A reduced pyridine nucleotides-diaphorase activity associated to the assimilatory nitrite reductase complex from Neurospora crassa

The Neurospora crassa assimilatory NAD(P)H-nitrite reductase complex has associated a NAD(P)H-diaphorase activity. 1. This NAD(P)H-diaphorase activity can use either mammalian cytochrome c, 2,6--dichlorophenol-indophenol, ferricyanide, or menadione as electron acceptor from the reduced pyridine nucleotides, and requires flavin adenine dinucleotide for maximal activity. 2. It is inhibited by p-hydroxymercuribenzoate, 1 muM, and it is unaffected by cyanide, sulfite, or arsenite at concentrations which completely inhibit the NAD(P)H-nitrite reductase activity. 3. Flavin adenine dinucleotide specifically protects the NAD(P)H-diaphorase activities, but not the NAD(P)H-nitrite reductase activities, against thermal inactivation. 4. In vitro preincubation of the Neurospora crassa nitrite reductase complex with reduced pyridine nucleotides plus flavin adenine dinucleotide inactivates the NAD(P)H-nitrite reductase activities, but does not affect the NAD(P)H-diaphorase activities, indicating that this nitrite reductase inactivation occurs in the part of the enzyme that contain the nitrite reducing center.     

Siroheme: a prosthetic group of the Neurospora crassa assimilatory nitrite reductase.

The Neurospora crassa assimilatory nitrite reductase (EC 1.6.6.4) catalyzes the NADPH-dependent reduction of nitrite to ammonia, a 6-electron transfer reaction. Highly purified preparations of this enzyme exhibit absorption spectra which suggest the presence of a heme component (wavelength maxima for oxidized senzyme: 390 and 578 nm). There is a close correspondence between nitrite reductase activity and absorbance at 400 nm when partially purified nitrite reductase preparations are subjected to sucrose gradient centrifugation. In addition, a role for an iron component in the formation of active nitrite reductase is indicated by the fact that nitrate-induced production of nitrite reductase activity in Neurospora mycelia in vivo requires the presence of iron in the induction medium. The heme chromophore present in Neurospora nitrite reductase preparations is reducible by NADPH. Complete reduction, however, requires the presence of added FAD. The NADPH-nitrite reductase activity of the enzyme is also dependent upon addition of FAD. A spectrally unique complex is formed between the heme chromophore and nitrite (or a reduction product thereof) when nitrite is added to NADPH-reducted enzyme. Carbon monoxide forms a complex with the heme chromophore of nitrite reductase with an intense alpha-band maximum at 590 nm and a beta-band of lower intensity at 550 nm. CO is an inhibitor of NADPH-nitrite reductase activity. Spectrophotometrically detectable CO complex formation and Co inhibition of enzyme activity share the following properties...     

Regulation of the Neurospora crassa assimilatory nitrate reductase.

Reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate reductase from Neurospora crassa was purified and found to be stimulated by certain amino acids, citrate, and ethylenediaminetetraacetic acid (EDTA). Stimulation by citrate and the amino acids was dependent upon the prior removal of EDTA from the enzyme preparations, since low quantities of EDTA resulted in maximal stimulation. Removal of EDTA from enzyme preparations by dialysis against Chelex-containing buffer resulted in a loss of nitrate reductase activity. Addition of alanine, arginine, glycine, glutamine, glutamate, histidine, tryptophan, and citrate restored and stimulated nitrate reductase activity from 29- to 46-fold. The amino acids tested altered the Km of NADPH-nitrate reductase for NADPH but did not significantly change that for nitrate. The Km of nitrate reductase for NADPH increased with increasing concentrations of histidine but decreased with increasing concentrations of glutamine. Amino acid modulation of NADPH-nitrate reductase activity is discussed in relation to the conservation of energy (NADPH) by Neurospora when nitrate is the nitrogen source.     

Purification and characterization of homogeneous assimilatory reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase from Neurospora crassa.

Neurospora crassa wild type STA4 NADPH-nitrate reductase (NADPH : nitrate oxidoreductase, EC 1.6.6.3) has been purified 5000-fold with an overall yield of 25--50%. The final purified enzyme contained 4 associated enzymatic activities: NADPH-nitrate reductase, FADH2-nitrate reductase, reduced methyl viologen-nitrate reductase and NADPH-cytochrome c reductase. Polyacrylamide gel electrophoresis yielded 1 major and 1 minor protein band and both bands exhibited NADPH-nitrate and reduced methyl viologen-nitrate reductase activities. SDS gel electrophoresis yielded 2 protein bands corresponding to molecular weights of 115 000 and 130 000. A single N-terminal amino acid (glutamic acid) was found and proteolytic mapping for the two separated subunits appeared similar. Purified NADPH-nitrate reductase contained 1 mol of molybdenum and 2 mol of cytochrome b557 per mol protein. Non-heme iron, zinc and copper were not detectable. It is proposed that the Neurospora assimilatory NADPH-nitrate reductase consists of 2 similar cytochrome b557-containing 4.5-S subunits linked together by one molybdenum cofactor. A revised electron flow scheme is presented. p-Hydroxymercuribenzoate inhibition was reversed by sulfhydryl reagents. Inhibitory pattern of p-hydroxymercuribenzoate and phenylglyoxal revealed accessible sulfhydryl and arginyl residue(s) as functional group(s) in the earlier part of electron transport chain as possibly the binding site of NADPH or FAD.     

