1. Current Research and Principal Research Interests
are interested in "Structural Biology on Biologically Important Membrane
Proteins and Supra-Complexs" and "Biological Structural Chemistry on Enzymatic
Reactions by Four-Dimensional Protein Crystallography". In order to
succeed in these projects, if needed, we also develop new technologies regarding
X-ray protein crystallography with synchrotron radiation.
1.1 Structural Biology on Oxygen-Evolving Photosystem
II Membrane Protein Supra-Complex
Photosystem II (PSII) is
a large membrane protein complex (molecular weight: >640kDa as one dimer)
located on thylakoid membrane of cyanobacteria and higher-plant cells, that
carries out light-induced electron transfer and water-splitting reactions,
leading ultimately to the formation of molecular oxygen. The molecular oxygen
is one of the most important materials for our life on the earth.
We reported in 2003 the crystal structure of PSII from Thermosynechococcus
vulcanus at 3.7Å resolution [1, 7].
We have recently succeeded in crystallizing PSII in
much higher diffraction quality, calculating new electron density maps at
3.3Å resolution, and constructing new structure models of four major
transmembrane (TM) subunits: D1, D2, CP47 and CP43, two small TM subunits
of cytochrome b-559, and three extrinsic soluble subunits:
33k- and 12k-Da subunits, and cytochrome c-550. In this stage
of crystal structure analysis, we determine the arrangement of PSII pigments,
such as chlorophyls (Chls), the water-splitting manganese (Mn) cluster, carotenoids
(Cars), and so on. The Chls in the reaction center (RC) play important
roles in the primary electron transfer pathway of PSII. One major question,
how to transfer the solar energies absorbed by antenna Chls held by CP47
and CP43 to the RC-Chls held by D1 and D2, was resolved by theoretical calculations
based on our structure model, and it was revealed that the four specific
Chls mediated the energy transfer from the antenna Chls to the RC .
The Cars have long been reported that isolated PSII-RC
contains two molecules of β-carotenes. Our crystal structural analysis
revealed two regions of electron density in the vicinity of RC that could
be attributed to two molecules of Cars. Although the density for one
of the two Cars is relatively weak and should be considered tentative, these
Cars are located in a cavity surrounded by D1, D2, CP43 and cytochrome b-559,
and are in a relatively close location with each other. The arrangement
of these putative Cars yields important functional consequences on the secondary
electron transfer pathway within PSII.
For understanding the reaction
mechanism of PSII, most important and interesting is the precise structure
of water-splitting and oxygen-evolving Mn cluster, which is remaining unclear
because of the relatively lower resolution of structure analysis.
At present, there are two major structure models in the world for the Mn
cluster containing four Mn atoms. To clarify the real structure of the Mn
cluster and the arrangement of PSII pigments relating to the secondary electron
transfer pathway mentioned above, to improve more and more the diffraction
quality of PSII crystals is the first target of our laboratory.
1.2 Biological Structural Chemistry on Enzymatic Reactions
of Photo-Reactive Nitrile Hydratase by Four-Dimensional Protein Crystallography
Nitrile hydratase (NHase)
catalyzes the hydration of nitriles to the corresponding amides (R-CN + H2O
→ R-CONH2) and is used for industrial production of acrylamide and nicotinamide.
NHase consists of two distinct subunits, α and β, each with a molecular
weight of 23kDa, and has a non-heme iron (III) as the catalytic center.
The iron center is coordinated by two main-chain nitrogen atoms and three
side-chain sulfur atoms of cystein residues in a conserved motif of a-subunit,
Cys(109)-Ser-Leu-Cys-Ser-Cys(114). This enzyme shows unique photo-reactivity.
In the nitrosylated inactive form of enzyme, one nitrogen monoxide (NO)
molecule occupy the sixth coordination site, and contrary, in the photo-activated
form, a water molecule replaces the NO molecule.
The crystal structure of nitrosylated NHase has been
elucidated by our group, and αCys112 and αCys114 are post-translationally
modified to cystein-sulfinic acid (Cys-SO2H) and cystein-sulfenic acid (Cys-SOH),
respectively. These modifications were also verified by mass spectrometry
and recently the modified cysteins were identified by FTIR spectroscopy to
be at their ionizing states, cystein-sulfinate and cystein-sulfenate .
