title: Evidence for oxygen binding at the active site of particulate methane monooxygenase.
abstract: Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that converts methane to methanol in methanotrophic bacteria. The enzyme consists of three subunits, pmoB, pmoA, and pmoC, organized in an α(3)β(3)γ(3) trimer. Studies of intact pMMO and a recombinant soluble fragment of the pmoB subunit (denoted as spmoB) indicate that the active site is located within the soluble region of pmoB at the site of a crystallographically modeled dicopper center. In this work, we have investigated the reactivity of pMMO and spmoB with oxidants. Upon reduction and treatment of spmoB with O(2) or H(2)O(2) or pMMO with H(2)O(2), an absorbance feature at 345 nm is generated. The energy and intensity of this band are similar to those of the μ-η(2):η(2)-peroxo-Cu(II)(2) species formed in several dicopper enzymes and model compounds. The feature is not observed in inactive spmoB variants in which the dicopper center is disrupted, consistent with O(2) binding to the proposed active site. Reaction of the 345 nm species with CH(4) results in the disappearance of the spectroscopic feature, suggesting that this O(2) intermediate is mechanistically relevant. Taken together, these observations provide strong new support for the identity and location of the pMMO active site.
authors.affiliation: Department of Molecular Biosciences, Northwestern University, Evanston, Illinois 60208, USA.
authors.forename: Megen A
authors.initials: MA
authors.lastname: Culpepper
authors.forename: George E
authors.initials: GE
authors.lastname: Cutsail
authors.forename: Brian M
authors.initials: BM
authors.lastname: Hoffman
authors.forename: Amy C
authors.initials: AC
authors.lastname: Rosenzweig
mesh_terms: D020134:Catalytic Domain; D020619:Methylococcus capsulatus / Q000737:chemistry / Q000201:enzymology*; D008958:Models, Molecular; D010100:Oxygen / Q000378:metabolism*; D010105:Oxygenases / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D010105:Oxygenases; C021555:methane monooxygenase; D010100:Oxygen