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 α₃β₃γ₃ 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 O₂ or H₂O₂ or pMMO with H₂O₂, an absorbance feature at 345 nm is generated. The energy and intensity of this band are similar to those of the μ-η²:η²-peroxo-Cu^II₂ 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 O₂ binding to the proposed active site. Reaction of the 345 nm species with CH₄ results in the disappearance of the spectroscopic feature, suggesting that this O₂ intermediate is mechanistically relevant. Taken together, these observations provide strong new support for the identity and location of the pMMO active site.