Electronic/molecular structure of enzyme heme pockets

我们建议详细研究功能相关的分子/电子结构和动力学 一系列血红素加氧酶、HO、酶及其与底物/反应的复合物的性质 使用高分辨率溶液2D/3D NMR，在可变的氧化/自旋/连接状态下的中间体。何， 在脊椎动物、植物和细菌中发现，通过一种共同的机制和一组中间体起作用， 血红素作为底物和辅因子，立体选择性地将血红素切割成α-胆绿素、铁和CO。 独特之处在于使用过氧化氢物质作为其活化形式，并且活性物质的结构性质 稳定物种的位点还没有得到很好的理解，除了远端内有序的水分子 氢键网络参与。我们选择了三种HO，来自人的ispzyme #1，hHO，和来自两种人的那些。 病原菌C.白喉杆菌（N.脑膜炎（NmHO），它们共享一个共同的折叠，但 对于参与氢键网络的残基表现出可变的序列同源性。目标 衍生物是无底物的或app-HO，静息态HO-氯化血红素-H/jO，HO-氯化血红素-CN作为模型， 不稳定的氧复合物，和HO-氯化血红素-OH作为反应性hydroperpxy物种的模型。因为除了 一种靶向HO衍生物是顺磁性的，重点是利用适当定制的 1D/2D/3D NMR提取超精细位移中丰富的独特信息。我们将开发一个新的， 直接反映轴向配体与氯化血红素之间氢键结合程度的高灵敏度NMR探针 和远端有序水/氢键网络，使用复合HO-氯化血红素-H2O/-OH对，并使用 该探针以及先前建立的程序，以提供 溶液结构我们的兴趣集中在局部解与低温晶体学的比较上 分子结构，特别注意扩展的H-键网络与一些显着 强大的氢键，以及这些网络中有序的水分子。我们强调比较 研究一个HO的各种衍生物之间，以及给定衍生物的不同HO之间， 阐明可变强度H-键与轴向配体性质之间的关系。另外我们 将表征HO，底物或中间体及其轴向配体对动力学的影响 与基材的进入和退出有关的性质。最后，对于NmHO，我们将描述 血红素取代基在其活性位点上的位置，决定了晶体结构 无序的C-末端发现折叠成活性位点在溶液中，并阐明C-末端的作用 和独特的活性位点Cys 113在多个，功能相关，微异质性。的详细 血红素加氧酶的分子结构和动力学性质的描述将提高我们的 了解哺乳动物酶的各种作用。对相似之处的说明， 细菌和哺乳动物血红素加氧酶之间的差异将改善设计的前景， 病原菌中酶的选择性抑制剂。

We propose the detailed study of functionally relevant molecular/electronic structural and dynamic properties of a series of heme oxygenase, HO, enzymes and their complexes with substrate/reaction intermediates in variable oxidation/spin/liagtion states, using high resolution solution 2D/3D NMR. HO, found in vertebrates, plants and bacteria, acts by a common mechanism and set of intermediates, using heme as both substrate and cofactor, to stereoselectively cleave heme into a-biliverdin, iron and CO. HO is unique in using the hydrpperoxy species as its activated form, and the structural properties of the active site that stabilize the species are not well understood except that ordered water molecules within a distal H-bond network are involved. We select three HOs, ispzyme #1 from human, hHO, and those from two pathogenic bacteria C. diphtheriae (CofHO) and N. meningitidis (NmHO), which share a common fold, but exhibit variable sequence homology for the residues involved in the H-bonding network. The target derivatives are substrate-free or app-HO, resting state HO-hemin-H/jO, HO-hemin-CN as a model for the unstable oxy complex, and HO-hemin-OH as a model for the reactive hydroperpxy species. Since all but one targeted HO derivative are paramagnetic, emphasis is placed on utilizing appropriately tailored 1D/2D/3D NMR to extract the wealth of unique information in hyperfine shifts. We will develop a new and highly sensitive NMR probe that directly reflect the degree of H-bonding between axial ligand to the hemin and the distal ordered-water/H-bond network, using the pair of complex HO-hemin-H2O/-OH, and to use this probe, as well as previously established procedures, to provide a detailed characterization of the solution structure. Our interests focus on comparison of local solution with cryogenic crystallographic molecular structure, with particular attention paid to extended H-bond networks with some remarkably robust H-bonds, and the ordered water molecules within these networks. We emphasize comparative studies among the various derivatives of one HO, and among the different HOs for a given derivative, to elucidate the relationship between variable strength H-bonds and axial ligand properties. In addition, we will characterize the influence of HO, substrate or intermediates and their axial ligands on dynamic properties related to entry and exit of substrate. Lastly, for NmHO, we will characterize the influence of heme substituents on its seating in the active site, determine the structure of the crystallographically disordered C-terminus found folded into the active site in solution, and illuminate the role of the C-terminus and the unique active site Cys113 in multiple, functionally relevant, microheterogeneities. The detailed description of the molecular structural and dynamic properties of heme oxygenase will improve our understanding of the varied roles of mammalian enzymes. The elucidation of the similarities and differences between bacterial and mammalian heme oxygenase will improve prospects for the design of selective inhibitors for the enzyme in pathogenic bacteria.