|Budget Amount *help
¥3,620,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥420,000)
Fiscal Year 2007: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2006: ¥1,800,000 (Direct Cost: ¥1,800,000)
1. Characterization of Soybean HO-1 (Gm HO1) and determination of the heme degradation mechanism. We obtained purified proteins of heme oxygenase-1 of plant-origin for the first time and clarified following matters :
(1)Stoichiometry of the heme binding, heme coordination numbers and ligand species, spin state of heme, characteristics of the ligand field of heme, pKa of the ligand water, have been determined. (2) The electron-donation system for the heme conversion to be NADPH/FNR/Fd, as well as ascorbate. Chemistry of heme conversion by Gm HO-1 is similar to that of known HOs but interaction of the distal residues with one of the intermediate, verdoheme, is unique. (3) The first detection of the ferric low-spin state of verdoheme-HO intermediate by EPR, during the reaction processes. (4) Estimation of contribution of the distal residues of Gm HO-1 to the heme degradation reaction by use of mutants made by site-specific mutagenesis. (1, 2 : reported on FEBS J, 2006 ; 3, submitted for BB
RC, 2008 ; 4, in preparation for submission)
2. Elucidation of causes discriminating the heme degradation velocity under ascorbate between rat and cyanobacterial HO-ls.
Site-specific mutagenesis of ratHO-1 at the proximal helices yielded eight mutants. Structural and kinetic analyses in the presence of ascorbate were performed on the heme-mutant complexes. As a result, it has been evidenced that the whole molecular movement of the heme-distal helix connection coupled with modulation of the redox potential of heme and regulates the heme degradation rate (submitted to Biochemistry, 2008).
3. Searches on the electron transmission pathways in the heme-rat HO-1 complexes. Twenty aromatic amino-acid residues were mutated with leucine and kinetics of each heme complexes were examined in detail. On the other hand, binding of the HO complexes to the electron donor, CPR, was measured by the quenching of Ravine fluorescence from CPR. As a result, the inlet of electrons was identified (in preparation for submission). Less