Characterization of the reductase domain of rat neuronal nitric oxide synthase generated in the methylotrophic yeast Pichia pastoris. Calmodulin response is complete within the reductase domain itself.
Rat neuronal NO synthase (nNOS) is comprised of a flavin-containing reductase domain and a heme-containing oxygenase domain. Calmodulin binding to nNOS increases the rate of electron transfer from NADPH into its flavins, triggers electron transfer from flavins to the heme, activates NO synthesis, and increases reduction of artificial electron acceptors such as cytochrome c. To investigate what role the reductase domain plays in calmodulin's activation of these functions, we overexpressed a form of the nNOS reductase domain (amino acids 724-1429) in the yeast Pichia pastoris that for the first time exhibits a complete calmodulin response. The reductase domain was purified by 2',5'-ADP affinity chromatography yielding 25 mg of pure protein per liter of culture. It contained 1 FAD and 0.8 FMN per molecule. Most of the protein as isolated contained an air-stable flavin semiquinone radical that was sensitive to FeCN6 oxidation. Anaerobic titration of the FeCN6-oxidized reductase domain with NADPH indicated the flavin semiquinone re-formed after addition of 1-electron equivalent and the flavins could accept up to 3 electrons from NADPH. Calmodulin binding to the recombinant reductase protein increased its rate of NADPH-dependent flavin reduction and its rate of electron transfer to cytochrome c, FeCN6, or dichlorophenolindophenol to fully match the rate increases achieved when calmodulin bound to native full-length nNOS. Calmodulin's activation of the reductase protein was associated with an increase in domain tryptophan and flavin fluorescence. We conclude that many of calmodulin's actions on native nNOS can be fully accounted for through its interaction with the nNOS reductase domain itself.
Gachhui
R
R
Department of Immunology, The Cleveland Clinic Research Institute, Cleveland, Ohio 44195, USA.
Presta
A
A
Bentley
D F
DF
Abu-Soud
H M
HM
McArthur
R
R
Brudvig
G
G
Ghosh
D K
DK
Stuehr
D J
DJ
D000818:Animals; D001483:Base Sequence; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D005415:Flavins / Q000737:chemistry; D005420:Flavoproteins / Q000737:chemistry; D007527:Isoenzymes / Q000737:chemistry*; D008969:Molecular Sequence Data; D009245:NADH Dehydrogenase / Q000737:chemistry*; D009474:Neurons / Q000201:enzymology; D019001:Nitric Oxide Synthase / Q000737:chemistry*; D010084:Oxidation-Reduction; D010843:Pichia / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D017931:DNA Primers; D005415:Flavins; D005420:Flavoproteins; D007527:Isoenzymes; D011994:Recombinant Proteins; D014364:Tryptophan; D019001:Nitric Oxide Synthase; D009245:NADH Dehydrogenase