Redox properties of human endothelial nitric-oxide synthase oxygenase and reductase domains purified from yeast expression system

Characterization of the redox properties of endothelial nitric-oxide synthase (eNOS) is fundamental to understanding the complicated reaction mechanism of this important enzyme participating in cardiovascular function. Yeast overexpression of both the oxygenase and reductase domains of human eNOS, i.e. eNOS(ox) and eNOS(red), has been established to accomplish this goal. UV-visible and electron paramagnetic resonance (EPR) spectral characterization for the resting eNOS(ox) and its complexes with various ligands indicated a standard NOS heme structure as a thiolate hemeprotein. Two low spin imidazole heme complexes but not the isolated eNOS(ox) were resolved by EPR indicating slight difference in heme geometry of the dimeric eNOS(ox) domain. Stoichiometric titration of eNOS(ox) demonstrated that the heme has a capacity for a reducing equivalent of 1-1.5. Additional 1.5-2.5 reducing equivalents were consumed before heme reduction occurred indicating the presence of other unknown high potential redox centers. There is no indication for additional metal centers that could explain this extra electron capacity of eNOS(ox). Ferrous eNOS(ox), in the presence of l-arginine, is fully functional in forming the tetrahydrobiopterin radical upon mixing with oxygen as demonstrated by rapid-freeze EPR measurements. Calmodulin binds eNOS(red) at 1:1 stoichiometry and high affinity. Stoichiometric titration and computer simulation enabled the determination for three redox potential separations between the four half-reactions of FMN and FAD. The extinction coefficient could also be resolved for each flavin for its semiquinone, oxidized, and reduced forms at multiple wavelengths. This first redox characterization on both eNOS domains by stoichiometric titration and the generation of a high quality EPR spectrum for the BH(4) radical intermediate illustrated the usefulness of these tools in future detailed investigations into the reaction mechanism of eNOS.