Mammalian nitric-oxide synthases are large modular enzymes that evolved from independently expressed ancestors. Calmodulincontrolled isoforms are signal generators; calmodulin activates electron transfer from NADPH through three reductase domains to an oxygenase domain. Structures of the reductase unit and its homologs show FMN and FAD in contact but too isolated from the protein surface to permit exit of reducing equivalents. To study states in which FMN/heme electron transfer is feasible, we designed and produced constructs including only oxygenase and FMN binding domains, eliminating strong internal reductase complex interactions. Constructs for all mammalian isoforms were expressed and purified as dimers. All synthesize NO with peroxide as the electron donor at rates comparable with corresponding oxygenase constructs. All bind cofactors nearly stoichiometrically and have native catalytic sites by spectroscopic criteria. Modest differences in electrochemistry versus independently expressed heme and FMN binding domains suggest interdomain interactions. These interactions can be convincingly demonstrated via calmodulin-induced shifts in high spin ferriheme EPR spectra and through mutual broadening of heme and FMNH ⅐ radical signals in inducible nitricoxide synthase constructs. Blue neutral FMN semiquinone can be readily observed; potentials of one electron couple (in inducible nitric-oxide synthase oxygenase FMN, FMN oxidized/ semiquione couple ‫؍‬ ؉70 mV, FMN semiquinone/hydroquinone couple ‫؍‬ ؊180 mV, and heme ‫؍‬ ؊180 mV) indicate that FMN is capable of serving as a one electron heme reductant. The construct will serve as the basis for future studies of the output state for NADPH derived reducing equivalents. Nitric-oxide synthases (NOSs)3 are a family of enzymes that generate nitric oxide (NO) from arginine, requiring 2 mol of O 2 and 1.5 mol of NADPH/mol of NO produced (1-3). The constitutive isoforms, eNOS and nNOS, are regulated by calcium/calmodulin (Ca ϩ2 /CaM) (4, 5) and additional inputs including phosphorylation of specific residues (6); the NO produced by constitutive isoforms functions as a molecular signal. A cytokine inducible isoform (iNOS) is calcium insensitive, and produces much larger fluxes of NO as a cytotoxin in immune response (4). Eukaryotic NOS isoforms are large modular enzymes. The monomer molecular mass is 120 -161 kDa; the dimer is the active form (7), and the dimerization interface includes the tetrahydrobiopterin (H 4 B) binding site in the oxygenase domain (8). The common elements are the heme and H 4 B containing the oxygenase domain and a complex reductase unit homologous to NADPH P450 reductase that consists of a NADPH binding domain, a FAD binding domain, and an FMN binding domain (9 -11). The reductase and oxygenase regions are linked by a polypeptide segment containing a CaM binding site (12). Evidence suggests that the oxygenase domain of one monomer is reduced by the reductase unit of the other (13) through an oxygenase domain surface that exposes the corner of the hem...

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