Following Heme Oxygenase Reaction at Atomic Scale

血红素加氧酶（HO）将血红素氧化降解为胆绿素。该研究项目的具体目标是通过测定所有催化重要反应中间体的高分辨率晶体结构来描绘酶作用的分子机制。我们选择了白喉棒状杆菌血红素加氧酶HmuO，我们的研究小组对其光谱和酶学性质进行了广泛的研究，在第一年，我们确定了HmuO血红素复合物的铁和亚铁形式的晶体结构，在1.4和1.5 μ m分辨率下确定了血红素降解的第一和第二步。我们发现HO催化所需的质子从溶剂水通过不同的管道之间的哺乳动物HO和HmuO的氧活化位点，尽管这两个HO蛋白之间的整体蛋白质折叠高度相似，在第二年，我们已经成功地获得了催化关键的氧形式的晶体，并确定其结构在1.85 A分辨率。由于氧形式的低稳定性，其他研究人员先前对氧HO的结晶尝试没有成功，我们的出版物是催化显著的氧形式的结构的第一份报告。该结构解决了血红素氧合酶的氧亲和力为何如此之高、血红素氧合酶如何阻止产物抑制、血红素基团的区域选择性羟基化与活性位点的哪些部分有关等问题，并解决了HmuO与最终产物胆绿素及其直接前体铁胆绿素的晶体结构。这些结构提供了线索，以了解从HO蛋白质的底物释放的机制，HO催化的限速步骤。

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.