LACK OF COPPER INSERTION INTO UNPROCESSED CYTOPLASMIC NITROUS-OXIDE REDUCTASE GENERATED BY AN R20D SUBSTITUTION IN THE ARGININE CONSENSUS MOTIF OF THE SIGNAL PEPTIDE

Metal insertion into an engineered cytoplasmic form of the multicopper enzyme N2O reductase (N2OR) (EC 1.7.99.6) of Pseudomonas stutzeri was studied. The reductase has an unusually long presequence of 50 amino acids for translocation into the periplasm. The signal peptide of N2OR shares a conserved twin-arginine sequence motif with the signal pepti des of other N2O reductases and a sizeable group of periplasmic or mem brane-bound enzymes, requiring cofactor insertion or processing. A cat alytically inactive reductase, N2ORR20D, that lacked Cu, accumulated i n the cytoplasm on mutation of the first arginine of this motif. The C u-A site of N2ORR20D could be reconstituted in vitro indicating that t he lack of metal was not due to a serious conformational restraint. Ou r findings locate the event of in vivo Cu insertion into N,OR in the p eriplasm or allow it to take place concomitant with protein translocat ion. (C) 1997 Elsevier Science B.V.