Nitrate from Neurospora crassa

Pan, S.-S. Nason, A. (1978). Purification and characterization of homogeneous assimilatory reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase from Neurospora crassa. Biochimica et Biophysica Acta 523, 297-313. 

Figure 1. Nitrate reductase from Neurospora crassa —composition and presumed electron transfer sequence (16, 18)...

Nitrite reduction in assimilatory nitrate-reducing Neurospora crassa, Torulopsis nitratophila, Azotobacter vinelandii, and Azotobacter chro-ococcum appears to be catalyzed by enzyme systems which require flavin and metals. The enzyme from N. crassa has been partially purified, and its molecular weight has been estimated to be 300,000 (344, 346, 351, 367). The enzyme reduces both nitrite and hydroxylamine to ammonia and utilizes NADH or NADPH as electron donor. It is reported to be a FAD-dependent enzyme and to contain iron, copper, and active thiol (346, 367). Three moles of NADH are oxidized per mole of nitrite reduced to ammonia. It has been suggested that the reduction of nitrite occurs in three steps, each involving two electrons. Thus, hyponitrite and hydroxylamine have been proposed as successive intermediates in the re-... 

Gorlatova, N., M. Tchorzewski, T. Kurihara, K. Soda, and N. Esaki. 1998. Purification, characterization, and mechanism of a flavin mononucleotide-dependent 2-nitro-propane dioxygenase from Neurospora crassa. Appl. Environ. Microbiol. 64 1029-1033. Corny, N., G. Wahl, A. Brune, and B. Schink. 1992. A strictly anaerobic nitrate-reducing bacterium growing with resorcinol and other aromatic compunds. Arch. Microbiol. 158 48-53. 

The molybdenum cofactor was liberated from D. gigas AOR, and under appropriate conditions was transferred quantitatively to nitrate reductase in extracts of Neurospora crassa nit-1 mutant) to yield active nitrate reductase 217). On the basis of molybdenum content, the activity observed for reconstitution with molybdenum cofactor of D. gigas was lower (25%) than the values observed for the procedure using extractable molybdenum cofactor of XO, used as reference. This result can now be put in the context of the difference in pterin present (MPT-XO and MCD-AOR) 218). 

The assimilatory enzyme from the mold Neurospora crassa has been intensively studied for over two decades, particularly by Nason and his collaborators. Thus, Nason and Evans (39) identified FAD as a prosthetic group in the enzyme Nicholas, Nason, and McElroy (40) showed that molybdenum was required for the synthesis of nitrate reductase Nicholas and Nason (41) suggested its presence in the enzyme Garrett and Nason (42) showed that a b-type cytochrome (cytochrome 6557) co-purifies with this nitrate reductase and Nason et al. (11) suggested, from in vitro complementation experiments with nitrate reductaseless mutants, that the enzyme consists of at least two components required for activity. These workers have suggested that the electron transfer pathway is ... 

N.r. of Neurospora crassa has M, 228,000. A N.r. mutant (Neurospora crassa nit-3) produces a subunit of N.r. containing cytochrome and Mo (M, 160,000) this does not react with NAD(P)H, but transfers electrons to nitrate from exogenous FADH2 or reduced methyl viologen. The other component is produced by Neurospora crassa nit-I it is unable to reduce nitrate, but catalyses the reduction of cytochrome c by NAD(P)H. 

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