Laboratory for Biological Structural Chemistry

Nobuo Kamiya (Professor)

http://www.sci.osaka-cu.ac.jp/chem/struct/index_e.html



1. Current Research and Principal Research Interests

         We 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 [3].


         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 [8].  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 of 140K.



          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 samples.
          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, Heiderberg (2005)

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).

4. "Multi-wavelength 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).

5. "Ligand-induced 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).

8. "Protonation 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, 11642-11650 (2003).

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).