XAS STUDIES OF COPPER MONOOXYGENASE ENZYMES

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 X射线吸收光谱测量将用于研究含铜单加氧酶包括肽基甘氨酸单加氧酶和多巴胺β单加氧酶的结构和机制。吸收边和EXAFS在铜K边将结合其他光谱方法，提供关键的机制测试。一个建议，即活性铜-二氧物种是一个Cu（II）-superoxo实体将进行测试的期望，这样的物种应该是EPR沉默还表现出氧化的吸收边。通过对M314 X，X= I，H，C和D突变体的XAS研究，将研究配位甲硫氨酸残基的重要作用。对pH依赖性进行了详细的研究，以检验活性位点碱是结合在CuM中心的配位水分子的假设。最后，用各种还原剂还原蛋白质的研究将探测铜中心之间的电子进入和再分配机制。

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.