Nitrous oxide (N2O) respiration by the denitrifying bacterium Pseudomonas stutzeri requires the synthesis of the multi-copper enzyme N2O reductase. nosZ, the structural gene for this enzyme, is part of a DNA region of approximately 8 kbp that carries several essential genes. Insertional mutagenesis of the region downstream of nosZ generates apoenzyme-synthesizing strains, which argues for the existence of functions for copper acquisition or copper processing, or both, for N2O reductase. The relevant DNA region of approximately 3.2 kbp was sequenced and found to consist of three genes, nosDFY, presumably within a single transcriptional unit. Cellular concentration, copper content, and location of the N2O reductase protein were studied with mutants which were affected in the three genes.
Interactions of the deduced gene products among each other and with the cytoplasmic membrane appear to be analogous to those of the components of osmotic-shock- sensitive bacterial transport systems. The 33.8-kDa product of the nosF gene belongs to the family of nucleotide-binding proteins [C. Higgins et al. (1986) Nature 323, 448-450]. Its amino acid sequence shows two canonical nucleotide-binding motifs, and the positional identity of amino acids to members of this family is around 30%. The 29.4-kDa product of the nosY gene is a hydrophobic protein with six predicted transmembrane helices and an export signal. The 48.2-kDa product of the nosD gene is a periplasmic component; it carries an export signal and is a hydrophilic protein. N2O reductase itself is a periplasmic enzyme. Our results provide evidence for an auxiliary system of Cu processing and suggest its involvement in the periplasmic biosynthesis of the Cu centers of N2O reductase.