Molecular interplay of an assembly machinery for nitrous oxide reductase

Emissions of the critical ozone-depleting and greenhouse gas nitrous oxide (N₂O) from soils and industrial processes have increased considerably over the last decades^1-3. As the final step of bacterial denitrification, N₂O is reduced to chemically inert N₂ (refs. ^1,4) in a reaction that is catalysed by the copper-dependent nitrous oxide reductase (N₂OR) (ref. ⁵). The assembly of its unique [4Cu:2S] active site cluster Cu_Z requires both the ATP-binding-cassette (ABC) complex NosDFY and the membrane-anchored copper chaperone NosL (refs. ^4,6). Here we report cryo-electron microscopy structures of Pseudomonas stutzeri NosDFY and its complexes with NosL and N₂OR, respectively. We find that the periplasmic NosD protein contains a binding site for a Cu⁺ ion and interacts specifically with NosL in its nucleotide-free state, whereas its binding to N₂OR requires a conformational change that is triggered by ATP binding. Mutually exclusive structures of NosDFY in complex with NosL and with N₂OR reveal a sequential metal-trafficking and assembly pathway for a highly complex copper site. Within this pathway, NosDFY acts as a mechanical energy transducer rather than as a transporter. It links ATP hydrolysis in the cytoplasm to a conformational transition of the NosD subunit in the periplasm, which is required for NosDFY to switch its interaction partner so that copper ions are handed over from the chaperone NosL to the enzyme N₂OR.