Preparation and some properties of homogeneous Neurospora crassa assimilatory NADPH-nitrite reductase.
The Neurospora crassa assimilatory NADPH-nitrite reductase (NAD(P)H: nitrite oxidoreductase, EC 1.6.6.4), which catalyzes the NADPH-dependent formation of ammonia from nitrite, has been purified to homogeneity as judged by polyacrylamide gel electrophoresis. The specific activity of the purified enzyme is 26.9 mumol nitrite reduced/min per mg protein, which corresponds to a turnover number of 7800 min(-1). The enzyme also has associated NADH-nitrite reductase, NADPH-hydroxylamine reductase and NADH-hydroxylamine reductase activities. The stoichiometry of 3 mol NADPH oxidized per mol nitrite reduced and ammonia formed has been confirmed. The visible absorption spectrum of the nitrite reductase reveals maxima at 280,390 (Soret) and 580 (alpha) nm. The latter bands are indicative of the occurrence of siroheme as a prosthetic group. The A280nm/A390nm ratio of 7.0 and the Soret/alpha ratio of 3.8 are compatible with values reported for other purified siroheme-containing enzymes. These results are discussed in terms of the comparative biochemistry of various enzymes involved in nitrite, hydroxylamine and sulfite metabolism in Neurospora crassa and other organisms.
D006898:Hydroxylamines; D007700:Kinetics; D008722:Methods; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D009572:Nitrite Reductases / Q000302:isolation & purification* / Q000378:metabolism; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000378:metabolism; D013045:Species Specificity; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet
D006898:Hydroxylamines; D009247:NADH, NADPH Oxidoreductases; D009572:Nitrite Reductases; D050862:Oxidoreductases Acting on Sulfur Group Donors