Purification and properties of nitrate reductase from Mitsuokella multiacidus.
Nitrate reductase of Mitsuokella multiacidus (formerly Bacteroides multiacidus) was solublized from the membrane fraction with 1% sodium deoxycholate and purified 40-fold by immunoaffinity chromatography on the antibody-Affi-Gel 10 column. The preparation showed a major band (86% of total protein) with enzyme activity and a minor band on polyacrylamide gel after disc electrophoresis in the presence of 0.1% Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a major band, the relative mobility of which corresponded to a molecular weight of 160,000, and two minor bands. The molecular weight of the enzyme was determined to be 160,000 by gel filtration on Bio-Gel A-1.5 m in the presence of 0.1% deoxycholate. Molybdenum cofactor was detected in the enzyme by fluorescence spectroscopy and by complementation of nitrate reductase from the nit-1 mutant of Neurospora crassa. The M. multiacidus enzyme catalyzed reduction of nitrate, chlorate, and bromate using methyl viologen as an electron donor. The maximal activity was found at pH 6.2-7.5 for nitrate reduction. Either methyl or benzyl viologen served well as the electron donor, but FAD, FMN, and horse heart cytochrome c were not effective. Ferredoxin from Clostridium pasteurianum supplied electron to the nitrate reductase. The purified enzyme had Km values of 0.13 mM, 0.12 mM, and 0.22 mM for nitrate, methyl viologen, and ferredoxin, respectively. The enzyme activity was inhibited by cyanide (85% at 1 mM), azide (88% at 0.1 mM), and thiocyanate (75% at 10 mM).
Yamamoto
I
I
Shimizu
H
H
Tsuji
T
T
Ishimoto
M
M
D001439:Bacteroides / Q000201:enzymology*; D002846:Chromatography, Affinity; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D008982:Molybdenum / Q000032:analysis; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D013379:Substrate Specificity
D008982:Molybdenum; D009565:Nitrate Reductases; D050901:Nitrate Reductase