Product analogue binding identifies the copper active site of particulate methane monooxygenase

Nature's primary methane-oxidizing enzyme, the membrane-bound particulate methane monooxygenase (pMMO), catalyses the oxidation of methane to methanol. Copper is required for pMMO activity, and decades of structural and spectroscopic studies have sought to identify the active site among three candidates: the CuB, CuC and CuD sites. Challenges associated with the isolation of active pMMO hindered identification of its catalytic centre; however, we have recently shown that reconstituting pMMO into native lipid nanodiscs stabilizes its structure and restores its activity. Here, such active samples were incubated with 2,2,2-trifluoroethanol, a product analogue that serves as a readily visualized active-site probe. Interactions between 2,2,2-trifluoroethanol and the CuD site were observed with pulsed electron nuclear double resonance spectroscopy and cryoelectron microscopy, implicating CuD and the surrounding hydrophobic pocket as the likely site of methane oxidation. Use of these orthogonal techniques on parallel samples is a powerful approach that can circumvent difficulties in interpreting metalloenzyme cryoelectron microscopy maps. Different locations have been proposed for the catalytic centre of particulate methane monooxygenase for methane oxidation to methanol. Now, cryoelectron microscopy structures and electron nuclear double resonance spectroscopic measurements of the enzyme with a product analogue identify CuD as the active site and provide insights into substrate binding.