SATO Michihiko Yamagata University, School of Medicine, Associate Professor, 医学部, 助教授 (00135344)
ZHANG Xuhong Yamagata University, School of Medicine, Instructor, 医学部, 助手 (10292442)
|Budget Amount *help
¥3,800,000 (Direct Cost : ¥3,800,000)
Fiscal Year 2006 : ¥1,100,000 (Direct Cost : ¥1,100,000)
Fiscal Year 2005 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Fiscal Year 2004 : ¥1,400,000 (Direct Cost : ¥1,400,000)
1.We cloned a cDNA for a Drosophila melanogaster homologue of mammalian heme oxygenase (HO) and constructed a bacterial expression system of a truncated, soluble form of its HO. The purified enzyme degraded hemin to biliverdin, CO and iron in the presence of reducing systems such as NADPH/cytochrome P-450 reductase and ascorbate.
2.Previously, HO-2 of cyanobacterium Synechocystis sp. PCC 6803 was reported to be inactive. To clarify the enzymatic nature of this protein we constructed a bacterial expression system for Syn HO-2 and successfully obtained purified protein. Accordingly, it was established that Syn HO-2 binds hemin stoichiometrically and converts it into biliverdin, CO and iron in the presence of oxygen and electrons, demonstrating that this protein is a true HO.
3.A higher plant HO, GmHO-1, of Glycine max (soybean), was prepared to evaluate the molecular features of its heme complex, the enzymatic activity, and the mechanism of heme conversion. Although the similarity in the amino acid sequence between GmHO-1 and HOs from other biological species is low, the heme bound to GmHO-1, in the ferric high-spin state, exhibits an acid-base transition and is converted to biliverdin, VO and iron in the presence of NADPH/ferredoxinreductase/ferredoxin, orascorbic acid. The mechanism of heme degradation by GmHO-1 appeared to be similar to that of known HOs, despite the low sequence homology.
4.The oxy form of HmuO, a HO of Corynebacterium diphtheriae, the precursor of the catalytically active ferric hydroperoxo species, was characterized by ligand binding kinetics, resonance Raman spectroscopy, and X-ray crystallography. In the crystal structure of the oxy form the Fe-O-O angle is 110° and the O-O bond is pointed toward the heme α-meso-carbon.
5.HO degrades verdoheme through a dual pathway using either O_2 or H_2O_2. Both reactions are initiated by the binding of O_2 or H_2O_2 to allow the first direct observation of degradation intermediates of verdoheme.