  National Insitute for Basic Biology

DIVISION OF BIOENERGETICS

Professor:: Yoshihiko Fujita

Associate Professor:: Sigeru Itoh

Research Associates:: Mamoru Mimuro; Katsunori Aizawa

JSPS-Post-doctral Fellow:: Tohru Ikeya

NIBB Visiting Scientist:: Sung-Jun Kim ¹⁾

Technical Staff:: Akio Murakami

( ¹⁾ from Kyngpook National University)

Primary processes of photosynthesis have been studied. A special attention has been paid for the regulation of PSI/ PSII stoichiometry in thylakoid electron transport system (ETS) in response to photosynthetic environments. Biophysical studies of the excitation energy trans-fer in light-harvesting system and the electron transfer in photosynthetic reaction center complexes have also been carried out.

I. Regulation of PSI/PSII stoichiometry in oxygenic photo-synthesis.

In our previous study, we proposed a hypothesis that (1) stoichiometry between the two photosystems is regulated in response to light regime for photosynthesis so as to maintain the efficiency of light-energy conversion in photosynthesis, and that (2) the stoichiometry is adjusted by regulation of PSI abundance, mainly regulation of PSI formation; redox state of electron component(s), probably cytochrome b₆-f complex, is monitored, and PSI formation is regulated by the signal from monitoring system (cf. Fig. 1). As one of trials for evaluating this hypothesis, the identification of the step to be regulated in PSI formation was carried out. Assay of mRNA activity for synthesis of PsaA/B polypeptides by the in vitro translation showed that the mRNA abundance re-mains constant irrespective of the PSI/ PSII regulation while rates of PsaA/B polypeptides sysnthesis varies by a factor of 2. Sensitivity of PsaA/B synthesis to the inhibitors for peptide elongation was different in differently regulated syn-thesis; the synthesis was less sensitive when the synthetic rate remained at low level under the light mainly exciting PSI, but the sensitivity became high when the synthesis was accelerated under the light mainly for PSII excitation. Results indicated that the apoprotein synthesis is regulated at the step of translation, and further suggest that the translation is suppressed under PSI light and is re-leased from the suppression under PSII light. Since apoprotein synthesis depends on Chl a supply, we also analyzed Chl a biosynthesis. The step before Chl a synthetase was not regulated. However, a marked accumulation of Pchlide upon suppression of PSI synthesis suggested a possibility for regulation of Chl a supply to each photosystem assembly. We suspect that the step(s) for formation of ribosome=mRNA complex or installation of Chl a into peptides is the target(s) for the regulation.

Fig. 1.
Schematic presentation of regulation of PSI/PSII stoichiometry and electron transport state. Shaded area in each circle indicates level of reduced state of each component. Reduced PSII and oxidized PSI are photochemically inactive, respectively, and large occurrence of such states causes low efficiency of photo-synthesis. PQ, plastoquinone; Cyt b ₆ - f, cytochrome b ₆ - f complex; PC, plastocyanin.

In the cyanophyte Synechocystis PCC 6714, NaCl- and C0₂-stresses for autotrophic growth were found to induce variation of PSI/PSII stoichiometry besides light regime. Again, PSI abundance varied in these cases. Further, the activity of cytochrome c oxidase, another terminal of ETS in thyalkoids (Ohki and Fujita (1995) Protoplasma in press), varied simultaneously in the same direction to PSI variation in all cases. Since these stresses affect the steady state of thylakoid ETS in a similar manner to the light regime, we suspect that the mechanism is common among regulations of PSI/PSII stoichiometry in response to three different environmental stresses, and further that cytochrome c oxidase activity is also regulated under the same mechanism.

II. Energy transfer in light-harvesting pigment system.

Energy transfer processes in photosynthetic pigment systems have been studied using steady-state and time-resolved spectroscopy. Carotenoid-containing pigment systems in algae and chlorosomes of photosynthetic green bacteria were studied. A new method for spectral analysis was also introduced to biological study.

Dynamics of excited state of B-carotene in n-hexane were analyzed in subpico second time-range. Decay kine-tics of the S₂ state were single-exponential with a lifetime of 195 fs ą10 fs, and a dynamic stokes shift was not observed in a 50-fs time range. Spectral analysis was made for peridinine-Chl a-protein complex newly isolated from the dinoflagellate Alexandorium cohorticula. Magnetic circular dichroism indicated that magnetic mixing of excited states of peridinine, S₁ and S₂, did not occur, and Chl a did not interact with other pigments. In the light-harvesting pigment-protein complex 11 of the siphonous green alga Bryposis maxima, siphonein and siphonaxanthin were found to trans-fer the energy directly to Chl a. The feature is the same as the energy transfer from fucoxanthin to Chl a in the fucoxanthin-containing pigment protein complex of brown algae, suggesting a general property of the energy transfer in carotenoid-Chl pigment-protein complex.

Based on the in vitro self assembly of Bchl c and the in vivo time-resolved fluorescence polarization spectrum in the ps time-range, a model was proposed for molecular structure of Bchl c in chlorosomes.

A new method for spectral analysis, principal multi-component spectral estimation method, was found to valid in application to biological system. The method is advantageous in that the analysis does not require the assumption on number of components and spectral shape.

