Deciphering the oxygen activation mechanism at the CuC 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 O 2 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 (DQH 2 ). We demonstrate that O 2 activation is in fact initiated by a Cu C ( ii )–DQH − species generated by deprotonation of DQH 2 . Our simulations capture the exclusive pathway for the sequential formation of the intermediates, Cu C ( ii) −O 2 •− , Cu C ( ii )−OOH − and H 2 O 2 , along the O 2 reduction pathway. Furthermore, H 2 O 2 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 O 2 activation and help resolve the enigmatic mechanism of pMMO. The catalytic mechanism of oxygen activation employed by particulate methane monooxygenase for the oxidation of methane has remained elusive. Now, computational simulations suggest an important role of the phenol co-substrate and a catalytic cycle is proposed.