The Bacterial Nitrate Reductases

Abstract

Nitrate reductase of Escherichia coli has been solubilized from particle fractions by a double treatment : first an alkali-acetone precipitation and then a solubilization in a buffered sodium deoxycholate. The enzyme has been purified 50-fold with a yield of 1 to 2O/,. Polyacrylamide-gel electrophoresis and ultracentrifugation show the preparation to be nearly homogeneous. The protein has a molecular weight of 320000 and an iso-electric point a t pH 4.2. The absorbance which increases continuously from 600 to 280 nm does not reveal the presence of a heme or a flavin group but the spectrum resembles that of some bacterial ferroproteins. The estimation of metals indicates 1.5 atoms Mo and 20 atoms Fe per mole. Approximately one labile sulfide is found per iron atom. It is likely that nitrate reductase A is an iron-sulfur protein containing molybdenum. The purified protein uses as substrates NO,-and C10,-and as electron donors reduced benzyl-and methyl-viologens, FMNH, and FADH, but not NADH or NADPH. It should be pointed out that the solubilization does not modify the enzymatic properties of nitrate reductase. CN-and N,-are strong inhibitors. Azide is a competitive inhibitor and the nitrate reductase affinity for this inhibitor is 1000 times greater than for nitrate. The type of inhibition observed and the metal chelating nature of the inhibitor suggest that a metal, Fe or Mo, or both, play a role in the formation of enzyme-substrate complex.Numerous bacterial strains are able to utilize nitrate as nitrogen source or as electron acceptor. In both roles nitrate, is first reduced to nitrite by nitrate reductase. This enzyme, when it is of type A [I], is localized in the cytoplasmic membrane with an electron carrier chain constituting what we call the nitrate respiratory system because of its many analogies with the system involved in aerobic respiration [2]. The membrane-bound complex thus formed has to be dissociated to allow study of its constituants. In the case of nitrate reductase, some methods of solubilization have been tried with success, as, for example, the fractionation of the particle fraction by heating employed by Tanigushi

Induction and Characterization of Chlorate-resistant Strains of Rosa damascena Cultured Cells

“…Nitrate reduction was estimated by the loss of nitrate from medium plus cell extract. Reduction of nitrate occurred from day 3 to day 6 (Fig. 6) with some variation among strains and experiments.…”

Isolation and preliminary characterization of a respiratory nitrate reductase from hydrocarbon-degrading bacterium Gordonia alkanivorans S7

“…Only several membrane-bound respiratory reductases from Escherichia coli, Aerobacter aerogenes, Haloarcula marismortui, Haloferax mediterranei, Paracoccus denitrificans, Pseudomonas chlororaphis and Pseudomonas aeruginosa have been isolated and characterized up to now [4,7,[9][10][11][12][13][14][15].…”

A nitrate reductase gene of the cyanobacterium Synechococcus PCC6301 inferred by heterologous hybridization, cloning and targeted mutagenesis

“…This polypeptide is believed to contain the pterin molybdenum cofactor (MacGregor et aL, 1974;Forget, 1974;Lund& DeMoss, 1976;Chaudhry & MacGregor, 1983;McPherson et aL, 1984;Wootton et aL, 1991). Probes from parts of narGH DNA show cross hybridization to genomes of diverse bacteria known to contain respiratory and/or assimilatory nitrate reductases .…”