BPS2026 – Structure and function of copper membrane monooxygenases

Aerobic microbial processes are important sources and sinks for greenhouse gases with methane-oxidizing bacteria (methanotrophs) consuming methane and ammonia-oxidizing bacteria (nitrifiers) releasing nitrous oxide. Methanotrophs and nitrifiers use copper-dependent membrane monooxygenases to carry out the first steps in their metabolisms: the conversions of methane to methanol by particulate methane monooxygenase (pMMO) and ammonia to hydroxylamine by ammonia monooxygenase (AMO). Due to the loss of enzymatic activity upon detergent solubilization from their native intracytoplasmic membranes (ICMs), elucidating the structures and mechanisms of pMMO and AMO has posed significant challenges. Both enzymes consist of three subunits, including PmoB/AmoB, PmoA/AmoA, and PmoC/AmoC. Despite the availability of multiple crystal and cryoelectron microscopy (cryo-EM) structures, the location and nature of the pMMO copper active site remain controversial. Attempts to study AMO have not been successful, leaving details of its molecular architecture and copper centers unknown. Cryo-EM structures of active pMMO in native lipid nanodiscs alone and in the presence of product analogs combined with advanced paramagnetic resonance characterization have provided new insights into the identity of the pMMO active site. In addition, pMMO and AMO have been visualized directly in their native ICMs at high resolution by cryo-EM single particle analysis. These structures reveal the arrangement of enzyme trimers in the membrane, details of the copper centers, bound lipids, and previously unobserved components. The ability to obtain molecular level insight within the native environment will enable further understanding of these and other environmentally important membrane-bound cuproenzymes.