2005 Fiscal Year Final Research Report Summary
Studies of heme degradation mechanism by heme oxygenase based on the crystal structures.
| Project/Area Number |
15590260
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General medical chemistry
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| Research Institution | Kurume University school of Medicine |
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Principal Investigator |
NOGUCHI Masato Kurume University school of medicine, Dept.Med.Biochem., Professor, 医学部, 教授 (10124611)
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| Co-Investigator(Kenkyū-buntansha) |
HIGASHIMOTO Yuichiro Kurume University school of medicine, Dept.Med.Biochem., Lecturer, 医学部, 講師 (40352124)
SAKAMOTO Hiroshi Kyushu Institute. of Technology, Dept.Bioscience and Bioinformatics, Associate Professor, 情報工学部, 助教授 (70309748)
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| Project Period (FY) |
2003 – 2005
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| Keywords | heme Oxygenase / heme degradation / α-hydroxyheme / verdoheme / biliverdin / crystallography / structural biology |
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| Research Abstract |
Heme oxygenase-1(HO-1)catalyzes the physiological degradation of heme at the expense of molecular oxygen using electrons donated by NADPH-cytochrome P450 reductase(CPR). We obtained the results(1)-(3)in this project. (1)we investigated the effect of NADP(H)on the interaction of HO-1 with CPR by surface plasmon resonance and analyzed by computer modeling of the HO-1/CPR complex. The guanidino group of Arg-185 is located within the hydrogen bonding distance of 2'-phosphate of NADPH, suggesting that Arg-185 contributes to the binding to CPR through an electrostatic interaction with the phosphate group. On the other hand, Lys-149 is close to a cluster of acidic amino acids near the FMN binding site of CPR. Thus, Lys-149 and Lys-153 appear to interact with CPR in such a way as to orient the redox partners for optimal electron transfer from FMN of CPR to heme of HO-1. (2)To investigate the electron transfer pathway from CPR, we examined the heme degradation by rat HO-1(rHO-1)using rat FMN-de
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pleted CPR. The FMN-depleted CPR was prepared by dialyzing the CPR mutant, Y140A/Y178A, against 2 M KBr. The first electron to reduce the ferric heme iron to the ferrous state must be supplied from FMN of CPR. The electrons for conversion of verdoheme to ferric biliverdin-iron chelate can be supplied from FAD or NADPH via heme propionates, and that the final electron to reduce ferric biliverdin, leading to the release of ferrous iron and biliverdin, should be provided from FMN of CPR. (3)O_2-dependent reactions of the ferric and ferrous forms of α-hydroxyheme complexed with water-soluble rat heme oxygenase-1 were examined by rapid-scan stopped-flow measurements. Ferric α-hydroxyheme reacted with O_2 to form ferric verdoheme with an O_2-dependent rate constant of 4x10^5 M^<-1>s^<-1> at pH 7.4 and 9.0. A decrease of the rate constant to 2.8x10^5 M^<-1>s^<-1> at pH 6.5 indicates that the reaction proceeds by direct attack of O_2 on the p-neutral radical form of α-hydroxyheme, which is generated by deprotonation of the α-hydroxy group. The reaction of ferrous α-hydroxyheme with O_2 yielded ferrous verdoheme in a biphasic fashion involving a new intermediate having absorption maxima at 415 and 815 nm. The rate constants for this two-step reaction were 68 and 145 s^<-1>. These results show that conversion of α-hydroxyheme to verdoheme is much faster than the reduction of coordinated iron(<1 s^<-1>)under physiological conditions suggesting that, in vivo, the conversion of ferric α-hydroxyheme to ferric verdoheme precedes the reduction of ferric α-hydroxyheme. Less
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Research Products
(27 results)
