Electronic/molecular structure of enzyme heme pockets

描述（由申请人提供）：我们提出使用高分辨率溶液2D/3D NMR详细研究一系列血红素加氧酶、HO、酶及其与可变氧化/自旋/连接状态下的底物/反应中间体的复合物的功能相关分子/电子结构和动力学性质。在脊椎动物、植物和细菌中发现的HO通过共同的机制和一组中间体起作用，使用血红素作为底物和辅因子，将血红素立体选择性地切割成α-胆绿素、铁和CO。HO独特之处在于使用氢过氧物质作为其活化形式，和稳定物种的活性位点的结构特性没有得到很好的理解，除了远端H-涉及到网络。我们选择了3个HO，来自人类的同工酶#1，hHO和来自2种病原菌C的HO。白喉杆菌（N.脑膜炎球菌（NmHO），其共享共同的折叠，但对于参与H-键合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或app-HO，静止状态HO-氯化血红素-H/jO，HO-氯化血红素-CN作为不稳定的氧复合物的模型，和HO-氯化血红素-OH作为反应性过氧化氢物种的模型。由于所有，但1有针对性的HO衍生物是顺磁性的，重点放在利用适当定制的1D/2D/3D NMR提取超精细位移的独特信息的财富。我们将开发一种新的和高灵敏度的NMR探针，直接反映轴向配体的氯化血红素和远端有序的水/氢键网络之间的氢键的程度，使用对复杂的HO-氯化血红素-H2O/-OH，并使用这种探针，以及以前建立的程序，提供一个详细的表征的解决方案的结构。我们的兴趣集中在本地解决方案与低温晶体分子结构的比较，特别注意到扩展的氢键网络与一些非常强大的氢键，和有序的水分子在这些网络。我们强调1 HO的各种衍生物之间的比较研究，并在不同的HO之间为一个给定的衍生物，阐明可变强度H-键和轴向配体性能之间的关系。此外，我们将表征HO，底物或中间体及其轴向配体对与底物进入和退出相关的动力学性质的影响。最后，对于NmHO，我们将表征血红素取代基对其在活性位点中的座位的影响，确定在溶液中发现折叠成活性位点的晶体学无序C-末端的结构，并阐明C-末端和独特的活性位点Cys 113在多个功能相关的微异质性中的作用。血红素加氧酶的分子结构和动力学特性的详细描述将提高我们对哺乳动物酶的各种作用的理解。阐明细菌和哺乳动物血红素加氧酶之间的相似性和差异将改善致病菌中酶的选择性抑制剂的设计前景。

DESCRIPTION (provided by applicant): 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 hydroperoxy 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 3 HOs, isozyme #1 from human, hHO, and those from 2 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 1 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 1 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.