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Emissions of the critical ozone-depleting and greenhouse gas nitrous oxide (N2O) from soils and industrial processes have increased significantly over the last decades 1-3 . As the final step of bacterial denitrification, the critical greenhouse gas N2O is reduced to chemically inert N2 (ref. 1,4) in a reaction catalyzed by the copper-dependent nitrous oxide reductase (N2OR) 5 . The assembly of its unique [4Cu:2S] active site cluster CuZ requires both the ATP-bindingcassette (ABC) complex NosDFY and the membrane-anchored copper chaperone NosL 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 features a binding site for a Cu + ion and interacts specifically with NosL in its nucleotide-free state, while its binding to N2OR requires a conformational change triggered by ATP binding.Mutually exclusive complex structures of NosDFY with NosL and N2OR reveal a sequential metal trafficking and assembly pathway for the most complex copper site known to date. In it, NosDFY acts as a mechanical energy transducer rather than 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.

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