Menaquinol-nitrate oxidoreductase of Bacillus halodenitrificans

Abstract

When grown anaerobically on nitrate-containing medium, Bacillus halodenitrificans exhibited a membranebound nitrate reductase (NR) that was solubilized by 2% Triton X-100 but not by 1% cholate or deoxycholate. Purification on columns of DE-52, hydroxylapatite, and Sephacryl S-300 yielded reduced methyl viologen NR (MVH-NR) with specific activities of 20 to 35 U/mg of protein that was stable when stored in 40% sucrose at -20°C for 6 weeks. 3-[(3-cholamidopr6pyl)dimethylammonio]-2-hydroxypropone-l-sulfonate (CHAPSO) and dodecyl-o-D-maltoside stimulated enzyme activity three-to fourfold. Membrane extractions yielded purified NR that separated after electrophoresis into a 145-kDa a subunit, a 58-kDa I8 subunit, and a 23-kDa y subunit. The electronic spectrum of dithionite-reduced, purified NR displayed peaks at 424.6, 527, and 557 nm, indicative of the presence of a cytochrome b, an interpretation consistent with the pyridine hemochrome spectrum formed. Analyses revealed a molybdenum-heme-non-heme iron ratio of 1:1:8 for the NR and the presence of molybdopterin. Electron paramagnetic resonance (EPR) signals characteristic of iron-sulfur centers were detected at low temperature. EPR also revealed a minor signal centered in the g = 2 region of the spectra. Upon reduction with dithionite, the enzyme displayed signals at g = 2.064, 2.026, 1.906, and 1.888, indicative of the presence of low-potential iron-sulfur centers, which resolve most probably as two [4Fe-4S]+1 clusters. With menadiol as the substrate for nitrate reduction, the Km for nitrate was 50-fold less than that seen when MVH was the electron donor. The cytochrome b557-containing enzyme from B. halodenitrificans is characterized as a menaquinol-nitrate:oxidoreductase.

Evidence for a novel nitrate reductase in the dissimilatory iron-reducing bacterium Geobacter metallireducens

“…Paracoccus denitrificans, Pseudomonas stutzeri, Ps. aeruginosa, Bacillis halodenitrificans ) [6][7][8][9][10][11][12][13][14]. Regardless of whether the final product is ammonia or dinitrogen (N2), the NRs (the enzymes that convert nitrate to nitrite) appear to be similar.…”

“…Regardless of whether the final product is ammonia or dinitrogen (N2), the NRs (the enzymes that convert nitrate to nitrite) appear to be similar. They consist of a large a subunit (the enzyme, 112-150 kDa) and a smaller /3 subunit (52-64 kDa) and are associated with cytochrome b (19-23 kDa) [6,[9][10][11][12][13][14]. The NR from E. coli has been cloned and sequenced.…”

Enzymatic Microtiter Plate-Based Nitrate Detection in Environmental and Medical Analysis

“…Respiratory nitrate reductases from bacterial sources like Escherichia coli (23), Paracoccus denitrificans (24), Bacillus halodentrificans (25), Pseudomonas aeruginosa (26), and Pseudomonas stutzeri (27) also accept artificial electron donors. The main requirement is that redox equivalents are effectively shuttled to the molybdopterin cofactor carrying the ␣-subunit of nitrate reductase (28).…”

Biochemical and spectroscopic characterization of the membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617

“…With the exception of the eukaryotic nitrate reductases, which are part of the sulfite oxidase family, all the prokaryotic nitrate reductases belong to the dimethyl sulfoxide (DMSO) reductase family of Mo-containing enzymes [7]. The respiratory nitrate reduction pathway involves the membrane-bound respiratory nitrate reductase (Nar), which has been isolated from a variety of organisms, including a few well-studied bacteria [8][9][10][11][12][13][14]. Nars are involved in the redox loop generating the proton motive force across biomembranes [15].…”