Functional domains of assimilatory NADH:nitrate reductase from Chlorella.
Assimilatory nitrate reductase from Chlorella is a homotetramer which contains one of each of the prosthetic groups FAD, heme, and molybdenum per subunit. Besides the reduction of nitrate by NADH, nitrate reductase also catalyzes the partial activities NADH:cytochrome c reductase, NADH:ferricyanide reductase, and reduced methyl viologen:nitrate reductase. Incubation of native nitrate reductase with either trypsin, Staphylococcus aureus V8 protease, or a natural inactivator protease from corn results in a loss of NADH:nitrate reductase and NADH:cytochrome c reductase activities but no loss of reduced methyl viologen:nitrate reductase activity. Incubation of nitrate reductase with V8 protease or corn inactivator protease resulted in two different products, each of which retained a different partial activity. Reduced methyl viologen:nitrate reductase activity was associated with a homotetrameric fragment of about 260 kDa which contained heme and molybdenum but no FAD. The molecular mass of native nitrate reductase determined under the same conditions was 375 kDa. NADH:ferricyanide reductase activity was associated with a monomeric species of approximately 30 kDa which contained FAD and the NADH-binding site. These results are consistent with a structure-function model of nitrate reductase which has the following features: FAD/NADH-binding domains exposed on the surface of the molecule, a protease-sensitive hinge region which connects the nitrate-reducing and NADH dehydrogenase moieties, and the quaternary structure maintained via association sites on the heme/molybdenum domain.
D000345:Affinity Labels / Q000378:metabolism; D001665:Binding Sites; D002708:Chlorella / Q000201:enzymology*; D002850:Chromatography, Gel; D003486:Cyanides / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D009243:NAD / Q000378:metabolism*; D009565:Nitrate Reductases / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism
D000345:Affinity Labels; D003486:Cyanides; D009243:NAD; D009565:Nitrate Reductases; D010447:Peptide Hydrolases