Studies on nitrate reductase of Clostridium perfringens. IV. Identification of metals, molybdenum cofactor, and iron-sulfur cluster.
Nitrate reductase of Clostridium perfringens was purified by an improved method using immuno-affinity chromatography. The purified preparation contained Mo, Fe, and acid-labile sulfide; the Mo content was 1 mol per mol and the Fe 3.7 mol per mol of the enzyme. The inactive enzyme obtained from cells grown in the presence of tungstate did not hold Mo but contained 1 mol of W. The content of Fe was not increased. The presence of molybdenum cofactor in the nitrate reductase was indicated by the formation of molybdopterin form A in the oxidation of the enzyme by iodine and by the complementation of NADPH-nitrate reductase with the heart-treated enzyme in the extract of Neurospora crassa nit-1. The Clostridium nitrate reductase had an absorption maximum at 279 nm and shoulders at 320, 380, 430, and 520 nm. This enzyme seems to contain an iron sulfur cluster since the reduced enzyme showed decreased absorption in visible region. The CD spectrum of the enzyme has a positive peak at 425 nm and negative ones at 310, 360, and 595 nm. It was compared with the CD spectrum of ferredoxin (2Fe-2S or 4Fe-4S cluster) and the nitrate reductase of Plectonema boryanum.
D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003016:Clostridium perfringens / Q000201:enzymology*; D003067:Coenzymes; D005288:Ferredoxins / Q000032:analysis*; D007120:Immunochemistry; D007506:Iron-Sulfur Proteins / Q000302:isolation & purification*; D008667:Metalloproteins / Q000032:analysis / Q000302:isolation & purification*; D008670:Metals / Q000032:analysis*; D000090006:Molybdenum Cofactors; D008983:Molybdoferredoxin / Q000032:analysis*; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000302:isolation & purification*; D011621:Pteridines / Q000032:analysis; D013057:Spectrum Analysis / Q000379:methods; D013440:Sulfides / Q000032:analysis
D003067:Coenzymes; D005288:Ferredoxins; D007506:Iron-Sulfur Proteins; D008667:Metalloproteins; D008670:Metals; D000090006:Molybdenum Cofactors; D008983:Molybdoferredoxin; D050901:Nitrate Reductase; D009565:Nitrate Reductases; D011621:Pteridines; D013440:Sulfides; C022261:molybdopterin