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
peptides
of other N2O reductases and a sizeable group of periplasmic or
membrane-bound
enzymes, requiring cofactor insertion or processing. A catalytically
inactive
reductase, N2ORR20D,
that lacked Cu, accumulated in the cytoplasm on mutation of the first
arginine
of this motif. The CuA site of N2ORR20D
could be reconstituted in vitro indicating that the lack of metal was
not
due to a serious conformational restraint. Our findings locate the
event
of in vivo Cu insertion into N2OR in the
periplasm
or allow it to take place concomitant with protein translocation.