Deciphering the oxygen activation mechanism at the Cu_C site of particulate methane monooxygenase

The enzymatic oxidation of methane to methanol was discovered in methanotrophs over 110 years ago. Nevertheless, the mechanism of action of particulate methane monooxygenase (pMMO) remains elusive, especially regarding O₂ activation and the nature of the active species of the enzyme. Here we decipher the catalytic cycle of pMMO in the presence of the physiological reductant duroquinol (DQH₂). 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. Furthermore, H₂O₂ activation by Cu_C(ii)−DQH⁻ is initiated by dissociation of DQH^• to yield Cu_C(i), followed by Cu_C(i)-catalysed O−O homolysis, en route to the formation of the Cu_C(ii)−O^•− species, which is responsible for C−H oxidations. These findings uncover the important roles of the phenol co-substrate for O₂ activation and help resolve the enigmatic mechanism of pMMO. [Figure not available: see fulltext.]