Assimilatory nitrate reductase from the green alga Ankistrodesmus braunii.
Assimilatory nitrate reductase (NAD(P)H-nitrate oxidoreductase, EC 1.6.6.2) from the green alga Ankistrodesmus braunii can be purified to homogeneity by dye-ligand chromatography on blue-Sepharose. The purified enzyme, whose turnover number is 623 s-1, presents an optimum pH of 7.5 and Km values of 13 microM, 23 microM and 0.15 mM for NADH, NADPH and nitrate, respectively. The NADH-nitrate reductase activity exhibits an iso ping pong bi bi kinetic mechanism. The molecular weight of the native nitrate reductase is 467 400, while that of its subunits is 58 750. These values suggest an octameric structure for the enzyme, which has been confirmed by electron microscopy. As deduced from spectrophotometric and fluorimetric studies, the enzyme contains FAD and cytochrome b-557 as prosthetic groups. FAD is not covalently bound to the protein and is easily dissociated in diluted solutions from the enzyme. Its apparent Km value is 4 nM, indicative of a high affinity of the enzyme for FAD. The results of the quantitative analyses of prosthetic groups indicate that nitrate reductase contains four molecules of flavin, four heme irons, and two atoms of molybdenum. The three components act sequentially transferring electrons from reduced pyridine nucleotides to nitrate, thus forming a short electron transport chain along the protein. A mechanism is proposed for the redox interconversion of the nitrate reductase activity. Inactivation seems to occur by formation of a stable complex of reduced enzyme with cyanide or superoxide, while reactivation is a consequence of reoxidation of the inactive enzyme. Both reactions imply the transfer of only one electron.
D000460:Chlorophyta / Q000201:enzymology*; D004579:Electron Transport; D004789:Enzyme Activation; D005182:Flavin-Adenine Dinucleotide / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D008982:Molybdenum / Q000494:pharmacology; D050896:Nitrate Reductase (NAD(P)H); D009565:Nitrate Reductases / Q000302:isolation & purification / Q000378:metabolism*
D046911:Macromolecular Substances; D005182:Flavin-Adenine Dinucleotide; D008982:Molybdenum; D009565:Nitrate Reductases; D050896:Nitrate Reductase (NAD(P)H)