Menaquinol-nitrate oxidoreductase of Bacillus halodenitrificans.
When grown anaerobically on nitrate-containing medium, Bacillus halodenitrificans exhibited a membrane-bound 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 degrees C for 6 weeks. 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxypropone-1-sulfonat e (CHAPSO) and dodecyl-beta-D-maltoside stimulated enzyme activity three- to fourfold. Membrane extractions yielded purified NR that separated after electrophoresis into a 145-kDa alpha subunit, a 58-kDa beta subunit, and a 23-kDa gamma 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.
Ketchum
P A
PA
Department of Biological Sciences, Oakland University, Rochester, Michigan 48309-4401.
Denariaz
G
G
LeGall
J
J
Payne
W J
WJ
D001407:Bacillus / Q000201:enzymology*; D002793:Cholic Acids / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005960:Glucosides / Q000494:pharmacology; D008970:Molecular Weight; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000737:chemistry* / Q000302:isolation & purification; D012965:Sodium Chloride / Q000494:pharmacology; D014812:Vitamin K / Q000031:analogs & derivatives / Q000378:metabolism
D002793:Cholic Acids; D003902:Detergents; D005960:Glucosides; D014812:Vitamin K; D012965:Sodium Chloride; C040358:dodecyl maltoside; C048531:chapso; D009565:Nitrate Reductases; D050901:Nitrate Reductase; C023885:menadiol