NATIONAL INSTITUTE FOR BASIC BIOLOGY

DIVISION OF BIOENERGETICS

Professor:

Yoshihiko Fujita

Associate Professor:

Sigeru Itoh

Research Associates:

Mamoru Mimuro
Katsunori Aizawa

Visiting Scientist:

Sung-Jun Kim *

JSPS-Post-doctral Fellow:

Tohru lkeya

Vlsiting Fellow:

Yuji Nakajima **

Technical Staff:

Akio Murakami

( * from Kyngpook National University)
( ** from Advanced Technology Research Center Mitsubishi Heavy Industries, LTD)

Oxygenic photosynthesis is an energy yielding metabolism that supports autotrophic life of plants. Primary process of photosynthesis, Iight-energy conversion, in thylakoids is the target for the study of this research division. The research has been conducted for regulatory aspects of thylakoid system in response to photosynthetic environments and also for molecular mechanism of light-capturing and conversion in functional molecules.

I. Regulation of PSI/PSII stoichiometry

Light-energy conversion in oxygenic photosynthesis is driven by the two light reactions of the two photosystems, PSI and PSII. Imbalance between the two light reactions causes lowering the efficiency of energy conversion. Our previous study with cyanophytes has revealed that plants have an ability for regulating the stoichiometry of the two photosystems so as to compensate imbalance between the two light reactions. Further, at least in the cyanophyte Synechocystis PCC 6714, the stoichiometry is regulated by the control of PSI formation, which occurs in response to the signal comming from the state of the electron transport between the two photosystems.

Controlling mechanism for PSI formation has been conducted in two ways; determining the target sites for regulation (1)in Chl a synthesis and (2)in apoprotein synthesis. Analysis for dynamics of intermediates of Chl a synthesis yielded following results (Fujita et al., in press): (1) Pchlide reduction is regulated in parallel with regulation of PSI formation. The activity is suppressed under conditions for suppression of PSI formation, such as light regime preferentially exciting PSI (PSI Iight). The suppression is released by the conditions for stimulation of PSI formation, such as light regime exciting preferentially PSII (PSII light). (2) Changes in the activity of Pchlide reduction are neither due to acceleration-deceleration of PSI synthesis nor due to differential occurrence of light-dependent Pchlide reduction. The enzyme activity itself is regulated. Analysis for synthesis of PsaA/B peptides, core peptides of PSI complex, with pulse-labeling method revealed that (1) inhibitors for peptide elongation suppresses PsaA/B synthesis differently in accelerated and decelerated PSI formations. Inhibition at low inhibitor concentrations was stronger in the former than that in the latter, suggesting that pausing of pepetide elongation becomes longer under conditions for suppression of PSI formation. (2) The inhibitor for initiation of translation showed similar effect. (3) Effect of chrolamphenicol suggested that initiation of translation is also regulated; it is suppressed by PSI Iight, and PSII light releases from suppression. Together with previous results, evidences obtained show that regulation of PSI formation in PSI/PSII adjustment occurs at the translation level of PsaA/B synthesis. Since Chl a stabilizes PsaA/B peptides, we assume that regulation of psaA/B expression at translation level is achieved at least by Chl a supply.

Changes in thylakoid composition in the green alga Chlamydomonas reinhardtii in response to light quality were determined under collaboration with Dr. A. Melis (Univ. Cal., Berkeley). Results indicated that the pattern of change was similar to that in the cyanophyte Synechocystis PCC 6714. Abundance of PSI was markedly changed while that of PSII and Cyt b6-f remained fairly constant, resulting higher PSI/PSII ratio under PSI Iight and lower ratio under PSI Iight. Changes in PSI/PSII improved photosynthetic efficiency under respective light regimes. Differently from cyanophyte system, a marked decrease in LHC, in parallel with PSI decrease, occurred under PSI Iight. Results suggest that Chl supply to assembling of Chl-protein complexes is a primary determinant for adjustment of PSI/PSII stoichiometry in this organism also.

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II. Mechanism of energy transfer in light-capturing pigment system

Examination was made for function of carotenoids in photosynthesis. Relationship between molecular structure and optical properties in the excited state was mainly analyzed. (1) The forbidden S1 state of neurosporene and spheroidene, which is responsible for the energy transfer to bacteriochlorophylls a, was directly measured by the one-photon absorption spectroscopy; the energy level was higher than and close to that of acceptors. (2) It was proved that the relaxation processes from the excited state of carotenoids follow the energy gap law of internal conversion. Presence of a keto carbonyl group induced a rapid internal conversion to the S1 state, which was favorable for energy transfer to chlorophylls a. This corresponds to the case for fucoxanthin in brown algae and diatom. (3) The S2 Iifetime of B-carotene was directly measured by the up-conversion method to be 195 +- 10 fs with an isotropy ratio of 0.39 +- 0.02. (4) Theoretical analysis of the energy transfer processes was carried out by calculation of the energy transfer matrix elements between neurosporene and bacteriochlorophyll a. The electron exchange mechnism is concluded to be less probable, even if the forbidden singlet state is involved. (5) Based on these results, the energy transfer through the dipole-multipole interaction from the Sl state to (bacterio)chlorophyll a is proposed.

