FUNCTIONAL DISSECTION AND SITE-DIRECTED MUTAGENESIS OF THE STRUCTURALGENE FOR NAD(P)H-NITRITE REDUCTASE IN NEUROSPORA-CRASSA

Neurospora crassa NAD(P)H-nitrite reductase, coded by the nit-6 gene, is a soluble, alpha(2)-type modimeric protein composed of 127-kDa poly peptide subunits. This multicenter oxidation-reduction enzyme utilizes either NADH or NADPH as electron donor and possesses as prosthetic gr oups two iron-sulfur (Fe4S4) clusters, two siroheme groups, and two FA D molecules. The native activity of the enzyme is the NAD(P)H-dependen t reduction of nitrite to ammonia. In addition, N. crassa nitrite redu ctase displays several partial activities in vitro, including a sirohe me-independent NAD(P)H-cytochrome c reductase activity and an FAD-inde pendent dithionite-nitrite reductase activity. These partial activitie s are presumed to be manifestations of discrete functional domains wit hin the protein. A full-length nit-6 cDNA was constructed and used in developing an expression system within E. coli capable of yielding hig h levels of NADPH-nitrite reductase activity. Maximal expression was o btained in nirB(-) E. coli cells grown anaerobically at 22 +/- 1 degre es C, in conjunction with co-expression of a plasmid-borne cysG gene ( encoding the rate-limiting enzyme in siroheme synthesis) and cotransfo rmation with plasmid pGroESL (encoding bacterial chaperonins GroES and GroEL). Dissection of gene segments encoding putative functional doma ins within the nit-6 gene was performed. Expression of a partial cDNA construct encoding the FAD-/NAD-binding domain yielded extracts with N ADPH-cytochrome c reductase activity but no NADPH-nitrite reductase ac tivity or dithionite-nitrite reductase activity. Expression of a cDNA construct encoding the (Fe4S4)-siroheme-binding domain resulted in ext racts possessing dithionite-nitrite reductase activity but no NADPH-ni trite reductase or NADPH-cytochrome c reductase activity. Analysis of site-directed mutations altering amino acid residues Cys-331 within th e FAD-/NAD-binding domain and Ser-755 within the (Fe4S4)-siroheme-bind ing domain of the nitrite reductase demonstrated that these residues w ere not essential for native or partial enzyme activity. Cys-757 withi n the (Fe4S4)-siroheme-binding domain was essential for native enzyme activity.