of the precursor D1 protein (pD1) is a typical example of post-translational regulation.

The extrinsic 33-kDa protein of photosystem II (PSII) was intramolecularly cross-linked by a zero-length cross-linker, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The resulting cross-linked 33-kDa protein rebound to urea/NaCl-washed PSII membranes, which stabilized the binding of manganese as effectively as the untreated 33-kDa protein. In contrast, the oxygen evolution was not restored by binding of the cross-linked protein, indicating that the binding and manganese-stabilizing capabilities of the 33-kDa protein are retained but its reactivating ability is lost by intramolecular cross-linking of the protein. From measurements of CD spectra at high temperatures, the secondary structure of the intramolecularly cross-linked 33-kDa protein was found to be stabilized against heat treatment at temperatures 20 degrees C higher than that of the untreated 33-kDa protein, suggesting that structural flexibility of the 33-kDa protein was much decreased by the intramolecular cross-linking. The rigid structure is possibly responsible for the loss of the reactivating ability of the 33-kDa protein, which implies that binding of the 33-kDa protein to PSII is accompanied by a conformational change essential for the reactivation of oxygen evolution. Peptide mapping, N-terminal sequencing, and mass spectroscopic analysis of protease-digested products of the intramolecularly cross-linked 33-kDa protein revealed that cross-linkings occurred between the amino group of Lys48 and the carboxyl group of Glu246, and between the carboxyl group of Glu10 and the amino group of Lys14. These cross-linked amino acid residues are thus closely associated with each other through electrostatic interactions.

The photosystem II (PSII) is unique in that it generates a high redox potential for utilizing water molecules as electron donors. The D1 protein constitutes the core of the photosystem and is thought to play essential roles in its structure and function (Nanba and Satoh 1987). Debus and colleagues (1988) used site-directed mutagenesis to successfully elucidate the participation of a specific amino acid residue on the D1 protein in the function of PSII. A strain of cyanobacteria, Synechocystis sp. PCC6803, was utilized in this study, because of its potential for transformation efficiency and its capacity for heterotrophic growth (Williams 1988). A nitrofurantoin treatment was used to enrich spontaneous mutants, since this reagent tends to kill organisms having normal photosynthetic activity (Shestakov et al. 1994).

The D1 protein (D1) of photosystem II (PSII) reaction center is synthesized as a precursor (pD1) and then processed at its carboxyl terminus to establish the function of water cleavage. The amino acid sequence of the carboxyl terminal extension excised by this process is poorly conserved except for a residue after the cleavage site at position of 345. We have constructed a vector for site-directed mutagenesis of the chloroplast psbA gene encoding D1 of the green alga, Chlamydomonas reinhardtii. The vector enables one to transform the chloroplasts of a psbA deletion mutant (Fud7) and directly select transformants for resistance to spectinomycin. Using this transforming vector, we have substituted Ser345 to Gly, Cys, Val and Phe in order to investigate effects of the amino acid side chain at this position on the processing rate. All of the resulting transformants exhibited the PSII activity as wild type and grew normally under photoautotrophic conditions even under strong light where rapid turnover of D1 protein is expected to occur. Western blotting analysis demonstrated that mature D1 accumulates in these transformants at wild type level. Pulse and chase labeling of chloroplast-encoded proteins using [35S] sulfate revealed that the processing of D1 precursor protein occurs in all four transformants as efficiently as in wild type, at least under the experimental conditions examined. The results suggest that either the amino acid side chain at position of 345 (+1 position) is not crucial to the enzymatic cleavage of pD1 in vivo or the apparent rate of processing in vivo is not limited by the enzymatic cleavage.

A previous study has demonstrated that the carboxyl-terminal (C-terminal) processing protease in spinach for the D1 precursor protein (pD1) of the photosystem II reaction center is a monomeric protein of about 45 kDa. Based on the amino acid sequence data of the purified protease, a cDNA clone encoding the enzyme has been identified and sequenced, from a spinach green leaf cDNA library. In order to determine the 5′ end of the transcript, the rapid amplification of cDNA end (5′-RACE) technique was applied. By these analyses, the full-length transcript was established to consist of 1906 nucleotides and a poly(A) tail, containing an open reading frame (ORF) corresponding to a protein with 539 amino acid residues. By comparing the amino acid sequence of the purified protease with that deduced from nucleotide sequence of the cDNA clones, the enzyme was shown to be furnished with an extra amino-terminal extension characteristic of both a transit peptide and a signal sequence. This suggests that the protease is synthesized in the cytosol and translocated into the lumenal space of thylakoids. The mature part of the enzyme consists of 389 amino acid residues and exhibits a significant sequence homology with two groups of proteins as demonstrated by a computer homology search, i.e. (1) the deduced sequence of a protein proposed to be the C-terminal processing protease for pD1 in Synechocystis sp. PCC 6803, based on genetic experiments and (2) proteases for C-terminal cleavage identified in Escherichia coli and Bartonella bacilliformis.

Next Section Abstract In order to analyze the structural requirement(s) for proteolytic cleavage, synthetic oligopeptides corresponding to the carboxyl-terminal (COOH-terminal) sequence of the precursor to the D1 protein (pD1) of the photosystem II reaction center, with or without substituted side chain(s) around the cleavage site, were subjected to enzymatic analysis with partially purified processing protease from spinach. The efficiency of action as a competitive inhibitor of the enzymatic cleavage of the COOH-terminal extension, as well as the capacity to serve as a substrate, was used as an indication of effective binding to the protease. Neither a COOH-terminal fragment consisting of the 9 amino acids that are cleaved from pD1 by the protease nor a COOH-terminal fragment of the mature protein consisting of 15 amino acids inhibited the enzymatic processing of pD1. By contrast, a COOH-terminal fragment of pD1 consisting of 24 amino acids, which included the sequences of both the COOH-terminal extension and the COOH-terminal 15 amino acids of the mature protein, was effective both as a competitive inhibitor and as a substrate. This result suggests that the structure formed by linkage between these two parts of the protein moiety is important in the substrate-enzyme interaction. Among substitutions around the cleavage site, the replacement of Leu-343 by Ala (L343A) specifically destroyed the ability of the oligopeptide to serve as either a substrate or an inhibitor, suggesting that the presence of the hydrophobic Leu residue is crucial for the formation of the recognition site. A series of six substitutions at Ala-345 had marked effects on the value of V, without affecting the binding affinity, as represented by K; the order of substitutions at residue 345 in terms of their effects on V was Ala, Ser, Phe, Cys > Gly > Val Pro. With a Pro residue at position 345, the oligopeptide was practically inactive as a substrate.