Following Heme Oxygenase Reaction at Atomic Scale

原子尺度的血红素加氧酶反应

• 批准号: 14380300
• 负责人: SAITO Masao
• 金额: $ 7.74万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14380300/
• 关键词: Heme; heme oxygenase; X-ray crystallography; Reaction mechanism; oxygen activation

Heme oxygenase (HO) oxidatively degrades heme to biliverdin. Specific aim of this research project is to delineate the molecular mechanism of the enzyme action through determination of the high resolution crystal structures of all the catalytically significant reaction intermediates. We have chosen HmuO, a heme oxygenase of Corynebacterium diphtheriae, the spectroscopic and enzymatic properties of which have been extensively studied by our research team.In the first year, we determined the crystal structures of ferric and ferrous forms of the heme complex of HmuO, the initial and the second steps of the heme degradation at 1.4 and 1.5 Å resolution. We have found that protons required for HO catalysis is channeled from the solvent water to the oxygen activation site through different conduit between mammalian HO and HmuO despite the high similarity in the overall protein fold between these two HO proteins.In the second year, we have successfully obtained the crystals of the catalytically critical oxy form and determined its structure at 1.85 A resolution. Due to low stability of the oxy form, previous crystallization attempts of oxy HO by other investigators had not been successful, and our publication is the first report of the structure of the catalytically significant oxy form. The structure has resolved long standing important questions on HO, including why oxygen affinity of heme oxygenase is very high, how heme oxygenase prevents product inhibition, and which parts of the active site are relevant to regio-selective hydroxylation of the heme group.The crystal structures of the HmuO complexes with the final product (biliverdin) and its immediate precursor (iron biliverin) have also been solved. These structures provide clues to understand the mechanism of the substrate release from the HO protein, the rate limiting step of the HO catalysis.

血红素加氧酶（HO）将血红素氧化降解为胆绿素。本研究项目的具体目的是通过测定所有具有催化意义的反应中间体的高分辨率晶体结构来描述酶作用的分子机制。我们选择了HmuO，一种白喉棒状杆菌的血红素加氧酶，我们的研究小组已经对其光谱和酶学性质进行了广泛的研究。在第一年，我们测定了HmuO的铁和铁形式的血红素复合物的晶体结构，在1.4和1.5 Å分辨率下血红素降解的初始和第二步。我们发现，尽管哺乳动物HO和HmuO蛋白的整体蛋白折叠高度相似，但HO催化所需的质子从溶剂水通过不同的管道输送到氧活化位点。第二年，我们成功地获得了催化临界氧形态的晶体，并以1.85 A的分辨率确定了其结构。由于氧形式的稳定性较低，以前其他研究者对氧HO的结晶尝试都没有成功，而我们的论文是第一次报道具有催化意义的氧形式的结构。该结构解决了长期以来关于HO的重要问题，包括为什么血红素加氧酶的氧亲和力非常高，血红素加氧酶如何阻止产物抑制，以及活性位点的哪些部分与血红素基团的区域选择性羟基化有关。HmuO配合物与最终产物（胆绿素）及其直接前体（胆绿素铁）的晶体结构也得到了解决。这些结构为了解HO蛋白的底物释放机制提供了线索，这是HO催化的限速步骤。

项目成果

Tomita, et al.: "Recent progress in biochemical studies of hemoprotein"Seikagaku. 75(7). 577-587 (2003)

富田等人：“血红素蛋白生化研究的最新进展”Seikagaku。

Denisov et al.: "Cryogenic absorption spectra of hydroperoxo-ferric heme oxygenase, the active intermediate of enzymatic heme oxygenation"FEBS Lett.. 532(1-2). 203-206 (2002)

Denisov 等人：“氢过氧铁血红素加氧酶的低温吸收光谱，酶促血红素加氧的活性中间体”FEBS Lett.. 532(1-2)。

Davydov et al.: "Kinetic isotope effects on the rate-limiting step of heme oxygenase catalysis indicate concerted proton transfer/heme hydroxylation"J.Am.Chem.Soc.. 125(52). 16208-16209 (2003)

Davydov 等人：“动力学同位素对血红素加氧酶催化限速步骤的影响表明协同质子转移/血红素羟基化”J.Am.Chem.Soc.. 125(52)。

Coyle, et al.: "FeNO structure in distal pocket mutants of myoglobin from resonance Raman spectroscopy"Biochemistry. 42(17). 4896-4903 (2003)

Coyle 等人：“来自共振拉曼光谱的肌红蛋白远端口袋突变体中的 FeNO 结构”生物化学。

Gonzalez et al.: "Nature of the displaceable heme-axial residue in the EcDos protein, a heme-based sensor from Escherichia coli"Biochemistry. 41(26). 8414-8421 (2002)

Gonzalez 等人：“EcDos 蛋白中可置换血红素轴残基的性质，一种来自大肠杆菌的基于血红素的传感器”生物化学。