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III. Mechnism of electron transfer in reaction center complex

Mechanism of the primary photoenergy conversion in photosynthesis was studied in plant and bacterial systems by the combination of modern spectroscopy, and the biochemical and genetical manipulation of the protein complexes. In the PSI reaction center complex, the time constant for the primary charge separation between the electron donor chlorophyll (P700) and the acceptor chlorophyll was measured to be 7 x 10^(-12) s. The following reaction with phylloquinone was shown to take place with a time constant of 38 x 10^(-12) s The rate varied when various artificial quinones were introduced into the reaction center complex in place of native phylloquinone. Functions of quinones in various reaction center preparations were also studied. It is shown that the fine adjustment of the energy levels of native electron transfer components enable the highly efficient, ultra-fast electron transfer reactions. We isolated and characterized the reaction center complex of green bacteria, which seems to be ancestry to PSI reaction center. The complex was shown to have a polypeptide composition simpler than that of plant PSI but to undergo almost similar activities. Unique nature of the iron-sulfur centers in this complex was shown by cryogenic EPR studies. The structurefunction relationship of the iron-sulfur centers of the photosynthetic bacterium Rhodobacter capsulatus was also investigated by the site-directed mutagenesis.

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Publication List:

lkegami, I., Itoh, S., Warren, P.G. and Golbeck, J.H. (1993) Reconstitution of the photosystem I secondary quinone acceptor (A1) in the P700-Fx core isolated from Synechococcus PCC6301. Plant Cell Physiol. 34, 849-853.

Katoh, T. Tanaka, A. and Mimuro, M. (1993) Isolation of fucoxanthin-chlorophyll a/c protein assembly from brown algae. In: Methods in Enzymology, Vol. 214, Packer, R. ed., Academic Press, New York, pp. 402-412.

Matsuura, K., Hirota, M., Shimada, K. and Mimuro, M. (1993) Spectral forms and orientation of bacteriochlorophylls c and a in chlorosomes of the green photosynthetic bacterium Chloroflex aurantiacus. Photochem. Photo-biol. 57, 92-97.

Mimuro, M. (1993) Identification of the terminal energy donor to the reaction center in the pigment system of green plants by time-resolved fluorescence spectroscopy at -196C. Plant Cell Physiof 34, 321-327.

Mimuro, M., Nagashima, U., Nagaoka, S., Takaichi, S., Yamazaki, I., Nishimura, Y. and Katoh, T. (1993) Direct detection of a low-lying singlet excited (2Ag) state of a linear carotenoid, neurosporene, in solutions. Chem. Phys. Lett 204, 101-105.

Mimuro, M., Takaichi, S., Yamamoto, Y., Ito, M., Nagaoka, S., Nishimura, Y., Yamazaki, I., Katoh, T. and Nagashima, U. (1993) The effect of molecular structure on the relaxation processes in carotenoids containing a carbonyl group. Chem. Phys. Lett. 213, 576-580.

Murakami, A. and Fujita, Y. (1993) Regulation of stoichiometry between PSI and PSII in response to light regime for photosynthesis observed with Synechocystis PCC 6714: Relationship between redox state of Cyi b6-f complex and regulation of PSI formation. Plant Cell Physiot 34, 1175-1180.

Nagashima, K.V.P., Itoh, S., Shimada, K. and Matsuura, K. (1993) Photooxidation of reaction center-bound cytochrome c and generation of membrane potential determined by carotenoid band shift in the purple photosynthetic bacterium, Rhodospirillum molischianum. Biochim. Biophys. Acta, 1140, 297-303.

Nagae, H., Kakitani, T., Katoh, T. and Mimuro, M. (1993) A theoretical study on the interaction between carotenoids and bacteriochlorophyll: Calculation of the extitation transfer matrix elements between neurosporene S2 and Sl states and Bchl a S2 and S1 states. J. Chem. Phys. 98, 8012-8023.

Nishimura, Y., Shimada, K., Yamazaki, I. and Mimuro, M. (1993) Energy transfer processes in Rhodobacter palustris grown under low-light conditions: Heterogenous composition of LH2 complexes and parallel energy flow pathways. FEBS Lett. 329, 319-323.

Oh-oka, H., Kakutani, S., Matsubara, H., Malkin, R. and Itoh, S. (1993) Isolation of the photoactive reaction center complex that contains three types of Fe-S centers and a cytochrome c subunit from the green sulfur bacterium Chlorobium limicola f thiosulfatophilum, strain Larsen. Plant CellPhysioL 34, 93-101.

Uehara, K., Hioki, Y. and Mimuro, M. (1993) The chlorophyll a aggregate absorbing near 685 nm is selectively formed in aqueous tetrahydrofuran. Photochem. PhotobioL 58, 127-132.

