In vitro complementation of assimilatory NAD(P)H-nitrate reductase from mutants of Chlamydomonas reinhardii.
In vitro complementation of the soluble assimilatory NAD(P)H-nitrate reductase (NAD(P)H:nitrate oxidoreductase, EC 1.6.6.2) was attained by mixing cell-free preparations of Chlamydomonas reinhardii mutant 104, uniquely possessing nitrate-inducible NAD(P)H-cytochrome c reductase, and mutant 305 which possesses solely the nitrate-inducible FMNH2- and reduced benzyl viologen-nitrate reductase activities. Full activity and integrity of NAD(P)H-cytochrome c reductase from mutant 104 and reduced benzyl viologen-nitrate reductase from mutant 305 are needed for the complementation to take place. A constitutive and heat-labile molybdenum-containing cofactor, that reconstitutes the NAD(P)H-nitrate reductase activity of nit-1 Neurospora crassa but is incapable of complementing with 104 from C. reinhardii, is present in the wild type and 305 algal strains. The complemented NAD(P)H-nitrate reductase has been purified 100-fold and was found to be similar to the wild enzyme in sucrose density sedimentation, molecular size, pH optimum, kinetic parameters, substrate affinity and sensitivity to inhibitors and temperature. From previous data and data presented in this article on 104 and 305 mutant activities, it is concluded that C. reinhardii NAD(P)H-nitrate reductase is a heteromultimeric complex consisting of, at least, two types of subunits separately responsible for the NAD(P)H-cytochrome c reductase and the reduced benzyl viologen-nitrate reductase activities.
D001594:Benzyl Viologen / Q000378:metabolism; D002696:Chlamydomonas / Q000201:enzymology*; D003067:Coenzymes / Q000378:metabolism; D008982:Molybdenum / Q000378:metabolism; D009154:Mutation; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism; D009492:Neurospora crassa / Q000201:enzymology; D050896:Nitrate Reductase (NAD(P)H); D009565:Nitrate Reductases / Q000235:genetics / Q000378:metabolism*
D003067:Coenzymes; D001594:Benzyl Viologen; D008982:Molybdenum; D009251:NADPH-Ferrihemoprotein Reductase; D009565:Nitrate Reductases; D050896:Nitrate Reductase (NAD(P)H)