Purification and Properties of the Neurospora crassa Assimilatory Nitrite Reductase

Abstract

Abstract The Neurospora crassa (5297a) assimilatory nitrite reductase (EC 1.6.6.4) catalyzes the NADPH-dependent formation of ammonia from nitrite, a 6 electron reduction. The enzyme has been purified 90-fold by ammonium sulfate fractionations, polyethylene glycol treatment (to remove nucleic acids), DEAE-cellulose, and molecular sieve chromatography. Associated with this NADPH-nitrite reductase activity are NADH-nitrite reductase activity, dithionite-nitrite reductase activity, NADPH-hydroxylamine reductase activity, and NADH-hydroxylamine reductase activity. All the activities are manifestations of a single protein complex as shown by their co-elution from DEAE-cellulose and Sephadex G-200 columns, and by their co-sedimentation during sucrose density gradient centrifugation. The molecular weight was calculated to be 290,000 using a relative s0.72520, w value of 9.4 determined by sucrose density gradient centrifugation and a Stokes radius of 76 A estimated from Sephadex G-200 gel filtration data. The absorption spectrum of a partially purified sample of nitrite reductase exhibited maxima at 405, 555, 585, and g600 nm showing some characteristics of other nitrite and sulfite reducing enzymes which have been studied. The apparent Km for nitrite is 0.01 mm whereas that for hydroxylamine is 2 orders of magnitude higher, 3 mm, suggesting that nitrite is the physiological substrate. The reduced pyridine nucleotide-dependent activities, which require FAD for maximal activity, are more heat-labile and more sensitive to inhibition by p-hydroxymercuribenzoate than the dithionite-dependent activity. All the activities of the nitrite reductase complex are sensitive to inhibition by cyanide, sulfide, sulfite, and o-phenanthroline. The enzyme activities are inhibited on preincubation with either reduced pyridine nucleotide electron donor (NADPH or NADH), provided FAD is present. The electron acceptors, nitrite and hydroxylamine, protect against this inactivation. No evidence of feedback inhibition was found with the variety of nitrogenous compounds tested.