Molybdenum cofactors from molybdoenzymes and in vitro reconstitution of nitrogenase and nitrate reductase.
A molybdenum cofactor (Mo-co) from xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) can be isolated from the enzyme by a technique that has been used to isolate an iron-molybdenum cofactor (FeMo-co) from component I of nitrogenase. N-Methylformamide is used for the extraction of these molybdenum cofactors. Mo-co from xanthine oxidase activates nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.2) in an extract from Neurospora crassa mutant strain Nit-1; however, FeMo-co is unable to activate nitrate reductase in strain Nit-1. Mo-co from xanthine oxidase is unable to activate nitrogenase in an extract of Azotobacter vinelandii mutant strain UW45. Inactive component I in this extract can be activated by FeMo-co. These results indicate that nitrate reductase and xanthine oxidase share a common molybdenum cofactor, but this cofactor is different from the molybdenum cofactor in nitrogenase.A. vinelandii synthesizes both Mo-co and FeMo-co. Mo-co is produced when the cells fix N(2) and also when they are repressed for nitrogenase synthesis by growth in a medium containing excess ammonium. However, FeMo-co is not produced when cells are grown in an ammonium-containing medium. Partially purified preparations of component I from A. vinelandii and Klebsiella pneumoniae contain both FeMo-co and Mo-co. The presence of both FeMo-co and Mo-co activities in partially purified preparations of component I explains previous reports of activation of inactive nitrate reductase in strain Nit-1 by acid-treated component I of nitrogenase. The Mo-co can be separated from FeMo-co in these preparations by chromatography on Sephadex G-100 in N-methylformamide. Both FeMo-co and Mo-co are sensitive to oxygen.
D001395:Azotobacter; D003067:Coenzymes / Q000302:isolation & purification / Q000378:metabolism*; D005288:Ferredoxins / Q000378:metabolism*; D008982:Molybdenum / Q000302:isolation & purification; D008983:Molybdoferredoxin / Q000378:metabolism*; D009492:Neurospora crassa; D009565:Nitrate Reductases / Q000378:metabolism*; D009591:Nitrogenase / Q000378:metabolism*; D010084:Oxidation-Reduction; D013329:Structure-Activity Relationship; D014969:Xanthine Oxidase / Q000032:analysis / Q000378:metabolism*
D003067:Coenzymes; D005288:Ferredoxins; D008983:Molybdoferredoxin; D008982:Molybdenum; D014969:Xanthine Oxidase; D009591:Nitrogenase; D009565:Nitrate Reductases