COPPER-CONTAINING NITRITE REDUCTASE FROM PSEUDOMONAS-AUREOFACIENS IS FUNCTIONAL IN A MUTATIONALLY CYTOCHROME-CD(1)-FREE BACKGROUND (NIRS-) OF PSEUDOMONAS-STUTZERI

The structural gene, nirK, for the respiratory Cu-containing nitrite r eductase from denitrifying Pseudomonas aureofaciens was isolated and s equenced. It encodes a polypeptide of 363 amino acids including a sign al peptide of 24 amino acids for protein export. The sequence showed 6 3.8% positional identity with the amino acid sequence of ''Achromobact er cycloclastes'' nitrite reductase. Ligands for the blue, type I Cu-b inding site and for a putative type-II site were identified. The nirK gene was transferred to the mutant MK202 of P. stutzeri which lacks cy tochrome cd1 nitrite reductase due to a transposon Tn5 insertion in it s structural gene, nirS. The heterologous enzyme was active in vitro a nd in vivo in this background and restored the mutationally interrupte d denitrification pathway. Transfer of nirK to Escherichia coli result ed in an active nitrite reductase in vitro. Expression of the nirS gen e 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 t hat both heterologous hosts synthesized a cytochrome cd1 without the d 1-group.