Isolation and spectral characterization of Photosystem II reaction center from Synechocystis sp. PCC 6803.

We isolated highly-purified photochemically active photosystem (PS) II reaction center (RC) complexes from the cyanobacterium Synechocystis sp. PCC 6803 using a histidine-tag introduced to the 47 kDa chlorophyll protein, and characterized their spectroscopic properties. Purification was carried out in a one-step procedure after isolation of PS II core complex. The RC complexes consist of five polypeptides, the same as in spinach. The pigment contents per two molecules of pheophytin a were 5.8 +/- 0.3 chlorophyll (Chl) a and 1.8 +/- 0.1 beta-carotene; one cytochrome b(559) was found per 6.0 Chl a molecules. Overall absorption and fluorescence properties were very similar to those of spinach PS II RCs; our preparation retains the best properties so far isolated from cyanobacteria. However, a clear band-shift of pheophytin a and beta-carotene was observed. Reasons for these differences, and RC composition, are discussed on the basis of the three-dimensional structure of complexes.

Characterization of highly purified photosystem I complexes from the chlorophyll d-dominated cyanobacterium Acaryochloris marina MBIC 11017.

Photochemically active photosystem (PS) I complexes were purified from the chlorophyll (Chl) d-dominated cyanobacterium Acaryochloris marina MBIC 11017, and several of their properties were characterized. PS I complexes consist of 11 subunits, including PsaK1 and PsaK2; a new small subunit was identified and named Psa27. The new subunit might replace the function of PsaI that is absent in A. marina. The amounts of pigments per one molecule of Chl d' were 97.0 +/- 11.0 Chl d, 1.9 +/- 0.5 Chl a, 25.2 +/- 2.4 alpha-carotene, and two phylloquinone molecules. The light-induced Fourier transform infrared difference spectroscopy and light-induced difference absorption spectra reconfirmed that the primary electron donor of PS I (P740) was the Chl d dimer. In addition to P740, the difference spectrum contained an additional band at 728 nm. The redox potentials of P740 were estimated to be 439 mV by spectroelectrochemistry; this value was comparable with the potential of P700 in other cyanobacteria and higher plants. This suggests that the overall energetics of the PS I reaction were adjusted to the electron acceptor side to utilize the lower light energy gained by P740. The distribution of charge in P740 was estimated by a density functional theory calculation, and a partial localization of charge was predicted to P1 Chl (special pair Chl on PsaA). Based on differences in the protein matrix and optical properties of P740, construction of the PS I core in A. marina was discussed.

Identification of the special pair of photosystem II in a chlorophyll d-dominated cyanobacterium.

The composition of photosystem II (PSII) in the chlorophyll (Chl) d-dominated cyanobacterium Acaryochloris marina MBIC 11017 was investigated to enhance the general understanding of the energetics of the PSII reaction center. We first purified photochemically active complexes consisting of a 47-kDa Chl protein (CP47), CP43' (PcbC), D1, D2, cytochrome b(559), PsbI, and a small polypeptide. The pigment composition per two pheophytin (Phe) a molecules was 55 +/- 7 Chl d, 3.0 +/- 0.4 Chl a, 17 +/- 3 alpha-carotene, and 1.4 +/- 0.2 plastoquinone-9. The special pair was detected by a reversible absorption change at 713 nm (P713) together with a cation radical band at 842 nm. FTIR difference spectra of the specific bands of a 3-formyl group allowed assignment of the special pair. The combined results indicate that the special pair comprises a Chl d homodimer. The primary electron acceptor was shown by photoaccumulation to be Phe a, and its potential was shifted to a higher value than that in the Chl a/Phe a system. The overall energetics of PSII in the Chl d system are adjusted to changes in the redox potentials, with P713 as the special pair using a lower light energy at 713 nm. Taking into account the reported downward shift in the potential of the special pair of photosystem I (P740) in A. marina, our findings lend support to the idea that changes in photosynthetic pigments combine with a modification of the redox potentials of electron transfer components to give rise to an energetic adjustment of the total reaction system.

Chlorophyllase as a serine hydrolase: identification of a putative catalytic triad.

Chlorophyllases (Chlases), cloned so far, contain a lipase motif with the active serine residue of the catalytic triad of triglyceride lipases. Inhibitors specific for the catalytic serine residue in serine hydrolases, which include lipases effectively inhibited the activity of the recombinant Chenopodium album Chlase (CaCLH). From this evidence we assumed that the catalytic mechanism of hydrolysis by Chlase might be similar to those of serine hydrolases that have a catalytic triad composed of serine, histidine and aspartic acid in their active site. Thus, we introduced mutations into the putative catalytic residue (Ser162) and conserved amino acid residues (histidine, aspartic acid and cysteine) to generate recombinant CaCLH mutants. The three amino acid residues (Ser162, Asp191 and His262) essential for Chlase activity were identified. These results indicate that Chlase is a serine hydrolase and, by analogy with a plausible catalytic mechanism of serine hydrolases, we proposed a mechanism for hydrolysis catalyzed by Chlase.