NATIONAL INSITUTE FOR BASIC BIOLOGY

National Institute for Basic Biology

Division of Biological Regulation and Photobiology

(Adjunct)

Professor (adjunct):
Kimiyuki Satoh
Associate Professor (adjunct):
Hirokazu Kobayashi
Research Associate:
Noritoshi Inagaki
NIBB Fellow:
Hiroshi Kuroda
Graduate Students:
Yoshihiro Narusaka*
Tatsuya Tomo*
Akihiko Tohri*
(*from Okayama University)


From an energetic point of view, the conversion of radiant energy into chemical energy in photosynthesis is the most important biological process on our planet. The highly efficient energy conversion in this process is ensured by the highly ordered organization of molecules in the photochemical reaction center, in a physical, chemical and biological sense. The project in this division is aiming to elucidate the dynamic as well as static organization of molecules in the photosystem II (PSII) reaction center (RC) of oxygenic photosynthesis which has a unique property to generate a strong oxidant leading to the extraction of electrons from water for the synthesis of organic compounds.

In the first approach, molecular organization of the PSII RC, which has been identified in our study, will be analyzed by biochemical and biophysical techniques which include crystallographic analysis, chemical modification & cross-linking analysis and optical & EPR spectroscopies. Structure-functional analysis for the constituent subunits will also be conducted using random and site-directed mutants of transformable algae, Synechocystis PCC 6803 and Chlamydomonas reinhardtii. The principal target of these analyses is the structure and molecular environment of the RC chlorophylls, P-680, which determine the redox potential of this photosystem.

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 PSII RC, D1 protein. Some of unique phenomena are involved in this metabolic process; i.e., photo-damage of the function of photochemical RC, specific degradation by proteases of a photo-damaged protein subunit, light-regulated gene expression at the translational level, post-translational cleavage of the C-terminal extension and incorporation of cofactors and subunits into multi-component pigment-protein complexes.


I. Structural organization of photosystem II reaction center

(1) The purified PSII RC contains D1 and D2 proteins, a and b subunits of cytochrome b-559 (Cytb-559) and the psbI gene product. It also contains a number of cofactors important in the primary processes of PSII, such as chlorophyll a (Chl a), pheophytin a (Pheo a) and b-carotene (b-Car), as well as cytochrome b-559 heme. Taking advantage of biased distributions of amino acids in the constituent protein subunits, we have conclusively demonstrated that they are present in an equimolar ratio in the isolated complex. We also have re-evaluated the pigment stoichiometry in the isolated PSII RC, by HPLC and ICP emission spectroscopy, to be 6/2/2/1 for Chl a/Pheo a/b-Car/Cytb-559.

(2) Pheo a molecule on the inactive branch of photochemical electron transport system in the isolated PSII RC was identified by fluorescence excitation spectroscopy at 77K, and confirmed by absorption, linear dichroism (LD) and magnetic circular dichroism spectra (in collaboration with Dr. Mimuro). 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. Two b-Car 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 the optical properties in the PSII RC.

(3) Pigments in the purified PSII RC were extracted with diethyl ether containing varied amount of water. As mentioned above, the preparation originally contained approximately six Chl a two b-Car and two Pheo a per one photochemically active Pheo a. The treatment with 30 - 50% water-saturated ether solution extracted one b-Car, as well as one Chl a absorbing at 677 nm, remaining 60% of the photochemical activity to reduce Pheo a. With 60 - 80% water-saturated ether, almost all of the b-Car in the RC was extracted. However, nearly 50% of the photochemical activity was retained in the complex, after the ether treatment. The absorption, fluorescence excitation and LD spectra demonstrated the presence of two spectral forms of b-Car in the PSII RC. The short-wavelength form of b-Car, with absorption peaks at 429, 458 and 489 nm, was selectively extracted with ether at the low water content, whereas the long-wavelength form, with peaks at 443, 473 and 507 nm, was extracted only at the higher water content. The extraction of carotenoid enhanced the photobleaching of Chl. Based on the photobleaching experiments it was suggested that Chl a forms with peaks at 667 and 675 nm are located close to the short-wavelength form of b-Car that can transfer excitation energy to the photoactive Pheo a on the D1 protein. Based on these observations, the topology of pigments in the PSII RC was discussed.

(4) Isolated PSII RC contains no functional plastoquinone, QA and QB. However, externally added quinones, such as dibromomethylisopropyl benzoquinone (DBMIB), serve as the electron acceptor. The present study provided evidence that DBMIB fully replaces the function of QA and rapidly oxidized Pheo- even at 77 K (in collaboration with Dr. Itoh). The use of DBMIB in the nanosecond spectroscopy at 77 K enabled us to inspect the spectrum of the P680+(DBMIB-) state that is fully reversible and contains neither the absorption change of the excited states nor the irreversible bleaching of the other pigments. The feature of the spectrum was discussed in relation to the difference spectra of 3P680/P680 and Pheo-/Pheo couples measured at 77-280 K.


