Identification of the Valence and Coordination Environmentof the Particulate Methane Monooxygenase Copper Centers by AdvancedEPR Characterization

摘要

Particulate methane monooxygenase (pMMO) catalyzes the oxidation of methane to methanol in methanotrophic bacteria. As a copper-containing enzyme, pMMO has been investigated extensively by electron paramagnetic resonance (EPR) spectroscopy, but the presence of multiple copper centers has precluded correlation of EPR signals with the crystallographically identified monocopper and dicopper centers. A soluble recombinant fragment of the pmoB subunit of pMMO, spmoB, like pMMO itself, contains two distinct copper centers and exhibits methane oxidation activity. The spmoB protein, spmoB variants designed to disrupt one or the other or both copper centers, as well as native pMMO have been investigated by EPR, ENDOR, and ESEEM spectroscopies in combination with metal content analysis. The data are remarkably similar for spmoB and pMMO, validating the use of spmoB as a model system. The results indicate that one EPR-active Cu(II) ion is present per pMMO and that it is associated with the active-site dicopper center in the form of a valence localized Cu(I)Cu(II) pair; the Cu(II), however, is scrambled betweenthe two locations within the dicopper site. The monocopper site observedin the crystal structures of pMMO can be assigned as Cu(I). ¹⁴N ENDOR and ESEEM data are most consistent with one of these dicopper-sitesignals involving coordination of the Cu(II) ion by residues His137and His139, the other with Cu(II) coordinated by His33 and the N-terminalamino group. ¹H ENDOR measurements indicate there is noaqua (HxO) ligand bound to the Cu(II),either terminally or as a bridge to Cu(I).