Electronic/molecular structure of enzyme heme pockets

描述(申请人提供)：我们提议使用高分辨率2D/3D核磁共振，详细研究一系列血红素加氧酶、HO、酶及其与底物/反应中间体在不同氧化/自旋/凝聚状态下的功能相关的分子/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，通过一种共同的机制和一系列中间体，使用血红素作为底物和辅因子，立体选择性地将血红素裂解成a-胆绿素、铁和一氧化碳。HO的独特之处在于它使用了氢过氧物种作为其活化形式，除了涉及远端H-键网络中的有序水分子外，稳定该物种的活性中心的结构性质还不是很清楚。我们选择了来自人类HHO的3个HOS，同工酶#1，以及来自2个致病细菌白喉弧菌(CofHO)和脑膜炎奈瑟菌(NmHO)的同工酶#1，它们有一个共同的折叠，但参与H-键网络的残基具有可变的序列同源性。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作为不稳定含氧络合物的模型，HO-氯化高铁血红素-OH作为活性羟基物种的模型。由于除1个靶向Ho衍生物外，所有的HO衍生物都是顺磁性的，因此重点是利用适当定制的1D/2D/3D核磁共振来提取丰富的超精细位移中的独特信息。我们将开发一种新的高灵敏的核磁共振探针，使用一对复杂的Ho-氯化血红素-H2O/-OH，直接反映轴向配体与氯化血红素和远端有序水/氢键网络之间的氢键程度，并使用该探针以及先前建立的程序，提供溶液结构的详细表征。我们的兴趣集中在局部溶液与低温晶体分子结构的比较上，特别关注具有一些非常健壮的氢键的扩展的氢键网络，以及这些网络中有序的水分子。我们着重比较研究了1HO的不同衍生物之间，以及同一衍生物的不同HOS之间的比较，以阐明可变强度的氢键与轴向配体性质的关系。此外，我们还将表征HO、底物或中间体及其轴向配体对底物进出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中心的位置的影响，确定在溶液中折叠到活性中心的结晶学上无序的C-末端的结构，并阐明C-末端和唯一的活性中心Cys113在多种功能相关的微异质性中的作用。对血红素加氧酶的分子结构和动力学性质的详细描述将有助于我们更好地理解哺乳动物酶的各种作用。阐明细菌和哺乳动物的血红素加氧酶的异同，将为设计针对病原菌的该酶的选择性抑制剂提供更好的前景。

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.