III. Electron transfer in reaction center complexes.

The study has been focussed on the electron transfer in photosystem I complex. Phylloquinone in PSI complex functions in the electron transfer from Chl a to iron-sulfur centers. After replacing phylloquinone with various artificial quinones, rates of the electron transfer from Chl a to respective quinones were determined in a pstimerange. The rates were found to be a function of free energy difference as predicted by the quantum mechanical Marcus theory. Protein environments regulate the quinone energy levels. The matching between the energy difference and the reorganization of reaction coordinates is a key for the optimal rate. Photodamage of PSI in vivo was also studied.

Reaction center complexes in the an-aerobic green sulfur bacteria were also studied. The highly purified complex from Chlorobium limicola consisted of only one kind of polypeptide and forms a "homo-dimer complex". The feature may be characteristic to primitive photo-synthetic system in these organisms.

Publication List:

Abe, S., Murakami, A., Ohki, K., Aruga, Y. and Fujita, Y. (1994) Changes in stoichiometry among PSI, PSII and Cyt b ₆ - f complexes in response to chromatic light for cell growth observed with the red alga Prophyra yezoensis. Plant Cell Physiol. 35:901-906.

Fujita, Y., Murakami, A., Aizawa, K. and Ohki, K. (1994) Short-term and long-term adaptation of the photosynthetic apparatus: Homeostatic proper-ties of thylakoids. In The Molecular Biology of Cyanobactria (D.A.Bryant ed.) Kluwer Academic Publishers, Dordrecht, pp.677-692.

Gu, T.-Q., Iwama, Y., Murakami, A., Adhikary, S.P. and Fujita, Y. (1994) Changes in the cytochrome c oxidase activity in response to light regime for photosynthesis observed with the cyanophyte Synechocystis PCC 6714. Plant Cell Physiol. 35:1135-1140.

Ikeya, T., Ohki, K., Takahashi, M. and Fujita, Y. (1994) Photosynthetic characteristics of marine Synechococcus spp. with special reference to light environments near the bottom of the euphotic zone of the open ocean. Marine Biol. 118: 215-221.

Iwaki, M. and Itoh, S. (1994) Reaction of reconstituted acceptor quinone and dynamic equilibration of electron transfer in the photosystem I reaction center. Plant Cell Physiol. 35: 983-993.

Kandori, H., Sasabe, H. and Mimuro, M. (1994) Direct determination of a lifetime of the S₂ state of B -carotene by femtosecond time-resolved fluorescence spectroscopy. J. Am. Chem. Soc. 116: 2671-2672.

Kumazaki, I., Iwaki, M., Ikegami, I., Kandori, H., Yoshihara, K. and Itoh, S. (1994) Rates of primary electron transfer reactions in the photosystem I reaction center reconstituted with different quinones as the secondary acceptor. J. Chem. Phys. 98: 1 1220-11225.

Mimuro, M., Hirota, M., Nishimura, Y., Moriyama, T., Yamazaki, I., Shimada, K. and Matsuura, K. (1994) Molecular organization of bacteriochlorophylls in chlorosomes of the green photosynthetic bacterium Chloroflexus aurantiacus: Studies of fluorescence depolarization accompanied with Photosyn. Res. 41: 181-191.

Mimuro, M., Nozawa, T., Tamai, N., Nishimura, Y. and Yamazaki, I. (1994) Presence and significance of minor antenna components in the energy transfer sequence of a green photosynthetic bacterium Chloroflexus aurantiacus. FEBS Lett. 340: 167-172.

Nakayama, K. and Mimuro, M. (1994) Chlorophyll forms and light energy transfer in light-harvesting chlorophyll a/b protein complexes from the siphonous green alga Bryopsis maxima. Biochim. Biophys. Acta 1184: 103-110.

Nakayama, K., Mimuro, M., Nishimura, Y., Yamazaki, I. and Okada, M. (1994) Kinetic analysis of energy transfer processes in LHCII isolated from a siphonous green alga Bryopsis maxima with use of a picosecond fluorescence spectroscopy. Biochim. Biophys. Acta 1188: 117- 124.

Ogata, T., Kodama, M., Nomura, S., Kobayashi, M., Nozawa, T., Katoh, T. and Mimuro, M. (1994) A novel peridinine-chlorophyll a protein (PCP) isolated from the dinoflagellate Alexandorium< cohorticula: A higher pigment content and plural spectral forms of peridinine and chl a. FEBS Lett. 356: 367-371.

Uehara, K., Mimuro, M., Ozaki, Y. and Olson, J.M. ( 1994) The formation and characterization of the in vitro polymeric aggregates of bacteriochlo-rophyll c homologs from Chlorobium limicola in aqueous solution in the presence of monogalactosyl digly-ceride. Photosyn Res. 41: 235-243.

Yamazaki, T., Nishimura, Y., Yamazaki, I., Hirano, M., Matsuura, K., Shimada, K. and Mimuro, M. (1994) Energy migration in a allophycocyanin B trimer with linker: Analysis by principal multicomponent spectral separation method. FEBS Lett. 353: 43-47.