RESOLUTION OF THE NIRD LOCUS FOR HEME D(1) SYNTHESIS OF CYTOCHROME CD(1) (RESPIRATORY NITRITE REDUCTASE) FROM PSEUDOMONAS-STUTZERI

The genetic organization of the nirD locus of Pseudomonas stutzeri ZoB ell, necessary for a catalytically active cytochrome cd(1) (EC 1.9.3.2 ), was determined. The locus comprises the unidirectionally transcribe d open reading frames nirFDLGH, downstream of nirMC of the nir gene cl uster, and immediately upstream of the norCB operon encoding nitric ox ide (NO) reductase (EC 1.7.99.7). Notable sequence relatedness was fou nd between NirF and cytochrome cd(1) (NirS), within NirDLGH, and betwe en NirM and NirC, suggesting several gene duplication events in this r egion. The derived NirF protein (391 amino acids, M(r) 43137) has 23.8 % identity (51.1% overall similarity) with NirS, but lacks the N-termi nal heme-c-binding domain of NirS. Insertional mutagenesis of the five open reading frames resulted in the loss of respiratory nitrite reduc tase activity in vivo and in vitro. Mutant strains, when induced with nitrate for denitrification, synthesized a periplasmic cytochrome cd(1 ) lacking heme d(1). The defect was caused by the inability of the cel l to synthesize heme d(1). The nirD locus is proposed to encode a mult imeric and multifunctional enzyme complex involved in the synthesis of heme d(1). Mutations in nirFDLGH lowered substantially the expression level of norCB. Nir(-) mutants, unable to generate NO in vivo, provid e indirect evidence for an NO sensor and an inducer role of NO for its cognate reductase.