Flavin and iron-sulfur containing ferredoxin-linked glutamate synthase from spinach leaves.
Ferredoxin-dependent glutamate synthase (native enzyme) [EC 1.4.7.1] of spinach has been purified to homogeneity in the presence of 2-oxoglutarate and sodium chloride and the properties of the enzyme have been studied. The molecular weight of the enzyme was estimated to be 140,000 by gel filtration. Subunit analysis by SDS-gel electrophoresis yielded a single protein band whose molecular weight was about 170,000. This purified enzyme showed a flavo-protein-like absorption spectrum having maxima at 279 and 438 nm with shoulders at 415 and 460 nm and a broad band around 360 nm. Fluorometric data indicated the presence of 2 mol of flavin per mol of the enzyme. Preliminary paper chromatography results indicated the presence of FAD and FMN in the purified enzyme. The enzyme also contained 4 mol of acid-labile sulfide and 4 g-atoms iron per mol of enzyme. In the absence of 2-oxoglutarate and/or sodium chloride, the purified enzyme was separated by either DE-52 cellulose chromatography or gel filtration with Ultrogel AcA 34 into two molecular forms (modified enzymes) with considerable inactivation. When reduced methyl viologen plus ferredoxin was used as the electron donor, the purified (native) enzyme showed high ferredoxin-dependent activity with a specific activity of 100 units/mg protein. Methyl viologen-dependent activity was negligible in the absence of ferredoxin. Kinetic properties and results of ESR studies were described. The results indicate that ferredoxin-linked glutamate synthase of spinach leaves is an iron-sulfur flavoprotein.
D000594:Amino Acid Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D001419:Bacteria / Q000201:enzymology; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D005486:Flavin Mononucleotide / Q000032:analysis; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D005415:Flavins / Q000032:analysis*; D007501:Iron / Q000032:analysis; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology*; D012256:Riboflavin / Q000032:analysis; D013045:Species Specificity; D013440:Sulfides / Q000032:analysis
D005415:Flavins; D013440:Sulfides; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D007501:Iron; D000594:Amino Acid Oxidoreductases; C042233:glutamate synthase (ferredoxin); D012256:Riboflavin