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.     

On the presence of a heme-binding domain homologous to cytochrome b(5) in Neurospora crassa assimilatory nitrate reductase

Assimilatory nitrate reductase has been purified with 55% recovery from a Neurospora crassa nmr-1 nit-6 mutant, using a modification of a published procedure. It possesses one heme per 240 000 g, and subunits of mol. wt. 68 000. Upon digestion with chymotrypsin, a heme-binding domain was isolated by gel filtration; its visible spectrum was highly similar to that of cytochrome b₅. On SDS gels, the fraction showed two heme-containing bands of ˜10 000 and 12 5000 daltons; their amino acid composition was not very different, suggesting that they originated from the same region of the polypeptide chain. After S-carboxymethylation, the mixture of bands was submitted to cyanogen bromide cleavage, and the fragments were separated by h.p.l.c. The two largest fragments yielded an identical sequence upon automated degradation. This sequence (39 residues with some gaps) could be easily aligned with that of cytochrome b₅ starting close to the N terminus. These results are discussed in terms of the possible quaternary structure of N. crassa nitrate reductase, whose heme-binding domain proves to be another member of the family of b₅-like cytochromes.     

The reduction of putidaredoxin reductase by reduced pyridine nucleotides

Putidaredoxin reductase (PdR), an FAD-containing protein, mediates the transfer of electrons from NADH to putidaredoxin in the cytochrome P-450cam-dependent oxidation of camphor. Using stopped-flow spectrophotometry, reduction of putidaredoxin reductase by NADH (70 microM) at 4 degrees C appeared to be a pseudo-first-order process with a rate constant in excess of 600 s-1. The reduction of putidaredoxin reductase by NADPH was much slower with a second-order rate constant of 530 s-1 M-1 at 4 degrees C. The reduction of the enzyme was monitored at several wavelengths: 455 nm to follow flavin reduction; 700 nm to follow the appearance of the long-wavelength charge-transfer complex; and 513 nm to detect the presence of a semiquinone form of the flavoprotein. There was no apparent semiquinone formation observed during reduction. The charge-transfer complex can be formed in the presence of NAD+, whereas, no charge-transfer band could be detected when PdR was reduced with NADPH. The titration of chemically or NADPH-reduced putidaredoxin reductase with either a stoichiometric or an excess amount of NAD+ resulted in the formation of a charge-transfer complex, indicating that the reduced form of PdR has a high affinity for NAD+ regardless of the method of reduction. The data presented indicate that putidaredoxin reductase is reduced without the formation of semiquinone intermediate and, upon reduction, forms a tight complex with NAD+. The Keq for the reduction of PdR by NADPH is 1.1 and the midpoint potential for this reaction is -317 +/- 5 mV.     

The stereospecificity of nitrate reductase for hydrogen removal from reduced pyridine nucleotides.

The stereospecificity of the hydrogen removal from reduced pyridine nucleotides catalyzed by nitrate reductase (NADH : nitrate oxidoreductase, EC 1.6.6.1, and NAD(P)H : nitrate oxidoreductase, EC 1.6.6.2) was investigated. A high degree of enzyme purification was required to obtain conclusive results. Improvements are described for the purification of nitrate reductase from Chlorella fusca and from spinach (Spinacea oleracea, L.) leaves. The latter enzyme is shown to contain a cytochrome. With highly purified nitrate reductase preparations from Cl. fusca, Neurospora crassa, Rhodotorula glutinis and spinach leaves the stereospecificity of the reaction was determined to be predominantly of the A-type in all cases.     

Neurospora crassa NAD(P)H-nitrite reductase. Studies on its composition and structure

Neurospora crassa nitrite reductase (Mr = 290,000) catalyzes the NAD(P)H-dependent 6-electron reduction of nitrite to ammonia via flavin and siroheme prosthetic groups. Homogeneous N. crassa nitrite reductase has been prepared employing conventional purification methods followed by affinity chromatography on blue dextran-Sepharose 4B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of homogeneous nitrite reductase reveals a single subunit band of Mr = 140,000. Isoelectric focusing of dissociated enzyme followed by sodium dodecyl sulfate-gel electrophoresis in the second dimension yields a single subunit spot with an isoelectric point at pH 6.8-6.9. Two-dimensional thin layer chromatography of acid-hydrolyzed nitrite reductase treated with 5-dimethylaminoaphthalene-1-sulfonyl chloride yields a single reactive NH2-terminal corresponding to glycine. An investigation of the prosthetic groups of nitrite reductase reveals little or no flavin associated with the purified protein, although exogenously added FAD is required for activity in vitro. An iron content of 9-10 Fe eq/mol suggests the presence of nonheme iron in addition to the siroheme moieties. Amino acid analysis yields 43 cysteinyl residues and sulfhydryl reagents react with 50 thiol eq/mol of nitrite reductase. The non-cysteinyl sulfur content, determined as 8.1 acid-labile sulfide eq/mol, is presumably associated with nonheme iron to form iron-sulfur centers. We conclude that N. crassa nitrite reductase is a homodimer of large molecular weight subunits housing an electron transfer complex of FAD, iron-sulfur centers, and siroheme to mediate the reduced pyridine nucleotide-dependent reduction of nitrite to ammonia.