XAS STUDIES OF COPPER MONOOXYGENASE ENZYMES

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。 用X射线吸收光谱分析方法研究了含铜单加氧酶的结构和作用机理，包括肽甘氨酸单加氧酶和多巴胺β单加氧酶。吸收边和铜K边的EXAFS将与其他光谱方法相结合，以提供关键的机理测试。活性铜-氧物种是铜(II)-超氧实体的提议将从这样一个物种应该是EPR沉默但显示氧化吸收边的期望中得到检验。通过对M314X、X=I、H、C和D突变体的XAS研究，将研究配位蛋氨酸残基的基本作用。将对pH依赖性进行详细的研究，以检验活性中心碱基是结合在CUM中心的配位水分子的假设。最后，对各种还原剂还原蛋白质的研究将探索电子进入和铜中心之间重新分配的机制。

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. X-ray absorption spectroscopic measurements will be used to investigate the structure and mechanism of copper containing monooxygenase enzymes including peptidylglycine monooxygenase and dopamine beta monooxygenase. Absorption edge and EXAFS at the Cu K-edge will be combined with other spectroscopic approaches to provide critical tests of mechanism. A proposal that the active copper-dioxygen species is a Cu(II)-superoxo entity will be tested from the expectation that such a species should be EPR silent yet exhibit an oxidized absorption edge. The essential role of a coordinated methionine residue will be investigated through XAS studies of M314X, X= I, H, C, and D mutants. Detailed studies on the pH dependence will be carried out to test the hypothesis that the active site base is a coordinated water molecule bound at the CuM center. Finally, studies of reduction of the protein with a variety of reductants will probe mechanisms of electron entry and redistribution between the copper centers.