The structural gene, nirK, for the respiratory Cu-containing
nitrite reductase from denitrifying Pseudomonas aureofaciens
was
isolated and sequenced. It encodes a polypeptide of 363 amino acids
including
a signal peptide of 24 amino acids for protein export. The sequence
showed
63.8% positional identity with the amino acid sequence of "Achromobacter
cycloclastes" nitrite reductase. Ligands for the blue, type I
Cu-binding
site and for a putative type-II site were identified. The nirK
gene
was transferred to the mutant MK202 of P. stutzeri which lacks
cytochrome
cd1
nitrite reductase due to a transposon Tn5 insertion in its
structural
gene, nirS. The heterologous enzyme was active in vitro and in
vivo
in this background and restored the mutationally interrupted
denitrification
pathway. Transfer of nirK to Escherichia coli resulted
in
an active nitrite reductase in vitro. Expression of the nirS
gene
from P. stutzeri in P. aureofaciens and E. coli
led
to nonfunctional gene products. Nitrite reductase activity of cell
extract
from either bacterium could be reconstituted by addition of heme
d1,
indicating that both heterologous hosts synthesized a cytochrome cd1
without the d1-group.