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Supplements to 1992

Aizawa, K. and Fujita, Y. (1992) Regulation of PSI formation induced by light quality observed with Synechocystis PCC 6714. In Research in Photosynthesis (Murata, N. ed., Kluwer Academic Publishers, Dordrecht), Vol. IV, pp. 329-332.

Fujita, Y., Murakami, A. and Aizawa, K. (1992) Light acclimation of thylakoid system in cyanophytes: Regulation of PSI formation in response to light regime. In Research in Photosynthesis (Murata, N. ed., Kluwer Academic Publishers, Dordrecht), Vol. IV, pp. 301-308.

Hoshina, S., Sue, S., Wada, K., Enami, I. and Itoh, S. (1992) Stabilization of iron-sulfur centers A and B in photosystem I particles by chemical crosslinking. In Research in Photosynthesis (Murata, N. ed., Kluwer Academic Publishers, Dordrecht), Vol. I, pp. 581-584.

Itoh, S., Mimuro, M. and lwaki, M. (1992) Organization of chlorophylls in photosystem I reaction center: Study by LD and fluorescence measurements in the ether-extracted particles which contain 11 chlorophyll/P700. In Research in Photosynthesis (Murata, N. ed., Kluwer Academic Publishers, Dordrecht), Vol. I, pp. 541-544.

Iwaki, M., Taksahashi, M., Shimada, K. and Itoh, S. (1992) Identification of the quinone binding site in PSI reaction center complex by photoaffinity labeling. In Research in Photosynthesis (Murata, N. ed., Kluwer Academic Publishers, Dordrecht) Vol. I, pp. 529-532.

Katoh, T. and Mimuro, M. (1992) S1 state of fucoxanthin involved in energy transfer to chlorophyll a in the light harvesting protein of brown algae. In Research in Photosynthesis (Murata, N. ed., Kluwer Academic Publishers, Dordrecht) Vol. I, pp. 227-230.

Kobayashi, M., Van de Meent, E.J., Oh-oka, H., Inoue, K., Itoh, S., Amesz, J. and Watanabe, T. (1992) Pigment composition of heriobacteria and green sulfur bacteria. In Research in Photosynthesis (Murata, N. ed., Kluwer Academic Publishers, Dordrecht), Vol. I, pp. 393-396.

Matsuura, K., Hirota, M., Shimada, K. and Mimuro, M. (1992) Pigment orientation and energy transfer kinetics in chlorosomes of green photosynthetic bacteria. In Research in Photosynthesis (Murata, N. ed., Kluwer Academic Publishers, Dordrecht) Vol. 1, pp. 113-116.

Mimuro, M. (1992) A Iong wavelength antenna in photosynthetic pigment system. In Research in Photosynthesis. (Murata, N. ed., Kluwer Academic Publishers, Dordrecht), Vol. 1, pp. 259-262.

Mimuro, M., Matsuura, K., Nishimura, Y., Shimada, K. and Yamazaki, I. (1992) Excitation energy transfer processes in green photosynthetic bacteria: Analysis in picosecond time domain. In Research in Photosynthesis (Murata, N. ed., Kluwer Academic Publishers, Dordrecht) Vol. I, pp. 17-24.

Murakami, A. and Fujita, Y. (1992) In vivo electron transport from Cyt b6-f complex to photosystem I complex in Synechocystis PCC 6714. In Research in Photosynthesis (Muratsa, N. ed., Kluwer Academic Publishers, Dordrecht), Vol. II, pp. 503-506.

Nakayama, K., Mimuro, M., Nishimura, Y. and Okada, M. (1992) Chlorophyll forms and excitation energy transfer pathways in light-harvesting chlorophyll a/b protein complexes from the siphonous green alga Bryopsis maxima. In Research in Photosynthesis (Murata, N. ed., Kluwer Academic Publishers, Dordrecht) Vol. I, pp. 219-222.

Oh-oka, H., Kakutani, S., Itoh, S., Matsubara, H. and Malkin, R. (1992) Isolation and characterization of the photoactive reaction center complex from the green sulfur bacterium Chlorobium lumicola. In Research in Photosynthesis (Murata, N. ed., Kluwer Acsademic Publishers, Dordrecht) Vol. I, pp. 385-388.

Ohki, K. and Fujita, Y. (1992) Lightdependent maintenance of active nitrogenase in the non-heterogenous cyanophyte Trichodesmium sp. NIBB 1067. In Research in Photosynthesis (Murata, N. ed., Kluwer Academic Publishers) Vol. IV, pp. 103-106.

Shimada, K., Nishimura, Y., Yamazaki, I. and Mimuro, M. (1992) Excitation energy flow in Roseobacter denitrificance (Erythrobacter sp. OCh 114) at low temperature. In Research in Photosynthesis (Murata, N. ed., Kluwer Academic Publishers, Dordrecht) Vol. I, pp. 137-140.