The synthesis of a functional nitrous oxide reductase requires an assembly apparatus for the insertion of the prosthetic copper. Part of the system is encoded by maturation genes located in Pseudomonas stutzeri immediately downstream of the structural gene for the enzyme. We have studied the transcriptional organization and regulation of this region and found a nosDFYLtatE operon structure. In addition to a putative ABC transporter, consisting of NosD, NosF, and NosY, the operon encodes a Cu chaperone, NosL, and a component of the Tat translocon, TatE. The nosD operon was activated in response to anaerobiosis and nitrate denitrification. The membrane-bound regulator NosR was required for operon expression; in addition, DnrD, a regulator of the Crp-Fnr family, enhanced expression under anaerobic conditions. This establishes a likely signal transduction sequence of NO -> DnrD -> nosR/NosR-> nosD operon. DnrD-dependent expression was also observed for the nnrS operon (located immediately downstream of the nosD operon), which encodes a putative heme-Cu protein (NnrS) and a member of the short-chain dehydrogenase family (ORF247). The NosF protein, encoded within the nosD operon, exhibits sequence similarity to ABC-type ATPases. It was fused to the Escherichia coli maltose-binding protein and overexpressed in soluble form. The fusion protein was purified and shown to have ATPase activity. NosF is the first maturation factor for which a catalytic function has been demonstrated in vitro.