Structure and Function of heme related proteins involved in intracellular signal transduction

在本工作中，我们对一种血红素降解酶--血红素加氧酶进行了研究。首先，我们的研究项目集中在哺乳动物血红素加氧酶HO-2的构成亚型上。重组HO-2在原核表达载体中高效表达。用新的方法成功纯化，排除了降解或变性酶等杂质。我们用这种高纯度的HO-2进行晶体筛选，寻找合适的结晶缓冲条件，但没有找到合适的条件。另一方面，我们已经制备了细菌血红素加氧酶HmuO的晶体，并试图阐明HmuO的晶体结构。根据HO-2和HmuO的光谱数据、光吸收光谱和共振拉曼光谱以及酶学分析，发现它们的血红素环境和反应机理与哺乳动物血红素加氧酶的诱导形式HO-1相同。X-射线晶体结构分析表明，HmuO晶体属于P21空间群，晶胞由三个分子组成。我们可以揭示出酶反应的第一和第二中间体--氧化态和还原态的结构。还原形式的晶体是通过向氧化形式的晶体中添加二亚硫酸钠作为电子供体而得到的。氧化态和还原态的晶体结构分辨率分别为1.4埃和1.7埃。通过对氧化态和还原态晶体结构的比较，首次发现了由于血红素铁电子态的变化而引起的大量结构变化。此外，还指出了氢键网络的存在，认为氢键网络在血红素降解反应中起到了质子供体的作用。现在我们正在努力了解HmuO的CO加合物的结构以及其他中间体，α-羟基血红素形式，马鞭铁血红素形式和胆汁素形式。

In this work, we studied about heme oxygenase, a heme degradation enzyme. First, our research project was focused on constitutive isoform of mammalian heme oxygenase, HO-2. Recombinant HO-2 overexpressed in E colli. was successfully purified with new method to exclude impurity such as degradated or denatured enzymes. We used this highly purified HO-2 for crystal screening to search an appropriate buffer condition of crystallization, but we could not find the condition. On the other hand, we had already made crystal of HmuO, a bacterial heme oxygenase, and tried to elucidate crystal structure of HmuO. On the basis of spectroscopic data, optical absorption and resonance Raman spectra, and enzymological analysis of HO-2 and HmuO, their heme environment and reaction mechanism were found to be identical to those of HO-1 , inducible form of mammalian heme oxygenase. X-ray crystal structure analysis revealed that the crystal of HmuO belonged to P21 space group and a unit cell of the crystal was composed of three molecules. We could reveal structure of oxidized form and reduced form, the first and second intermediate of the enzyme reaction. The reduced form of crystal was made by the addition of sodium dithionite as an electron donor to the oxidized form crystal. Resolutions of the crystal structures were 1.4 and 1.7 angstroms for oxidized form and reduced form, respectively. From the comparison of crystal structures in oxidized and reduced forms, substantial amount of structural changes provoked by changing of heme iron electronic state were discovered for the first time. Additionally, presence of an hydrogen bonding network, which is assumed to work as a proton donor in the heme-degradation reaction, was pointed out here. Now our efforts are thrusting to realize the structure of CO adduct of HmuO as well as other intermediate, alpha-hydroxyheme form, verdoheme form, and billiverdin form.