Simultaneous occupancy of Cu _C and Cu _D in the ammonia monooxygenase active site

Ammonia monooxygenase (AMO), a copper-dependent membrane enzyme, catalyzes the first and rate-limiting step of nitrification: the oxidation of ammonia to hydroxylamine. Despite its central role in the global nitrogen cycle and its biotechnological relevance, structural characterization of AMO has lagged behind that of its homolog, particulate methane monooxygenase (pMMO), due to the slow growth rates of ammonia-oxidizing bacteria and the instability of AMO upon purification. Recent cryoEM studies of <i>Nitrosomonas europaea</i> AMO and <i>Methylococcus capsulatus</i> (Bath) pMMO in native membranes revealed new structural features, including two adjacent copper-binding sites in the transmembrane region, Cu_C and Cu_D, believed to constitute the active site. Although multiple structures were determined under various conditions, simultaneous occupancy of Cu_C and Cu_D was never observed, leaving their potential functional interplay unresolved. Here we report the 2.6 Å resolution cryoEM structure of AMO from <i>Nitrosospira briensis</i> C-128 in isolated native membranes. This structure reveals the first instance of simultaneous copper occupancy of the Cu_C and Cu_D sites, along with occupancy of the periplasmic Cu_B site. Electron paramagnetic resonance (EPR) spectroscopic data indicate that the Cu_B site is primarily occupied by Cu(ii), while Cu_C and Cu_D are primarily occupied by diamagnetic ions, presumably Cu(i). Notably, a lipid molecule is bound between the Cu_C and Cu_D sites, separating them by ∼8.0 Å. The results underscore the importance of studying these enzymes in their native environments across species to resolve conserved and divergent molecular features.