Unraveling the Valence State and Reactivity of Copper Centers in Membrane-Bound Particulate Methane Monooxygenase.

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 (DQH 2 ). 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 H 2 O 2 formation and the generation of Cu(II)-O •- species. Finally, our simulations demonstrate that the natural reductant ubiquinol (CoQH 2 ) 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.