Although the cystein modifications have been found in several proteins previously,
NHase is the first protein found containing both Cys-SO2- and Cys-SO- as
the metal ligands. Considering that the catalytic mechanism of NHase
resides in the unique structure of metallocenter, various model complexes
have been synthesized and characterized in the world. However, the
role of modifications in catalytic mechanism remains unknown.
To elucidate the catalytic mechanism of NHase directly
by time-resolved four-dimensional protein crystallography, we constructed
a new experimental set-up at the third-generation synchrotron radiation
facility, SPring-8. Using the large-angle oscillation technique at the RIKEN
beamline, BL45XU, we succeeded to observe the dynamic process of NO release
from the iron center of NHase with a time-resolution of 30 min at a temperature
One set of X-ray diffraction
data was collected first from a nitrosylated NHase crystal in the dark at
90K. After photo-illumination was started and sample temperature was raised
to 140K, several data collections corresponding to progressive reaction
stages were continued in three hours, and the data sets were analyzed independently.
We found that the electron density distributions for NO molecule and Oδ
atoms of αCys114 cystein-sulfenate decreased very quickly within 30 min,
while that for Oδ1 and Oδ2 of αCys112 cystein-sulfinate more slowly around
2 hr (unpublished results). Based on the results obtained, we are considering
that the cystein-sulfenate activated by the photo-induced NO release may
relate to the hydration of nitrile compounds, the substrate of NHase.
Further investigations are in progress including the four-dimensional structure
analysis using crystals of substrate-nitrosylated NHase complex as initial
Following the NHase
case mentioned above, we have started to search other enzymes suitable for
the four-dimensional structure analysis. To gain deeper understanding
of enzymatic reactions, to apply the technique newly developed to many enzymes
is the second target of our laboratory.
2. Selected Publications
1. "3D Crystal Structure of the Photosystem II Core", in "Photosystem
II: The Light-Driven Water:Plastoquinone Oxidoreductase" edited by Wydrzynski
T. J. and Satoh K., Shen, J-R. and Kamiya, N., pp. 449-467, Springer,
2. "Structure of
P-protein of the glycine cleavage system: implications for nonketoic hyperglycinemia",
Nakai, T. Nakagawa, N., Maoka, N., Masui, R., Kuramitsu, S., Kamiya, N.,
EMBO J., 24, 1523-1536 (2005).
3. Vasil'ev S,
Shen JR, Kamiya N, Bruce D., "The orientations of core antenna chlorophylls
in photosystem II are optimized to maximize the quantum yield of photosynthesis.",
FEBS Lett. 561, 111-116 (2004).
anomalous diffraction method for I and Xe atoms using ultra-high energy X-rays
from SPring-8", Takeda, K., Miyatake, H., Park, S.- Y., Kawamoto, M., Kamiya,
N., and Miki, K., J. Appl. Cryst. 37, 925-933 (2004).
confromational changes and a reaction intermediate in branched-chain 2-oxo
acid dehydrogenase (E1) from Thermus thermophilus HB8, as revealed by X-ray
crystallography", Nakai, T., Nakagawa, N., Maoka, N., Masui, R., Kuramitsu,
S., and Kamiya, N., J. Mol. Biol. 337, 1011-1033 (2004).
6. "Crystal structure
of Thermus thermophilus HB8 H-protein of the glycine cleavage system, resolved
by a six-dimensional molecular-replacement method", Nakai, T., Ishijima,
J., Masui, R., Kuramitsu, S., and Kamiya, N., Acta Cryst. sec
D59, 1610-1618 (2003).
7. "Crystal Structure
of oxygene-evolving photosystem II from Thermosynechococcus vulcanus at
3.7 Å resolution", Kamiya, N. and Shen, J-R., Proc. Natl.
Acad. Sci. USA 100, 98-103(2003).
Structures of Cys-Sulfinic Acids in the Photosensitive Nitrile Hydratase
Revealed by Fourier Transform Infrared Spectroscopy", Noguchi, T., Nojiri,
M., Takei, K., Odaka, M., and Kamiya, N., Biochemistry 42,
9. "The Bio-Crystallography
beamline (BL41XU) at SPring-8", Kawamoto, M, Kawano, Y., and Kamiya,
N., Nucl. Instr. and Meth. A467-468, 1375-1379 (2001).