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Abstract

Particulate methane monooxygenase (pMMO) plays a critical role in catalyzing the conversion of methane to methanol, constituting the initial step in the C1 metabolic pathway within methanotrophic bacteria. However, the membrane-bound pMMO’s structure and catalytic mechanism, notably the copper’s valence state and genuine active site for methane oxidation, have remained elusive. Based on the recently characterized structure of membrane-bound pMMO, extensive computational studies were conducted to address these long-standing issues. A comprehensive analysis comparing the quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulated structures with cryo-EM data indicates that both the Cu_C and Cu_D sites tend to stay in the Cu(I) valence state within the membrane environment. Additionally, the concurrent presence of Cu(I) at both Cu_C and Cu_D sites leads to the significant reduction of the ligand-binding cavity situated between them, making it less likely to accommodate a reductant molecule such as durohydroquinone (DQH₂). Subsequent QM/MM calculations reveal that the Cu_D(I) site is more reactive than the Cu_C(I) site in oxygen activation, en route to H₂O₂ formation and the generation of Cu(II)–O^•– species. Finally, our simulations demonstrate that the natural reductant ubiquinol (CoQH₂) assumes a productive binding conformation at the Cu_D(I) site but not at the Cu_C(I) site. This provides evidence that the true active site of membrane-bound pMMOs may be Cu_D rather than Cu_C. These findings clarify pMMO’s catalytic mechanism and emphasize the membrane environment’s pivotal role in modulating the coordination structure and the activity of copper centers within pMMO.