Mechanism of heme degradation by heme oxygenase

血红素加氧酶降解血红素的机制

• 批准号: 10044233
• 负责人: YOSHIDA Tadashi
• 金额: $ 6.02万
• 依托单位: YAMAGATA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A).
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10044233/
• 关键词: Heme oxygenase; Heme degradation; Heme degradation enzyme; Hmu O; Activation of oxygen; CO; biliverdin; Bilirubin; Hmu O; 酸素活性化機構; ヘムオキシグナーゼ; オキシグナーゼ; ヘム; ピリルビン

(1)To identify the axial heme ligand of heme oxygenase-2 (HO-2), we prepared His45 to Ala (H45A) mutant. H45A was completely devoid of the heme dedradation activity. A 5-coordinate-type ferrous NO EPR spectrum was observed for the heme-H45A complex. These indicate that His 45 is the proximal ligand of heme oxygenase-2. (2)The OィイD22ィエD2 and CO reactions with the heme, hydroxyheme, and verdoheme complexes of HO were studied. The OィイD22ィエD2 affinities for heme and hydroxyheme are very high, but the CO affinities are only 1-6-fold higher than the OィイD22ィエD2 affinities. Thus, HO discriminates much more strongly against CO binding than myoglobin. (3)On the basis of Raman spectra of OィイD22ィエD2-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ィイD22ィエD2 formes hydrogen-bond interactions with distal amino acid residue. (4)We investigated the mechanism of the conversion of hydroxyhemin to verdoheme and confirmed that our previous conclusion that this step requires one reducing equivalent along with molecular oxygen. (5)We analyzed the first step of the heme degradation. We found that the molecular oxygen bound to heme-HO complex is stabilized by an H-bond and that hydroperoxy-HO genarated by cryoreduction catalyzes the formation of hydroxyheme. (6)We established the expression system of bacterial HO (Hmu O). Hmu O binds hemin stoichiometrically to form a hexacoordinate high spin hemin-Hmu O complex. When ascorbic acid is used as the electron donor, Hmu O converted hemin to biliverdin with hydroxyhemin and verdoheme as intermediates. Other enzymatic and protein-chemical properties closely resembled those of mammalian HOs.

(1)To为了鉴定血红素加氧酶-2（HO-2）的轴向血红素配体，我们制备了His 45 to Ala（H45 A）突变体。H45 A完全没有血红素降解活性。血红素-H45 A复合物的EPR谱为5配位型亚铁NO。这表明His 45是血红素加氧酶-2的近端配体。（2）研究了HO的血红素、羟基血红素和绿血红素配合物与O、O、D_（22）、O、D_（22）和CO的反应。O-血红素D22-血红素D2对血红素和羟基血红素的亲和力非常高，但CO亲和力仅比O-血红素D22-血红素D2亲和力高1-6倍。因此，HO比肌红蛋白更强烈地歧视CO结合。(3)On基于O-O-D22-D2-键合形式的血红素-HO络合物的拉曼光谱，提出了高度弯曲的Fe-O-O几何构型。然而，结合氧与远端氨基酸残基的相互作用还没有确定，为了澄清这一点，我们进行了EPR测量的钴（II）卟啉HO络合物，并显示，结合-O的邻位D22邻位D2形成氢键与远端氨基酸残基的相互作用。(4)We研究了羟基氯化血红素转化为绿血红素的机理，并证实了我们以前的结论，即该步骤需要一个还原当量沿着分子氧。(5)We分析了血红素降解的第一步。我们发现，血红素-HO复合物中的氧分子通过氢键稳定，低温还原生成的过氧氢HO催化羟基血红素的形成。(6)We建立了细菌HO（Hmu O）的表达系统。Hmu O化学计量地结合氯化血红素以形成六配位高自旋氯化血红素-Hmu O复合物。以抗坏血酸为电子给体时，Hmu O将氯化血红素转化为胆绿素，中间产物为羟基氯化血红素和绿血红素。其他酶和蛋白质化学性质与哺乳动物HO非常相似。

项目成果

Chu, Grace C. et al.: "Crystallization and preliminary X-ray diffraction analysis of a recombinant bacterial heme oxygenase (Hmu O) from Corynebacterium diphtheriae."J. Struct. Biol.. 126. 171-174 (1999)

Chu, Grace C. 等人：“来自白喉棒杆菌的重组细菌血红素加氧酶 (Hmu O) 的结晶和初步 X 射线衍射分析。”J.

Chu GC et.al.: "Crystallization and preliminary X-ray diffraction analysis of a recombinant bacterial heme oxygenase (Hmu O) from Corynebacterium diphtheriae"J. Stract. Biol. 126. 171-174 (1999)

Chu GC 等人：“来自白喉棒状杆菌的重组细菌血红素加氧酶 (Hmu O) 的结晶和初步 X 射线衍射分析”J。

Ishikawa K et al.: "Identification of histidine 45 as the axial heme ligand of heme oxygenase-2"J. Biol. Chem.. 273. 4317-4322 (1998)

Ishikawa K 等人：“鉴定组氨酸 45 作为血红素加氧酶-2 的轴向血红素配体”J。

Ishikawa, Kazunobu et al.: "Identification of histidine 45 as the axial heme iron ligand of heme ozygenase-2."J. Biol. Chem.. 273. 4317-4322 (1998)

Ishikawa, Kazunobu 等人：“鉴定组氨酸 45 作为血红素 Ozygenase-2 的轴向血红素铁配体。”J.

Davydof RM et al.: "Hydroperoxy-heme oxygenase generated by cryoreduction catalyzed the formation of α-meso-hydroxyheme as detedted by EPR and ENDOR"J. Am: Chem. Soc.. 121・45. 10656-10657 (1999)

Davydof RM 等人：“通过 EPR 和 ENDOR 检测，通过冷冻还原产生的氢过氧血红素加氧酶催化 α-内消旋-羟基血红素的形成”J. Soc. 121・45 (1999)。