| Project/Area Number |
10044233
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| Research Category |
Grant-in-Aid for Scientific Research (A).
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Structural biochemistry
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| Research Institution | YAMAGATA UNIVERSITY |
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Principal Investigator |
YOSHIDA Tadashi DEPARTMENT OF BIOCHEMISTRY, PROFESSOR, SCHOOL OF MEDICINE, YAMAGATA UNIVERSITY, 医学部, 教授 (10004673)
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| Co-Investigator(Kenkyū-buntansha) |
FUJII Hiroshi INSTITUTE FOR MOLECULAR SCIENCE, ASSOCIATE PROFESSOR, 分子科学研究所, 助教授 (80228957)
SATO Michihiko DEPARTMENT OF BIOCHEMISTRY, INSTRUCTOR, SCHOOL OF MEDICINE, YAMAGATA UNIVERSITY, 医学部, 助教授 (00135344)
ZHANG Xuhong DEPARTMENT OF BIOCHEMISTRY, INSTRUCTOR, SCHOOL OF MEDICINE, YAMAGATA UNIVERSITY, 医学部, 助手 (10292442)
MIGITA Taiko SCHOOL OF ALLIED HEALTH SCIENCES, YAMAGUCHI UNIVERSITY, ASSOCIATE PROFESSOR, 医療技術短期大学部, 助教授 (90159161)
OLSOH John S Rice University, ライス大学, 教授
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥9,400,000 (Direct Cost: ¥9,400,000)
Fiscal Year 1999: ¥4,300,000 (Direct Cost: ¥4,300,000)
Fiscal Year 1998: ¥5,100,000 (Direct Cost: ¥5,100,000)
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| Keywords | Heme oxygenase / Heme degradation / Heme degradation enzyme / Hmu O / Activation of oxygen / CO / biliverdin / Bilirubin / Hmu O / 酸素活性化機構 / ヘムオキシグナーゼ / オキシグナーゼ / ヘム / ピリルビン |
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| Research Abstract |
(1)To identify the axial heme ligand of heme oxygenase-2 (HO-2), we prepared His45 to Ala (H45A) mutant. H45A was completely devoid of the heme dedradation activity. A 5-coordinate-type ferrous NO EPR spectrum was observed for the heme-H45A complex. These indicate that His 45 is the proximal ligand of heme oxygenase-2. (2)The OィイD22ィエD2 and CO reactions with the heme, hydroxyheme, and verdoheme complexes of HO were studied. The OィイD22ィエD2 affinities for heme and hydroxyheme are very high, but the CO affinities are only 1-6-fold higher than the OィイD22ィエD2 affinities. Thus, HO discriminates much more strongly against CO binding than myoglobin. (3)On the basis of Raman spectra of OィイD22ィエD2-bound form of the heme-HO complex, a highly bent Fe-O-O geometry has been proposed. However, the interaction of bound oxygen with the distal amino acid residue has not been identified.
To clarify this, we have carried out EPR measurements of the cobalt(II) porphyrin HO complex and revealed that the bound-OィイD22ィエD2 formes hydrogen-bond interactions with distal amino acid residue. (4)We investigated the mechanism of the conversion of hydroxyhemin to verdoheme and confirmed that our previous conclusion that this step requires one reducing equivalent along with molecular oxygen. (5)We analyzed the first step of the heme degradation. We found that the molecular oxygen bound to heme-HO complex is stabilized by an H-bond and that hydroperoxy-HO genarated by cryoreduction catalyzes the formation of hydroxyheme. (6)We established the expression system of bacterial HO (Hmu O). Hmu O binds hemin stoichiometrically to form a hexacoordinate high spin hemin-Hmu O complex. When ascorbic acid is used as the electron donor, Hmu O converted hemin to biliverdin with hydroxyhemin and verdoheme as intermediates. Other enzymatic and protein-chemical properties closely resembled those of mammalian HOs.
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