CHARACTERIZATION OF THE REDUCTASE DOMAIN OF RAT NEURONAL NITRIC-OXIDESYNTHASE 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 re ductase domain and a heme-containing oxygenase domain, Calmodulin bind ing to nNOS increases the rate of electron transfer from NADPH into it s flavins, triggers electron transfer from flavins to the heme, activa tes NO synthesis, and increases reduction of artificial electron accep ters such as cytochrome c. To investigate what role the reductase doma in plays in calmodulin's activation of these functions, we overexpress ed a form of the nNOS reductase domain (amino acids 724-1429) in the y east Pichia pastoris that for the first time exhibits a complete calmo dulin response, The reductase domain was purified by 2',5'-ADP affinit y 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 is olated contained an air-stable flavin semiquinone radical that was sen sitive to FeCN6 oxidation. Anaerobic titration of the FeCN6-oxidized r eductase domain with NADPh indicated the flavin semiquinone re-formed after addition of 1-electron equivalent and the flavins could accept u p to 3 electrons from NADPH. Calmodulin binding to the recombinant red uctase protein increased its rate of NADPH-dependent flavin reduction and its rate of electron transfer to cytochrome c, FeCN6, or dichlorop henolindophenol to fully match the rate increases achieved when calmod ulin 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 action s on native nNOS can be fully accounted for through its interaction wi th the nNOS reductase domain itself.