XAS STUDIES OF COPPER MONOOXYGENASE ENZYMES

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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 cente