Purification and properties of nitrite reductase from Escherichia coli K12

Abstract

NADH-nitrite oxidoreductase (EC 1.6.6.4) was purified to better than 95 % homogeneity from batch cultures of Escherichia coli strain OR75Ch15, which is partially constitutive for nitrite reductase synthesis. Yields of purified enzyme were low, mainly because of a large loss of activity during chromatography on DEAE-cellulose. The quantitative separation of cytochrome c-552 from nitrite reductase activity resulted in an increase in the specific activity of the enzyme: this cytochrome is not therefore an integral part of nitrite reductase. The subunit molecular weights of nitrite reductase and of a haemoprotein contaminant, as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, were 88000 and 80000 respectively. The sedimentation coefficient was calculated to be in the range 8.5-9.5S, consistent with a mol.wt. of 190000. It is suggested therefore that the native enzyme is a dimer with two identical or similar-sized subunits. Purest samples contained 0.4mol of fiavin/mol of enzyme, but no detectable haem. Catalytic activity was totally inhibited by 20,uM-p-chloromercuribenzoate and 1 mM-cyanide, slightly inhibited by IpM-sulphite and lOmM-arsenite, but was insensitive to mM-2,2'-bipyridine, 4mM-1,10-phenanthroline and 10mM-NaN3. Three molecules of NADH were oxidized for each NO2-ion reduced: the product of the reaction is therefore assumed to be NH4+. The specific activity of hydroxylamine reductase increased at each step in the purification of nitrite reductase, and the elution profiles for these two activities during chromatography on DEAE-Sephadex were coincident. It is likely that a single enzyme is responsible for both activities.

Characterization of the expression and activity of the periplasmic nitrate reductase of Paracoccus pantotrophus in chemostat cultures

“…Residual nitrate concentration was measured using a Dionex anion-exchange HPLC system with a UV detector, whereby a standard of known concentration was used to facilitate the quantification of nitrate concentration. Nitrite concentrations were determined spectrophotometrically by a colorimetric change assay (Coleman et al, 1978). From assays of nitrite concentration in the culture vessel at steady state (above) nitrite accumulation rates were calculated and determined as a function of biomass at steady state.…”

Characterization of a nitrate-respiring bacterial community using the nitrate reductase gene (narG) as a functional marker

“…Isolates were tested for their ability to evolve gas from nitrate, reaction to Gram stain and production of fluorescent pigments on King's A and B media (Stolp & Gadkari, 1992). The ability to accumulate nitrite from nitrate was tested using a chemical nitrite assay based on that described by Coleman et al (1978). Cultures were grown anaerobically to stationary phase, and 1 ml samples were mixed with 89 ml of 1 % (w/v) sulphanilamide in 1 M HCl and 20.11 + + + 2 2 2 20.12 + + 2 2 2 20.13 + + 2 2 2 *Cultures were grown anaerobically into stationary phase in liquid minimal medium containing 10 mM nitrate.…”

Identification and cloning of genes involved in anaerobic sulfite reduction by Salmonella typhimurium

“…The hemoprotein subunit contains a novel heme group, called siroheme, in its catalytic center. Siroheme is also present in nitrite reductase, an anaerobically expressed enzyme that catalyzes the six-electron transfer reduction of nitrite to ammonia (6)(7)(8). The cysG gene, in which mutations lead to defective nitrite and biosynthetic sulfite reduction, is essential for siroheme biosynthesis (12).…”