Kimiyuki Satoh

Associate Professor:
Hirokazu Kobayashi

Research Associates:
Satoru Tokutomi
Noritoshi Inagaki

Graduate Students:
Kyoichi Isono *
Yoshihiro Narusaka *
Tatsuya Tomo *

(*from Okayama University)

The efficient energy transformation in the primary processes of photosynthesis is ensured by the highly ordered organization of molecules in the photochemical reaction centers, in a physical, chemical and biological sense. The project in this division is aiming to elucidate the organization of photosystem II reaction center of oxygenic photosynthesis which has a unique property to generate a strong oxidant for utilizing water molecules as electron donor.

In the first approach, the basic molecular structure of the reaction center, which has been isolated in our study, will be analyzed by several methods which include crystallographic analysis, chemical modification and cross-linking analysis and physical methods. Structure-functional analysis will also be conducted on the reaction center proteins using random and site-directed mutagenesis for transformable algae, Synechocystis PCC 6803 and Chlamydomonas reinhardtti. The target of these analyses is the structure and molecular environment of P-680, the primary donor, which determine the redox potential of this system.

In the second approach, the effort will be focused on the elucidation of molecular mechanism of light-regulated metabolic turnover of a subunit of the reaction center, the D1 protein. The process involves some of apparently unique steps; i. e., Iight-regulated gene expression at the translational level, post-translational cleavage of the C-terminal extension of protein and the incorporation of cofactors and subunits into multi-component pigment-protein complexes.

I. Structural. organization of photosystem 11 reaction center.

The structure and molecular interactions of the primary donor (P-680) in the photosystem II reaction center have been investigated by detecting light-induced FT-IR difference spectra upon the formation of its triplet state. From the band positions of the keto and carbomethoxy C=O stretches, the hydrogen-bonding properties of the two chlorophylls of P-680 were found to be asymmetrical; in one chlorophyll both the keto and carbomethoxy C=O groups form hydrogen bonds, while in the other chlorophyll the keto C=O is not hydrogen-bonded whereas the carbomethoxy C=O probably is hydrogen-bonded. Considering the orientation of P-680 analyzed by EPR and the structure of bacterial reaction center determined by X-ray crystallography together with the sequence homology between the D1 and D2 subunits of photosystem II and the L and M subunits of purple bacteria, a model of the P-680 structure and its interactions with apoproteins has been proposed (collaborative research with Drs Noguchi and Inoue, RIKEN). Site-directed modification of the protein subunits responsible for this hydrogen-bonding interaction (Ser-191 and Thr-192) is now in progress in order to prove this hypothesis.

Chemical cross-linking analysis has also been conducted for the isolated photosystem II reaction center to analyze the gross structure; i. e., the nearest neighbors of the constituent subunits and amino acid residues in cross-contact in the reaction center complex.

II. Dynamic aspects of the organization of photosystem II reaction center

The D1 subunit of photosystem II reaction center has a C-terminal extension consisting of 9-16 amino acid residues. Post-translational removal of this extension is absolutely required for constituting the machinery of oxygen evolution in photosystem II. The enzyme involved in this processing has been purified and the N-terminal partial amino acid sequence was determined.

The recognition signal was analyzed for the enzyme using substituted synthetic oligopeptides corresponding to the C-terminal sequence of precursor protein. A series of systematic substitutions around the cleavage site (between Ala-344 and Ala-345) were synthesized to analyze the function of specific amino acid and the sequence in the recognition. The efficiency of these oligopeptides as a substrate and their effectiveness as an inhibitor were examined. The conclusion from this analysis is that the secondary structure formed by the presence of specific amino acids around the cleavage site, i. e., Asp-342 and Ile-343, is important in the recognition.

The synthesis of D1 precursor protein is regulated by light at the stage of translation. The mechanism of this light regulation was analyzed using isolated pea chloroplasts. The isolated chloroplasts was shown to accumulate translation intermediate(s) of D1 protein in the presence of externally added ATP in the dark. The result of analysis suggested that the translation and/or stable accumulation of D1 protein require(s) factor(s) caused by illumination, in addition to energy supply by ATP.

III. Biophysical analyses on the molecular event during photo-transformation of phytochrome.

Phytochrome is a photoreceptor in green plants responsible for a variety of morphogenetic responses including lightregulated gene expression. Molecular event during its phototransformation from an inactive to an active forms, Pr and Pfr, respectively, was studied by using three different biophysical techniques. (1) Primary event on absorption of quanta is proposed to be isomerization of the chromophore composed of an open-tetrapyrrol. We had detected a photoproduct from Pr with rise time of 24ps by sub-picosecond flash photolysis, which has been proven to be a novel one formed directly from the excited state and probably resulted from isomerization (collaborative research with Drs Kandori and Yoshihara, IMS). (2) As the second event, we are proposing proton migration from the chromophore to the protein moiety based on resonance Raman scattering, which may be a trigger of the conformational change. We found that a proton associates with the C-ring nitrogen of the chromophore in Pr dissociates prior to the formation of a meta-intermediate by low-temperature resonance Raman scattering (collaborative research with Drs Mizutani and Kitagawa, IMS. Mizutani et al., 1994, published). (3) Ultraviolet resonance Raman scattering was measured to figure out the details in the conformational change of the protein moiety. Hydrophobicity around (a) Trp(s) in the chromophoric domain, possibly around Trp365 and (or) Trp567, increases on phototransformation. The content of a-helices and nonregular structure which are less populated in the chromophoric domain than the other domains, are almost unchanged by the phototransformation (collaborative research with Drs Mizutani, Kaminaka and Kitagawa, IMS).

Publication List:

Mizutani, Y., Tokutomi, S., Kaminaka, S. and Kitagawa, T. (1993) Ultraviolet resonance Raman spectra of pea intact, large and small phytochromes: Difference in molecular topography of the red- and far-red-absorbing forms. Biochemistry32, 6916-6922.

Noguchi, T., Inoue, Y. and Satoh, K. (1993) FT-IR studies on the triplet state of P in the photosystem II reaction center: Triplet equilibrium within a chlorophyll dimer. Biochemistry32, 7186-7195.

Satoh, K. (1993) Isolation and properties of the photosystem II reaction center. In The Photosynthetic Reaction Center (J. Norris and J. Deisen-hofer, eds.). Academic Press, New York, pp. 289-318.

Satoh, K. (1993) Molecular organization of the photochemical apparatus of oxygenic photosynthesis. In Frontiers of Photo-biology (A. Shima et al., eds.). Elsevier Sciences Publishers, Amsterdam, pp. 3-11.

Taguchi, F., Yamamoto, Y., Inagaki, N. and Satoh, K. (1993) Recognition signal for the C-terminal processing protease of D1 precursor protein in the photosystem II reaction center: An analysis using synthetic oligopeptides. FEBS Lett. 326, 227-231.

Taniguchi, M., Kuroda, H. and Satoh, K. (1993) ATP-dependent protein synthesis in isolated pea chloroplasts: Evidence for accumulation of a translation intermediate of the D1 protein. FEBS Lett. 317, 57-61.

Tomo, T., Enami, I. and Satoh, K. ( 1993) Orientation and nearest neighbor analysis of psbl gene product in the photosystem II reaction center complex using bifunctional cross-linkers. FEBSLett. 323, 15-18.