title: Purification and properties of 5,10-methylenetetrahydrofolate reductase, an iron-sulfur flavoprotein from Clostridium formicoaceticum.
abstract: Methylenetetrahydrofolate reductase in Clostridium formicoaceticum has been purified to a specific activity of 140 mumol min-1 mg-1 when assayed at 37 degrees C, pH 7.2, in the direction of oxidation of 5-methyltetrahydrofolate with benzyl viologen as electron acceptor. The purified enzyme is judged to be homogeneous by polyacrylamide disc-gel electrophoresis and gel filtration. The enzyme which is an octamer has a molecular weight of about 237,000 and consists of four each of two different subunits having the molecular weights 26,000 and 35,000. The octameric enzyme contains per mol 15.2 +/- 0.3 iron, 2.3 +/- 0.2 zinc, 19.5 +/- 1.3 acid-labile sulfur, and 1.7 FAD. The UV-visible absorbance spectrum has a peak at 385 nm and a shoulder at 430 nm and is that of a flavoprotein containing iron-sulfur centers. The reductase, which is sensitive to oxygen, must be handled anaerobically and is stabilized by 2 mM dithionite. It catalyzes the reduction of methylene blue, menadione, benzyl viologen, rubredoxin, and FAD with 5-methyltetrahydrofolate and the oxidation of reduced ferredoxin and FADH2 with 5,10-methylenetetrahydrofolate. No activity was observed with pyridine nucleotides. It is suggested that the physiologically important reaction catalyzed by the enzyme is the reduced ferredoxin-dependent reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate.
mesh_terms: D045103:5,10-Methylenetetrahydrofolate Reductase (FADH2); D000429:Alcohol Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D003013:Clostridium / Q000201:enzymology*; D007506:Iron-Sulfur Proteins / Q000302:isolation & purification; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D013379:Substrate Specificity
chemical_list: D007506:Iron-Sulfur Proteins; D046911:Macromolecular Substances; D000429:Alcohol Oxidoreductases; D045103:5,10-Methylenetetrahydrofolate Reductase (FADH2)