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的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 肽基甘氨酸？羟基化单加氧酶（PHM）是一种非偶联的双核Cu蛋白，催化β-羟基化，所有肽基甘氨酸底物的C-末端甘氨酸的碳。在催化机制中，PHM结合并激活O2以从肽基甘氨酸底物中提取H原子。预催化O2中间体的晶体结构是可用的，这表明在活性位点处的末端结合的Cu-O2物种。我们建议追求单晶铜K-边XAS调查这个预催化中间体，以确定其几何和电子结构。我们计划进行一个时间序列的光还原调查的前催化中间体，以确定其稳定性后，暴露于X射线束。我们还建议调查的完全氧化（CuIICuII）和完全还原（CuICuI）状态的PHM的结晶形式，以表征其几何结构和配体场强度使用的组合单晶XAS和计算方法（密度泛函理论（DFT）和时间依赖性DFT方法）。这些光谱和计算的调查将使电子结构的测定在PHM底物羟基化机制的相关国家。近边多重散射分析将被用来区分两个铜网站PHM中存在的，以确定在预催化中间体中的O2的铜的结合模式。这些数据也将使确定的作用的O2在分子内的电子转移从CuH网站的CuM网站。结合Cu K边数据、EXAFS、近边多重散射分析、密度泛函理论和含时密度泛函理论计算，将有助于更好地理解PHM对O2的结合、活化和底物羟基化作用。

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.