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Query: EC:1.7.1.2 (nitrate reductase)
 3,861 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

NADH:nitrate reductase (EC 1.6.6.1) from Chlorella vulgaris has been purified 640-fold with an over-all yield of 26% by a combination of protamine sulfate fractionation, ammonium sulfate fractionation, gel chromatography, density gradient centrifugation, and DEAE-chromatography. The purified enzyme is stable for more than 2 months when stored at minus 20 degrees in phosphate buffer (pH 6.9) containing 40% (v/v) glycerol. After the initial steps of the purification, a constant ratio of NADH:nitrate reductase activity to NADH:cytochrome c reductase and reduced methyl viologen:nitrate reductase activities was observed. One band of protein was detected after polyacrylamide gel electrophoresis of the purified enzyme. This band also gave a positive stain for heme, NADH dehydrogenase, and reduced methyl viologen:nitrate reductase. One band, corresponding to a molecular weight of 100, 000, was detected after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme contains FAD, heme, and molybdenum in a 1:1:0.8 ratio. One "cyanide binding site" per molybdenum was found. No non-heme-iron or labile sulfide was detected. From a dry weight determination of the purified enzyme, a minimal molecular weight of 152, 000 per molecule of heme or FAD was calculated. An s20, w of 9.7 S for nitrate reductase was found by the use of sucrose density gradient centrifugation and a Stokes radius of 89 A was estimated by gel filtration techniques. From these values, and the assumption that the partial specific volume is 0.725 cc/g, a molecular weight of 356, 000 was estimated for the native enzyme. These data suggest that the native enzyme contains a minimum of 2 molecules each of FAD, heme, and molybdenum and is composed of at least three subunits.
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PMID:Reduced nicotinamide adenine dinucleotide-nitrate reductase of Chlorella vulgaris. Purification, prosthetic groups, and molecular properties. 16 92

1. In respiratory nitrate reductase I of Klebsiella aerogenes, 0.24 atom of molybdenum, eight iron-sulfur groups and four tightly bound, non-heme iron atoms per molecule of enzyme (Mr 260 000) are found. 2. EPR spectra at 83 degrees K of oxidized and reduced nitrate reductase I show complex lines at g = 2.02 and g = 1.98, which are more intense in the reduced than in the oxidized enzyme. The resonances, the shape and intensity of which are rather temperature insensitive, are attributed to two species of paramagnetic molybdenum. In dithionite-reduced enzyme all these lines are saturated at the same microwave power of 15 mW. This is not the case in oxidized enzyme, where the resonance at g = 2.02 is hard to saturate. Addition of nitrate to dithionite-reduced reductase I decreases the intensity of the EPR lines to about that of oxidized enzyme. The participation of molybdenum in the electron transfer process has been discussed. 3. At 18 degrees K the oxidized enzyme exhibits an axial-symmetrical signal with g parallel = 2.10 and g = 2.03, and a signal with unknown symmetry at g = 2.015. Upon reduction by dithionite, a ferredoxin type of signal is observed with g values at 2.05, 1.95 and 1.88, while the g = 2.015 signal disappears. Reoxidation by nitrate causes a concomitant disappearance of the ferredoxin type of signal and reappearance of the g = 2.015 signal; hence iron-sulfur centres participate in the transfer of electrons to nitrate. 4. Nitrate reductase II, containing only two (Mr 117 000 and 57 000) of the three subunits found in nitrate reductase I and lacking the tightly bound iron, does not exhibit the axial-symmetrical signal (g = 2.10 and 2.03). Thus, it suggested that this signal in nitrate reductase I stems from an iron centre in the low-molecular weight subunit (Mr 52 000). 5. Inhibition studies confirm the participation of metals in the transfer of electrons from reduced benzylviologen to nitrate and show that the binding sites for these substrates are different.
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PMID:Characterization of the respiratory nitrate reductase of Klebsiella aerogenes as a molybdenum-containing iron-sulfur enzyme. 17 Sep 83

The reduction of ferricytochrome c by two molybdenum(V)-cysteine complexes has been investigated as a model for electron transfer in the molybdenum enzymes sulfite oxidase and nitrate reductase. The reduction by the dioxo-bridged Mo(V)-cysteine complex, di-mu-oxo-bis-[oxo(L-cysteinato)molybdate(V)] (I), is relatively slow and its rate is first order in cyt cIII and zero order in I (k = (1.09 +/- 0.10) times 10(-3) sec minus 1, pH 7.5, 20 degrees). The reduction by the monoxo-bridged complex, mu-oxo-bis[oxodihydroxo(L-cysteinato)molybdate(V)] (II), is extremely rapid and its rate is first order in both reactants (k = (2.6 +/- 0.7) times 10(7) M minus 1 sec minus 1, pH 7.0, 25 degrees). Above pH 7.5, the reduction by II follows biphasic kinetics due to the fast reduction of a low pH form of cyt cIII and a slower reduction of a high pH form (at pH 10.0, 25 degrees, k = 2.9 times 10(6) M minus 1 sec minus 1 for the low pH form and k = 7.2 times 10(4) M minus 1 sec minus 1 for the high pH form). Reaction mechanisms for reductions by both I and II are proposed and the biological implications of the results, both for sulfite oxidase and mechanisms of electron transfer to cytochrome c, are discussed.
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PMID:Model studies for molybdenum enzymes. The reduction of cytochrome c by molybdenum(V)-cysteine complexes. 24 Mar 86

