Nitrous oxide reductase (N 2 OR) is the terminal enzyme of the denitrification pathway of soil bacteria that reduces the greenhouse gas nitrous oxide (N 2 O) to dinitrogen. In addition to a binuclear Cu A site that functions in electron transfer, the active site of N 2 OR features a unique tetranuclear copper cluster bridged by inorganic sulfide, termed Cu Z . In copper-limited environments, N 2 OR fails to function, resulting in truncation of denitrification and rising levels of N 2 O released by cells to the atmosphere, presenting a major environmental challenge. Here we report studies of nosL from Paracoccus denitrificans, which is part of the nos gene cluster, and encodes a putative copper binding protein. A Paracoccus denitrificans ΔnosL mutant strain had no denitrification phenotype under copper-sufficient conditions but failed to reduce N 2 O under copper-limited conditions. N 2 OR isolated from ΔnosL cells was found to be deficient in copper and to exhibit attenuated activity. UV-visible absorbance spectroscopy revealed that bands due to the Cu A center were unaffected, while those corresponding to the Cu Z center were significantly reduced in intensity. In vitro studies of a soluble form of NosL without its predicted membrane anchor showed that it binds one Cu( I ) ion per protein with attomolar affinity, but does not bind Cu( II ). Together, the data demonstrate that NosL is a copper-binding protein specifically required for assembly of the Cu Z center of N 2 OR, and thus represents the first characterised assembly factor for the Cu Z active site of this key environmental enzyme, which is globally responsible for the destruction of a potent greenhouse gas.