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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.

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.

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.

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.

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.

All-transβ-carotene-5,6-epoxide has been found in the thylakoid membranes of spinach and of the cyanobacterium Synechococcus vulcanus Copeland. The epoxide was extracted from the thylakoid membranes with acetone, and was isolated by high-performance liquid chromatography (HPLC). The structure of the epoxide was identified by means of mass, Raman, and electronic absorption spectroscopy. Changes in the amount of the epoxide, as a result of epoxidation and (apparent) de-epoxidation reactions in the membranes, were traced by analysis of extracts on HPLC. In isolated thylakoid membranes, only the epoxidation reaction took place. The reaction was caused by irradiation or by the addition of ferricyanide, suggesting that electron transport reactions in the membranes are involved in the epoxidation. In intact spinach leaves, however, both epoxidation and de-epoxidation took place; the extent of epoxidation correlated with the intensity of light incident on the leaves. The epoxidation and de-epoxidation of all-transβ-carotene are contrasted with those of xanthophylls (in the violaxanthin cycle).