SINGLE CRYSTAL AND SOLUTION XAS STUDIES OF NON-COUPLED BINUCLEAR COPPER O2 ACTIV

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 氨基甘氨酸羟化单加氧酶(PHM)是一种非偶联的双核铜蛋白，催化所有氨基甘氨酸底物的C-末端甘氨酸的β-碳的立体特异性羟基化。在催化机理中，PHM结合并激活O2，从肽甘氨酸底物中提取氢原子。得到了预催化O2中间体的晶体结构，这表明在活性中心存在末端键合的铜-O2物种。我们建议对这种预催化中间体进行单晶CuK边XAS研究，以确定其几何结构和电子结构。我们计划对预催化中间体进行时间序列光还原研究，以确定其在X射线照射下的稳定性。我们还建议用单晶XAS和计算方法(密度泛函理论和含时密度泛函方法)相结合的方法来研究晶体形式的PHM的完全氧化(CuIICuII)和完全还原(CuICuI)态，以表征它们的几何结构和配位体的场强。这些光谱和计算研究将能够确定PHM底物羟化机理中的相关状态的电子结构。近边多重散射分析将被用来区分PHM中存在的两个铜中心，以确定O2与前催化中间体中铜的结合方式。这些数据还将有助于确定O2在分子内电子从CuH位到CUM位的转移中的作用。结合对CuK边数据的分析、EXAFS、近边多重散射分析、密度泛函和含时密度泛函计算，将有助于更好地理解PHM对O2的结合、活化和底物羟化作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Peptidylglycine ?-hydroxylating monooxygenase (PHM) is a non-coupled binuclear Cu protein, which catalyzes the stereospecific hydroxylation of the ?-carbon of the C-terminal glycine of all peptidylglycine substrates. In the catalytic mechanism, PHM binds and activates O2 for H-atom abstraction from the peptidylglycine substrate. Crystal structure of a pre-catalytic O2 intermediate is available, which demonstrates an end-on bound Cu-O2 species at the active site. We propose to pursue single-crystal Cu K-edge XAS investigations on this pre-catalytic intermediate to determine its geometric and electronic structure. We plan to carry out a time-series photoreduction investigation on the pre-catalytic intermediate to determine its stability upon exposure to the x-ray beam. We also propose to investigate the fully oxidized (CuIICuII) and fully reduced (CuICuI) states of PHM in the crystalline form to characterize their geometric structure and the ligand field strength using a combination of single-crystal XAS and computational methods (density functional theory (DFT) and time-dependent DFT methods). These spectroscopic and computational investigations will enable the electronic structure determination of relevant states in the PHM substrate hydroxylation mechanism. Near-edge multiple scattering analysis will be used to differentiate the two Cu sites present in PHM to determine the binding mode of O2 to Cu in the precatalytic intermediate. These data will also enable the determination of the role of the O2 in the intramolecular electron transfer from the CuH site to the CuM site. The combined analysis of Cu K-edge data, EXAFS, Near-edge multiple scattering analysis, DFT and time-dependent DFT calculations will lead to a better understanding of O2 binding, activation and substrate hydroxylation by PHM.