Bacterial nitrous oxide (N 2 O) reductase is the terminal oxidoreductase of a respiratory process that generates dinitrogen from N 2 O. To attain its functional state, the enzyme is subjected to a maturation process which involves the protein-driven synthesis of a unique copper-sulfur cluster and metallation of the binuclear Cu A site in the periplasm. There are seven putative maturation factors, encoded by nosA, nosD, nosF, nosY, nosL, nosX, and sco. We wanted to determine the indispensable proteins by expressing nos genes from Pseudomonas stutzeri in the nondenitrifying organism Pseudomonas putida. An in silico study of denitrifying bacteria revealed that nosL, nosX (or a homologous gene, apbE), and sco, but not nosA, coexist consistently with the N 2 O reductase structural gene and other maturation genes. Nevertheless, we found that expression of only three maturation factors (periplasmic protein NosD, cytoplasmic NosF ATPase, and the six-helix integral membrane protein NosY) together with nosRZ in trans was sufficient to produce catalytically active holo-N 2 O reductase in the nondenitrifying background. We suggest that these obligatory factors are required for Cu-S center assembly. Using a mutational approach with P. stutzeri, we also studied NosA, the Cu-containing outer membrane protein previously thought to have Cu insertase function, and ScoP, a putative membrane-anchored chaperone for Cu A metallation. Both of these were found to be dispensable elements for N 2 O reductase biosynthesis. Our experimental and in silico data were integrated in a model of N 2 O reductase maturation.