Membrane properties modulate methane oxidation by particulate methane monooxygenase.

The copper-dependent membrane monooxygenases particulate methane monooxygenase (pMMO) and ammonia monooxygenase (AMO) oxidize methane to methanol and ammonia to hydroxylamine, respectively. These enzymes, which are important targets for biotechnology, reside in intracytoplasmic membranes (ICMs) where they form densely packed hexagonal arrays. While cryoEM structures of pMMO and AMO in ICMs have revealed closely-associated lipids, little is known about how specific lipids and membrane morphologies influence activity. Here we show through cryoelectron tomography (cryoET) that three species of methane- and ammonia-oxidizing bacteria exhibit different types of ICM ultrastructure. Reconstitution of Methylococcus capsulatus (Bath) pMMO into liposomes replicated the array structure, allowing a systematic dissection of how liposome diameter and composition affect activity. Proteoliposome activity is inversely correlated with liposome size, suggesting that pMMO activity may be higher in membranes with increased surface curvature. Further, a comparison of lipids isolated from methanotrophs (native lipids), phosphatidylcholine (PC), and phosphoethanolamine (PE) showed that PE confers increased activity, with maximal activity observed for unsaturated PEs. Methane solubility measurements indicate that these enhancements are specific to pMMO. Cardiolipin further increases activity, consistent with its enrichment in M. capsulatus (Bath) cells. To assess pMMO-pMMO interactions in the ICMs, a 6 Å resolution cryoelectron microscopy (cryoEM) structure of three neighboring pMMO trimers was determined, revealing their arrangement in the array as well as specific residues and lipids mediating interaction interfaces. Taken together, these findings provide insight into the impact of the membrane environment on pMMO function and establish a platform for examining pMMOs and AMOs in tunable lipid environments.