II. Dynamic aspects of molecular organization

(1) The light-regulated synthesis of the D1 protein in PSII RC was analyzed using isolated pea chloroplasts. In the presence of externally added ATP in darkness, isolated chloroplasts accumulated two proteins of about 22 and 24 kDa which precipitate with specific antibodies raised against the D1 protein. By chasing in the light, these proteins disappeared concomitant with the appearance of the precursor and the mature forms of D1 protein. The pulse-chase experiment in the light and the polysome analysis unequivocally concluded that these two components were the paused intermediates of D1 protein. An unexpected observation was that these translational intermediates accumulated in illuminated chloroplasts by the combined addition of ATP and inhibitors of photosynthetic electron transport, such as atrazine, 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 3,5-dibrome-4-hydroxybenzonitrile. This indicates that the synthesis of the full-length D1 protein requires a factor(s) generated by the operation of photosynthetic electron transport. The accumulation of the full-length D1 protein, however, was induced even in the presence of inhibitory concentration of atrazine in the light when both 2,6-dichlorophenolindophenol and ascorbate, a system donating electrons to the photosystem I (PSI), were also present, and in darkness as well upon the addition of a reductant, dithiothreitol (DTT), suggesting that the translation of D1 protein is regulated via redox change of a component(s) around the PSI.

The accumulation of these two intermediates was also observed by pulse-labeling in in vitro run-off translation system in darkness using thylakoid-bound polysomes and oxidized S100 fraction prepared from pea chloroplasts. However, the synthesis of full-length D1 protein was achieved in this system by the addition of DTT-reduced stroma fraction. The fractionation of DTT-reduced stroma by a gel-filtration column demonstrated that the major activity corresponded to the molecular mass of about 40 kDa. The fractionation by DEAE anion-exchange chromatography and by ammonium sulfate precipitation of the active fraction indicated that the purification of this redox-responding regulatory factor is possible by the standard biochemical procedures, although further steps of purification is evidently required at present.

(2) The D1 protein of PSII RC is synthesized as a precursor that is processed by cleavage at the C-terminus. The C-terminal processing is necessary for the assembly of water-oxidizing machinery (Mn-cluster). The enzyme involved in this process was purified from sonicated extracts of thylakoids, by a method that includes chromatography on QAE-anion exchange, hydroxylapatite, Cu-chelating affinity and gel-filtration columns. Based on the amino acid sequence data of the purified protease, cDNA clones encoding the enzyme was identified and sequenced, from a spinach green leaf cDNA library. By these analysis, 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 consisted of 150 amino acids which is 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 (Fig. 1). The enzyme can be expressed in E. coli in its active form and purified in homogeneity.

Three sorts of compounds were used as substrate for the analysis of enzymatic activity, i.e., (i) synthetic oligopeptides corresponding to the C-terminal sequence of the precursor D1 protein, (ii) in vitro transcribed/translated precursor D1 protein, and (iii) in vivo synthesized precursor D1 protein in thylakoid membranes. A series of substitutions at Ala-345, for example, were shown to have marked effects on the value of Vmax, in the previous analysis. Mixed culture experiments using genetically-engineered Chlamydomonas mutants substituted at this position (from Ser to Val or Gly) demonstrated that these substitutions seriously affect the viability of this organism. No specific inhibitor, except for substituted C-terminal oligopeptides of precursor D1 protein, could be found for the enzyme. The identification of the catalytic center of this unique enzyme is now in progress.

Fig. 1.
A schematic drawing of maturation steps and function of C-terminal processing protease (CtpA) for the precursor D1 protein. The CtpA is synthesized in cytosol as a precursor with the N-terminal extension, consisting of a transit sequence (blue; T) and a signal sequence (red; S), for translocation into thylakoid lumen. The signals on the pCtpA are excised in two steps during the import, as shown in the figure. The mature CtpA cleaves off C-terminal extension of the pD1 protein as an integral event for the organization of oxygen evolving machinery in the PSII. Inset shows a hydropathy plot of the spinach pCtpA by a program of Kyte and Doolittle (1982). Two arrows indicate putative cleavage sites for the maturation of pCtpA.


Publication List:
Inagaki, N., Yamamoto, Y., Mori, H. and Satoh, K. (1996) Carboxyl-terminal processing protease for the D1 precursor protein: cloning and sequencing of the spinach cDNA. Plant Mol. Biol. 30, 39-50.
Itoh, S., Iwaki, M., Tomo, T. and Satoh, K. (1996) Dibromothymoquinone (DBMIB) replaces the function of QA at 77 K in the isolated photosystem II reaction center (D1-D2-cytochrome b559) complex: Difference spectrum of the P680+(DBMIB-) state. Plant Cell Physiol. 37, 833-839.
Kuroda, H., Kobashi, K., Kaseyama, H. and Satoh, K. (1996) Possible involvement of a low redox potential component(s) downstream of photosystem I in the translational regulation of the D1 subunit of the photosystem II reaction center in isolated pea chloroplasts. Plant Cell Physiol. 37, 754-761.
Narusaka, Y., Murakami, A., Saeki, M., Kobayashi, H. and Satoh, K. (1996) Preliminary characterization of a photo-tolerant mutant of Synechocystis sp. PCC 6803 obtained by in vitro random mutagenesis of psbA2. Plant Sci. 115, 361-366.
Satoh, K. (1996) Introduction to the photosystem II reaction center -Isolation and biochemical and biophysical characterization-. in "Oxygenic Photosynthesis: The Light Reactions" (D.R. Ort & C.F. Yocum eds), Kluwer Academic Publishers (Dordrecht), Vol. 4, pp.193-211.
Takahashi, Y., Utsumi, K., Yamamoto, Y., Hatano, A. and Satoh K. (1996) Genetic engineering of the processing site of D1 precursor protein of photosystem II reaction center in Chlamydomonas reinhardtii. Plant Cell Physiol. 37, 161-168.

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Last Modified: 12:00, June 27, 1997