Nitrate reductase (nar) A, B and E mutants of Escherichia coli with plasmids carrying Klebsiella pneumoniae nitrogen fixation (nif) genes reduced acetylene independently of added molybdate, but nar D mutants showed pleiotropic dependence on the concentration of added molybdate for expression of both nar and nif. No complementation of nar mutations by nif occurred; nitrite but not nitrate repressed nif in nar hosts. Derepression of nif occurred in molybdenum-deficient nar D (nif) strains since nitrogenase peptides were present. nifB mutants, thought to have a lesion in the pathway of molybdenum to nitrogenase, as well as nif deletion mutants, had normal nitrate reductase activity.
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PMID:Expression of Klebsiella pneumoniae nitrogen fixation genes in nitrate reductase mutants of Escherichia coli. 32 14

The cnx- group of mutants of Aspergillus nidulans lacks xanthine dehydrogenase (xanthine: NAD+ oxidoreductase, EC 1.2.1.37) and nitrate reductase (EC 1.6.6.3) activities and are thought to be defective in the synthesis of a molybdenum-containing cofactor, 'cnx', common to xanthine dehydrogenase and nitrate reductase [Pateman, J.A., Rever, B.M., Cove, D.J. and Roberts, D.B. (1964) Nature (Lond.) 201, 58-60]. The cnx cofactor has a role in maintaining the aggregated multimeric structure of nitrate reductase [MacDonald, D.W., Cove, D.J. and Coddington, A. (1974) Mol. Gen. Genet. 128, 187-199]. We report here that, in cnx- mutants grown under conditions inducing xanthine dehydrogenase I, a species cross-reacting with antisera to the native enzyme and of half its molecular weight is present, together with cross-reacting molecules of similar molecular weight to the native enzyme. This suggests that the cnx cofactor has a role in maintaining the aggregated structure of xanthine dehydrogenase I. Both cross-reacting species are capable of passing reducing equivalents from NADH to a tetrazolium salt, showing that the cnx cofactor is not necessary for enzymic activity towards NADH.
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PMID:The genetic control of molybdoflavoproteins in Aspergillus nidulans. A xanthine dehydrogenase I half-molecule in cnx- mutant strains of Aspergillus nidulans. 33 Jan 63

When Escherichia coli was grown in the presence of tungstate, inactive forms of two molybdoenzymes, nitrate reductase and formate dehydrogenase, accumulated and were converted to their active forms upon incubation of cell suspensions with molybdate and chloramphenicol. The conversion to the active enzymes did not occur in cell extracts. When incubated with [(99)Mo]molybdate and chloramphenicol, the tungstate-grown cells incorporated (99)Mo into protein components which were released from membranes by procedures used to release nitrate reductase and formate dehydrogenase and which migrated with these activities on polyacrylamide gels. Although neither activity was formed during incubation of the crude extract with molybdate, (99)Mo was incorporated into protein components which were released from the membrane fraction under the same conditions and were similar to the active enzymes in their electrophoretic properties. The in vitro incorporation of (99)Mo occurred specifically into these components and was equal to or greater than the amount incorporated in vivo under the same conditions. Molybdenum in preformed, active nitrate reductase and formate dehydrogenase did not exchange with [(99)Mo]molybdate, demonstrating that the observed incorporation depended on the demolybdo forms of the enzymes. We conclude that molybdate may be incorporated into the demolybdo forms both in vivo and in vitro; some unknown additional factor or step, required for active enzyme formation, occurs in vivo but not in vitro under the conditions employed.
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PMID:In vitro incorporation of molybdate into demolybdoproteins in Escherichia coli. 37 97

