Electronic/molecular structure of enzyme heme pockets

描述（由申请人提供）：我们建议使用高分辨率的2D/3D NMR溶液，详细研究一系列血红素加氧酶，HO，酶及其与底物/反应中间体在可变氧化/自旋/连接状态下的配合物的功能相关的分子/电子结构和动力学性质。HO存在于脊椎动物、植物和细菌中，通过一种共同的机制和一组中间体，利用血红素作为底物和辅助因子，立体选择性地将血红素切割成a-胆绿素、铁和CO。HO的独特之处在于，它使用氢过氧物质作为其激活形式，除了涉及远端氢键网络中的有序水分子外，稳定这些物质的活性位点的结构特性还没有得到很好的理解。我们选择了3个HOs，来自人类的同工酶#1，以及来自2种致病菌白喉C. （CofHO）和脑膜炎N. （NmHO）的同工酶，它们具有共同的折叠，但在h键网络中涉及的残基表现出可变的序列同源性。目标衍生物为无底物或app-HO，静息态HO-hemin-H/jO，不稳定氧配合物HO-hemin-CN模型，反应性羟基物种HO-hemin-OH模型。由于除了1个目标HO衍生物外，所有的HO衍生物都是顺磁性的，因此重点放在利用适当定制的1D/2D/3D NMR来提取超精细偏移中的丰富独特信息。我们将开发一种新的高灵敏度NMR探针，直接反映轴向配体与血红蛋白之间的氢键程度和远端有序水/氢键网络，使用络合物HO-hemin-H2O/-OH对，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，特别关注具有一些非常坚固的氢键的扩展氢键网络，以及这些网络中的有序水分子。我们着重比较研究了1 - HO的各种衍生物之间，以及给定衍生物的不同羟基之间的比较研究，以阐明变强度氢键与轴向配体性质之间的关系。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上的位置的影响，确定在溶液中折叠到活性位点的晶体无序c端结构，并阐明c端和独特的活性位点Cys113在多种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认识。阐明细菌和哺乳动物血红素加氧酶之间的异同，将为病原菌中该酶的选择性抑制剂的设计提供前景。

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.