| Co-Investigator(Kenkyū-buntansha) |
ZHANG Xuhong YAMAGATA UNIVERSITY SCHOOL OF MEDICINE, DEPARTMENT OF BIOCHEMISTRY, INSTRUCTOR, 医学部, 助手 (10292442)
SATO Michihiko YAMAGATA UNIVERSITY SCHOOL OF MEDICINE, CENTRAL LABORATORY FOR RESEARACH AND EDUCATION, ASSOCIATE PROFESSOR, 医学部, 助教授 (00135344)
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| Budget Amount *help |
¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 2003: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 2002: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
Heme oxygenase catalyzes the regiospecific oxidation of hemin to biliverding IXα with concomitant liberation of CO and iron by three sequential mono-oxygenase reaction. (1) We studied the conversion of heme to α-meso-hydroxyheme by EPR. ENDOR, and optical absorption spectroscopy. The results obtained by these studies demonstrate that hydroperoxo ferric-HO is indeed the reactive species, directly forming the α-meso-hydroxyheme by attack of the distal OH of hydroperoxo moiety at the heme α-carbon. D140A mutant failed to convert heme to α-meso-hydroxyheme, confirming our previous finding that the H-bonding network within the distal pocket of HO is disrupted. (2) We investigated the stereoselectivity of each of the two reaction steps from meso-hydroxyhemin to verdoheme and versoheme to biliverdin, by using a truncated form of rat HO-1 and the chemically synthesized four isomers of meso-hydroxythemin and verdoheme. HO-1 converted all four isomers of meso-hydroxyhemin to the corresponding is
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omers of verdoheme. In contrast, only verdoheme IXα was converted to the corresponding biliverdin IXα. We conclude that the third step, but not the second, is stereoselective for the α-isomer substrate. (3) An efficient bacterial expression system of cyanobacterium Synechocystis sp. PCC 6803 HO gene, ho-1, has been constructed, using a synthetic gene. A soluble protein was expressed at high levels and was highly purified, for the first time. The spectroscopic characters as well as the catabolic activities strongly suggest that, in spite of very high conservation of the primary structure, the heme pocket structure of Synechocystis HO-1 is different from that of rat HO. (4) Crystal structure of the ferric and ferrous heme complexes of HemO, a 24-kDA HO of Corynebacterium diphtheriae, have been refined to 1.4 and 1.5 A resolustion, respectively. The heme pocket architecture is responsible for stabilization of the ferric hydroperoxo-active intermediated by preventing premature hydrolytic O-O bond cleavage. Less
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