Molybdenum cofactor activity was found in the soluble fraction of cell-free extracts of Escherichia coli grown aerobically in media supplemented with molybdate. Cofactor was detected by its ability to complement the nitrate reductase-deficient mutant of Neurospora crossa, nit-1, resulting in the vitro formation of nitrate reductase activity. Acid treatment of E. coli extracts was not required for release of cofactor activity. Cofactor was able to diffuse through a membrane of nominal 2,000-molecular-weight cutoff and was insensitive to trypsin. The cofactor was associated with a carrier molecule (approximately 40,000 daltons) during gel filtration and sucrose gradient centrifugation, but was easily removed from the carrier by dialysis. The carrier molecule protected the cofactor from inactivation by heat or oxygen. E. coli grown in molybdenum-free media, without and with tungsten, synthesized a metal-free "empty" cofactor and its tungsten analog, respectively, both of which were subsequently activated by the addition of molybdate. Empty and tungsten-containing cofactor complemented the nitrate reductase subunits in the nit-1 extract, forming inactive, but intact, 7.9S nitrate reductase. Addition of molybdate to the enzyme complemented in this manner restored nitrate reductase activity.
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PMID:Characterization of molybdenum cofactor from Escherichia coli. 38 15

In animals the terminal step in the pathway for degradation of sulphur-containing amino acids is the oxidation of sulphite to sulphate. This reaction is catalysed by the enzyme sulphite oxidase. The enzyme contains molybdenum and a cytochrome b5 type haem, is localized in the mitochondrial intermembrane space and transfers electrons from sulphite to cytochrome c on the inner membrane. The sulphite oxidase protein has a molecular weight of 110 000 (chicken) to 122 000 (human) and exists as a dimer of identical subunits. The haem and molybdenum cofactors are present on separate domains of the molecule. The structure of the molydbenum cofactor has not been worked out in detail, but this cofactor is known to be present in many other molybdoenzymes including xanthine oxidase and nitrate reductase. Three cases of genetic sulphite oxidase deficiency in humans have been reported. The three affected children displayed mental retardation, neurological abnormalities and dislocated ocular lenses. The biochemical basis for lack of enzyme activity in each case has been studied. All three have been shown to lack the sulphite oxidase protein, but in one case this appears to be secondary to a defect in synthesis of the molybdenum cofactor. Sulphite oxidase deficiency has been produced in the rat by administration of high levels of tungsten. Sulphite oxidase-deficient animals are particularly susceptible to the toxic effects of sulphite and atmospheric sulphur dioxide.
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PMID:The oxidation of sulphite in animals systems. 39 60

Assimilatory nitrate reductase (EC 1.6.6.1 NADH:nitrate oxidoreductase) from Chlorella vulgaris purified by affinity chromatography was found to be homogeneous as judged by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel and by analytical ultracentrifugal techniques. The molecular weight of the intact enzyme and that of the enzyme dissociated in 6 M GuHCl, determined by sedimentation equilibrium studies, were 280,000 +/- 10,000 and 90,000 +/- 5,000, respectively. Comparable values were obtained using the S20,w value and the D20,w values in Svedberg's equation. The D20,w values were determined by laser light-scattering measurements. Active enzyme centrifugation showed that the monomer is an active species. A quantitative re-evaluation of the prosthetic groups present (FAD, heme, and molybdenum) was also made and was consistent with the conclusion that the active monomer contains three subunits as previously deduced by Solomonson et al. ((1975) J. Biol. Chem. 250, 4120). Electron micrographs showed images which corresponded to three subunits, supporting the data obtained by hydrodynamic studies. The enzyme is not cigar-shaped, as previously surmised, but has a roughly globular structure.
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PMID:Physical studies on assimilatory nitrate reductase from Chlorella vulgaris. 50 Jun 68

Nitrate reductase, released from the membrane fraction of Escherichia coli by a neutral heat treatment, was purified to homogeneity by gel filtration chromatography. The purified enzyme behaved as an associating-dissociating system, exhibiting concentration-dependent sedimentation constants which ranged from 24 S at high concentrations in the ultracentrifuge down to 10 S at low concentrations in sucrose gradients. The molecular weight determined at high concentrations by sedimentation equilibrium was 880,000 +/- 30,000. Large and small enzyme species were detected on polyacrylamide disc gels run with diluted samples of enzyme. The ratio of the two species was concentration-dependent and the dissociation was reversible. The purified enzyme appeared to be homogeneous and monodisperse in the ultracentrifuge, on sucrose gradients, during gel filtration on Bio-Gel and on polyacrylamide gels, but it had a heterogeneous subunit composition as determined by sodium dodecyl sulfate gel electrophoresis. Enzyme species with different subunit compositions were partially resolved by gel filtration. The fractions with the highest specific activity contained subunits of 150,000 and 55,000 daltons in a ratio of approximately 1:1. Other fractions contained reduced amounts of the 55,000-dalton subunit and correspondingly increased amounts of 51,000-, 45,000-, and 10,000-dalton subunits, suggesting that the heterogeneity was the result of proteolytic degradation of the 55,000-dalton subunit. The enzyme contained approximately 12 non-heme irons, 12 acid-labile sulfides, 24 cysteine residues, and 1 molybdenum per 200,000 daltons.
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PMID:Association-dissociation behavior and subunit structure of heat-released nitrate reductase from Escherichia coli. 77 Apr 63


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