Metal insertion into an engineered cytoplasmic form of the multicopper enzyme N 2 O reductase (N 2 OR) (EC 1.7.99.6) ofPseudomonas stutzeri was studied. The reductase has an unusually long presequence of 50 amino acids for translocation into the periplasm. The signal peptide of N 2 OR shares a conserved twin-arginine sequence motif with the signal peptides of other N 2 O reductases and a sizeable group of periplasmic or membrane-bound enzymes, requiring cofactor insertion or processing. A catalytically inactive reductase, N 2 OR R 2 0 D , that lacked Cu, accumulated in the cytoplasm on mutation of the first arginine of this motif. The Cu A site of N 2 OR R 2 0 D could be reconstituted in vitro indicating that the lack of metal was not due to serious conformational restraint. Our findings locate the event of in vivo Cu insertion into N 2 OR in the periplasm or allow it to take place concomitant with protein translocation.