Molucular mechanisms of oxygen activation at the three steps in heme oxygenase reaction

血红素加氧酶反应三步氧活化的分子机制

• 批准号: 09480158
• 负责人: YOSHIDA Tadashi
• 金额: $ 1.79万
• 依托单位: YAMAGATA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09480158/
• 关键词: ヘムオキシゲナーゼ; オキシゲナーゼ; 酵素活性化機構; ヘム; ヘムの分解; CO; ビリベルジン; ビリルビン; 酸素活性化機構

(1)The resonance Raman spectra for alpha-hydroxyheme and verdoheme complexes of heme oxygenase (HO) was measured. We found that the ferric alpha-hydroxyheme and ferrous verdoheme complexes showed atypical Raman patterns, which are interpreted as the result of the symmetry lowering of the porphyrin-conjugating pi-electron system. (2)To identify the axial heme ligand of HO-2, we prepared His45 to Ala (H45A) and Hisi 52 to Ala (Hi 52A) mutants. H45A was completely devoid of the heme dedradation activity. A 5-coordinate-type ferrous NO EPR spectrum was observed for the heme-H45A complex, On the contrary, H152A mutant exhibited spectroscopic and enzymatic properties identical to those of wild-type. His132 of HO-1 was also not important for the heme degradation. (3)The O_2 and CO reactions with the heme, hydroxyheme, and verdoheme complexes of HO were studied. The 02 affinities for heme and hydroxyheme are very high, but the CO affinities are only 1-6-fold higher than the O_2 affinities. Thus, HO discriminates much more strongly against CO binding than myoglobin. The CO affinities of the verdoheme complexes are about 10,000 times weaker than those of the heme complex. (4)On the basis of Raman spectra of O_2-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_2 forms hydrogen-bond interactions with distal amino acid residue.

(1)测定了血红素加氧酶(HO) α -羟血红素复合物和血红素复合物的共振拉曼光谱。我们发现铁- α -羟基血红素和铁-羟基血红素配合物表现出非典型的拉曼模式，这被解释为卟啉共轭pi电子系统对称性降低的结果。(2)为了鉴定HO-2的轴向血红素配体，我们制备了His45 To Ala （H45A）和Hisi 52 To Ala （Hi 52A）突变体。H45A完全没有血红素降解活性。血红素- h45a复合物具有5位型亚铁NO EPR谱，而H152A突变体具有与野生型相同的光谱和酶学特性。HO-1的His132对血红素降解也不重要。(3)研究了O_2和CO与HO的血红素、羟血红素和铁血红素配合物的反应。血红素和羟基血红素的o2亲和度很高，而CO亲和度仅为O_2亲和度的1-6倍。因此，与肌红蛋白相比，HO对CO结合的区别要强烈得多。血红素络合物的CO亲和力比血红素络合物弱约1万倍。(4)根据血红素- ho配合物o_2键合形式的拉曼光谱，提出了高弯曲的Fe-O-O几何结构。然而，结合氧与远端氨基酸残基的相互作用尚未确定。为了澄清这一点，我们对钴（II）卟啉HO配合物进行了EPR测量，发现结合- o_2与远端氨基酸残基形成氢键相互作用。

项目成果

Fujii H.et al.: "Cobalt porphyrin hene oxygenase complex, EIR evidences for the distal hene pocket hydrogen bonding" J.Ame.Chem.Soc.130・32. 8251-8252 (1998)

Fujii H.等人：“钴卟啉烯加氧酶复合物，EIR 证明远端烯袋氢键合”J.Ame.Chem.Soc.130・32（1998）。

Ishikawa, Kazunobu: "Identification of histidine 45 on the axial heme iron ligand of heme oxygenase-2" J.Biol.Chem.273・8. 4317-4322 (1998)

Ishikawa，Kazunobu：“血红素加氧酶 2 的轴向血红素铁配体上组氨酸 45 的鉴定”J.Biol.Chem.273·8（1998）。

Ikeda-Saito, Masao et al.: "Heme oxygenase : central enzyme of oxygen-dependent heme catabolism and carbon monoxide synthesis." In Oxygen Homeostasis and Its Dynamics (Ishimura, Y.et al eds.) , Spring-Verlag, Tokyo. 304-314 (1997)

Ikeda-Saito、Masao 等人：“血红素加氧酶：氧依赖性血红素分解代谢和一氧化碳合成的中心酶。”

Fujii, Hiroshi et al.: "Heme degradation mechanisms by heme oxygenase : conversion of alpha-meso-hydroxyheme to verdoheme IXalpha." In Oxygen Homeostasis and Its Dynamics (Ishimura, Y.et al eds.) , Spring-Verlag, Tokyo. 315-321 (1997)

Fujii、Hiroshi 等人：“血红素加氧酶的血红素降解机制：α-内消旋-羟基血红素转化为 verdoheme IXalpha。”

Ishikawa, K.et al.: "Identification of Hibidine 45 as the anal heme ligane of heme oxygenase-2" J.Biol.Chem.273・8. 4317-4322 (1998)

Ishikawa, K. 等：“Hibidine 45 作为血红素加氧酶 2 的肛门血红素配体的鉴定”J.Biol.Chem.273・8 4317-4322 (1998)。