The oxidation of methane to methanol is not only the first committed step in the metabolism of methanotrophs, but also vital in C-1 chemistry as the means of producing fuels and chemicals. Methane monooxygenase (MMO) enzymes, including soluble MMO (sMMO) and particulate MMO (pMMO), hosted by methanotrophs, catalyze the O₂-dependent conversion of methane to methanol and thus play a vital role in the global-carbon-cycle. The mechanisms of O₂ activation by sMMO are well-studied, and the various reactive intermediates have been characterized by experiments. By contrast, the mechanism of action of pMMO remains elusive, especially regarding O₂ activation and the nature of the active species of the enzyme. Extensive efforts have been put into structural studies and biochemistry of pMMO. However, to date, no oxygenated intermediates have been characterized or observed spectroscopically along the O₂ activation pathway in pMMO.

With these challenges in mind, we took herein into account the physiological reductant of duroquinol (DQH₂), and performed multiscale simulations (MD and QM/MM) to decipher the monocopper-mediated O₂ activation mechanism and the catalytic cycle of pMMO in a realistic manner. Herein, we demonstrate that O₂ activation is in fact initiated by a Cu_C(II)−DQH^– species generated by deprotonation of DQH₂. Our simulations capture the exclusive pathway for the sequential formation of the intermediates, Cu_C(II)−O₂^•–, Cu_C(II)−OOH^– and H₂O₂, along the O₂ reduction pathway. Further H₂O₂ activation by the co-substrate ligated Cu_C(II)−DQH^– is initiated by DQH^• dissociation to yield Cu_C(I), and leads to the O−O hemolysis followed by the formation of Cu_C(II)−O^•– active species that is responsible for C−H oxidations. In particular, we found that the Cu_C(I) species is in high energy relative to the Cu_C(II)−DQH^– species, suggesting that the Cu_C(II)−DQH^– species may have some protective role analogous to copper(II)−tyrosyl complex in LPMOs.

In summary, this study not only identified the key Cu_C site responsible for catalysis, but also deciphered the vital role of the phenol co-substrate for O₂ activation at the Cu_C site. All these findings have far-reaching implications in pMMO catalysis and may inspire further experimental investigations of this important metalloenzyme. To read more about our work: Peng, W., Qu, X., Shaik, S., Wang, B. “Deciphering the Oxygen Activation Mechanism at the Cu_C Site of Particulate Methane Monooxygenase” in Nature Catalysis 2021 (doi: https://doi.org/10.1038/s41929-021-00591-4)