The Copper Centers of Particulate Methane Monooxygenase: Differentiation of C- and E-Clusters

This dissertation investigates the approximately 15 copper ions of pMMO from M. capsulatus by a variety of spectroscopic, biochemical and molecular biological techniques. The first chapters describe spectroscopy studies of the C- and E-clusters of the protein. The pMMO protein is prepared in a number of chemical states which access the differentially oxidized of the C- and E-clusters. Investigation of these states by X-ray Absorption Spectroscopy (XAS) reveals unique oxidation states of pMMO copper ions upon anaerobic preparation, and oxidation with air, pure dioxygen, or hydrocarbon suicide substrate. Structural implications for the copper ions of the protein are discussed, and a model for the ability of acetylene to highly oxidize pMMO is explored. The differentiation of the copper ions of pMMO is explored by the use of proteolytic enzymes and chemical treatment with N,N,N',N'-Ethylenediamine-tetraacetic acid (EDTA), monitoring the ability of pMMO to form a Cu(II)-ferrocyanide adduct upon treatment with ferricyanide and subsequent electron transfer. Soluble, copper-binding domains are found, as well as specific centers that are buried in the transmembrane domain. The inability of an EDTA-treated preparation to regenerate a fully oxidized form of the protein indicates that the copper ions bound by pMMO do interact with one via electron transfer. The ability to assess the potential ligands of the copper ions of pMMO is determined by Electron Spin Echo Envelop Modulation (ESEEM) spectroscopy, revealing potential interactions between histidine residues and the enzyme active site. Subsequent chapters probe reported characteristic of the pMMO active site, thought to be located within the PmoA sub-unit. First, the identity of potential ligands for copper ions bound by the PmoA protein is assessed by a homology of pmoA. Next, the role of ion in pMMO is explored by treatment with NO(g), and it is concluded that the resulting signals in the g≈4.0 region of the Electron Paramagnetic Resonance (EPR) spectrum were due to an iron-nitrosyl adduct. The identity of the iron is assigned to adventitious iron bound to pMMO. A final chapter probes the overall structure and geometry of the active site by examining the regio- and stereoselectivity of pMMO-mediated hydroxylation and epoxidation chemistry.