| Co-Investigator(Kenkyū-buntansha) |
TOMITA Takeshi Institute for Multidisciplinaiy Research for Advanced Materials, Research Associate, 多元物質科学研究所, 助手 (20302242)
FUJII Hiroshi Okazaki National Research Institutes, Center for Integrative Bioscience, Associate Professor, 統合バイオサイエンスセンター, 助教授 (80228957)
SHIRO Yoshitsugu Riken, Biophysical Chemistry Laboratory, Chief Scientist, 生体物理化学研究室, 主任 (70183051)
MATSUI Toshitaka Institute for Multidisciplinary Research for Advanced Materials, Research Associate, 多元物質科学研究所, 助手 (90323120)
HIROTSU Shoko Institute for Multidisciplinary Research for Advanced Materials, Research Associate, 多元物質科学研究所, 助手 (50333901)
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| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 2001: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
In this work, we studied about heme oxygenase, a heme degradation enzyme. First, our research project was focused on constitutive isoform of mammalian heme oxygenase, HO-2. Recombinant HO-2 overexpressed in E colli. was successfully purified with new method to exclude impurity such as degradated or denatured enzymes. We used this highly purified HO-2 for crystal screening to search an appropriate buffer condition of crystallization, but we could not find the condition. On the other hand, we had already made crystal of HmuO, a bacterial heme oxygenase, and tried to elucidate crystal structure of HmuO. On the basis of spectroscopic data, optical absorption and resonance Raman spectra, and enzymological analysis of HO-2 and HmuO, their heme environment and reaction mechanism were found to be identical to those of HO-1 , inducible form of mammalian heme oxygenase. X-ray crystal structure analysis revealed that the crystal of HmuO belonged to P21 space group and a unit cell of the crystal was composed of three molecules. We could reveal structure of oxidized form and reduced form, the first and second intermediate of the enzyme reaction. The reduced form of crystal was made by the addition of sodium dithionite as an electron donor to the oxidized form crystal. Resolutions of the crystal structures were 1.4 and 1.7 angstroms for oxidized form and reduced form, respectively. From the comparison of crystal structures in oxidized and reduced forms, substantial amount of structural changes provoked by changing of heme iron electronic state were discovered for the first time. Additionally, presence of an hydrogen bonding network, which is assumed to work as a proton donor in the heme-degradation reaction, was pointed out here. Now our efforts are thrusting to realize the structure of CO adduct of HmuO as well as other intermediate, alpha-hydroxyheme form, verdoheme form, and billiverdin form.
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