NATIONAL INSTITUTE FOR BASIC BIOLOGY
DIVSION OF BIOLOGICAL REGULATION AND PHOTOBIOLOGY (ADJUNCT)
- Kimiyuki Satoh
- Associate Professor:
- Hirokazu Kobayashi
- Research Associates:
- Satoru Tokutomi
- 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
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
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
- 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
- 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).
- 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.