Methanotrophic bacteria oxidize methane to methanol in the first step of their metabolic pathway. Whereas current catalysts that can selectively activate the 105 kcal mol-1 C-H bond in methane require high temperatures and pressures, methanotrophs perform this chemistry under ambient conditions using methane monooxygenase (MMO) enzymes. In most methanotrophs, this chemically challenging reaction is catalyzed by particulate methane monooxygenase (pMMO), a copper-dependent, integral membrane enzyme. pMMO is composed of three subunits, PmoA, PmoB, and PmoC, arranged in a trimeric complex.1 Despite extensive research and the availability of multiple crystal structures, the location and nature of the pMMO copper active site remain controversial. Studies are further complicated by issues with retaining enzymatic activity upon detergent solubilization and purification. Reconstitution of pMMO into bicelles, which mimic the membrane environment, recovers methane oxidation activity, indicating that activity loss is not caused by the removal of catalytic copper ions.2 Electron paramagnetic resonance (EPR) spectroscopic data acquired on Methylococcus capsulatus (Bath) cells show that the same copper centers are present in vivo and in the isolated pMMO.3 Moreover, EPR data together with native top-down mass spectrometry (nTDMS) analysis of pMMO in nanodiscs indicate that PmoB and PmoC each contain a single copper ion.3-4 Finally, recent studies suggest that additional protein components, including the copper protein PmoD,5 may be critical for pMMO activity. Taken together, this work provides new insight into enzymatic oxidation of methane by a complex integral membrane metalloenzyme.