Shunsuke Ohashi

Effects of the Lack of Phosphatidylglycerol on the Donor Side of Photosystem II

Our previous studies with the pgsA mutant of the cyanobacterium Synechocystis sp. PCC6803 (hereafter termed pgsA mutant), which is defective for the biosynthesis of phosphatidylglycerol (PG), revealed an important role for PG in the electron acceptor side of photosystem II (PSII), especially in the electron transport between plastoquinones QA and QB. This study now shows that PG also plays an important role in the electron donor side of PSII, namely, the oxygen-evolving system. Analyses of purified PSII complexes indicated that PSII from PG-depleted pgsA mutant cells sustained only approximately 50% of the oxygen-evolving activity compared to wild-type cells. Dissociation of the extrinsic proteins PsbO, PsbV, and PsbU, which are required for stabilization of the manganese (Mn) cluster, followed by the release of a Mn atom, was observed in PSII of the PG-depleted mutant cells. The released PsbO rebound to PSII when PG was added back to the PG-depleted mutant cells, even when de novo protein synthesis was inhibited. Changes in photosynthetic activity of the PG-depleted pgsA mutant cells induced by heat treatment or dark incubation resembled those of ΔpsbO, ΔpsbV, and ΔpsbU mutant cells. These results suggest that PG plays an important role in binding extrinsic proteins required for sustaining a functional Mn cluster on the donor side of PSII.

Purification and characterization of photosystem I complex from Synechocystis sp. PCC 6803 by expressing histidine-tagged subunits

We generated Synechocystis sp. PCC 6803 strains, designated F-His and J-His, which express histidine-tagged PsaF and PsaJ subunits, respectively, for simple purification of the photosystem I (PSI) complex. Six histidine residues were genetically added to the C-terminus of the PsaF subunit in F-His cells and the N-terminus of the PsaJ subunit in J-His cells. The histidine residues introduced had no apparent effect on photoautotrophic growth of the cells or the activity of PSI and PSII in thylakoid membranes. PSI complexes could be simply purified from the F-His and J-His cells by Ni2+-affinity column chromatography. When thylakoid membranes corresponding to 20 mg chlorophyll were used, PSI complexes corresponding to about 7 mg chlorophyll could be purified in both strains. The purified PSI complexes could be separated into monomers and trimers by ultracentrifugation in glycerol density gradient and high activity was recorded for trimers isolated from the F-His and J-His strains. Blue-Native PAGE and SDS-PAGE analysis of monomers and trimers indicated the existence of two distinct monomers with different subunit compositions and no contamination of PSI with other complexes, such as PSII and Cyt b(6)f. Further analysis of proteins and lipids in the purified PSI indicated the presence of novel proteins in the monomers and about six lipid molecules per monomer unit in the trimers. These results demonstrate that active PSI complexes can be simply purified from the constructed strains and the strains are very useful tools for analysis of PSI.

Unique photosystems in Acaryochloris marina

A short overview is given on the discovery of the chlorophyll d-dominated cyanobacterium Acaryochloris marina and the minor pigments that function as key components therein. In photosystem I, chlorophyll d′, chlorophyll a, and phylloquinone function as the primary electron donor, the primary electron acceptor and the secondary electron acceptor, respectively. In photosystem II, pheophytin a serves as the primary electron acceptor. The oxidation potential of chlorophyll d was higher than that of chlorophyll a in vitro, while the oxidation potential of P740 was almost the same as that of P700. These results help us to broaden our view on the questions about the unique photosystems in Acaryochloris marina.

An overview on chlorophylls and quinones in the photosystem I-type reaction centers

Minor but key chlorophylls (Chls) and quinones in photosystem (PS) I-type reaction centers (RCs) are overviewed in regard to their molecular structures. In the PS I-type RCs, the prime-type chlorophylls, namely, bacteriochlorophyll (BChl) a′ in green sulfur bacteria, BChl g′ in heliobacteria, Chl a′ in Chl a-type PS I, and Chl d′ in Chl d-type PS I, function as the special pairs, either as homodimers, (BChl a′)2 and (BChl g′)2 in anoxygenic organisms, or heterodimers, Chl a/a′ and Chl d/d′ in oxygenic photosynthesis. Conversions of BChl g to Chl a and Chl a to Chl d take place spontaneously under mild condition in vitro. The primary electron acceptors, A0, are Chl a-derivatives even in anoxygenic PS I-type RCs. The secondary electron acceptors are naphthoquinones, whereas the side chains may have been modified after the birth of cyanobacteria, leading to succession from menaquinone to phylloquinone in oxygenic PS I.

Conversion of Chl a into Chl d by Heat-Treated Papain

In 1943, Chl d was first reported as a minor pigment in several red macroalgae. In 1996, a novel cyanobacterium, Acaryochloris marina, was isolated from colonial ascidians, and A. marina was found to contain Chl d as the dominant chlorophyll. However, the biosynthesis pathway of Chl d has not been clarified yet. Chl d is thought to be synthesized from Chl a, like Chl b, while no experimental evidence was given. In 2004, we came across the formation of Chl d from Chl a in acetone or ethanol containing 10 % water in the presence of papain (EC 3.4.22.2) at room temperature in the dark. Chl d was not formed when papain was absent. Here we report the conversion of Chl a into Chl d in aqueous acetone at room temperature in the presence of papain treated at 96°C for 20 min. The yield of Chl d from Chl a did not change, when the heat-treated papain was used. The result indicate that conversion of Chl a into Chl d was not enzymatically caused by papain, but by something else contained in commercially available papain.

The Oxidation Potential of Chl a Is the Lowest — A New Scheme for O2 Evolution in PS II

Oxygenic photosynthesis universally uses Chl a for P680. For water oxidation, higher oxidation potential of chlorophyll is supposed to be favorable. We found, however, Chl a had lower oxidation potential than Chls b and d. Phes a, b and d showed remarkably higher potentials than Chls. The results suggest that Phes, not Chls, might be favorable to water oxidation, although Phes has not yet been used in P680. Further, conventional scheme for water oxidation in PS II has a fundamental problem; redox potential of the Mn-complex is fixed during S-cycle. To explain the enigma, we propose a unique model for O2 evolution, where the stepwise positive shifts of oxidation potentials of the Mn-complex take place to create the great high oxidation power to oxidize water. Lower oxidation states may accept holes from P680+, but the highest oxidation state cannot do this and should utilize photon energy to attain the final state to oxidize water.

Redox Potential of Chlorophyll d

Chl d is a major pigment in a novel oxygenic prokaryote Acaryochloris marina and functions as the primary electron donor P740 in PS I. The midpoint potential of P740 was reported to be +335 mV, which is significantly more negative than that of P700, and thus Chl d had been supposed to possess a lower oxidation potential than Chl a. Here, we report that the oxidation potential of Chl d was +0.88 V vs. SHE in acetonitrile, which was higher than that of Chl a (+0.81 V), and lower than that of Chl b (+0.94 V). The oxidation potential order, Chl b< Chl d < Chl a, can be explained by inductive effect of substituent groups on the conjugated p-electron system on the macrocycle. The results will help us to broaden our views on questions about photosystems in A. marina.