Nitric-oxide synthases (NOSs) are flavo-heme enzymes whose electron transfer reactions are controlled by calmodulin (CaM). The NOS flavoprotein domain includes a ferredoxin-NADP ؉ reductase (FNR)-like module that contains NADPH-and FADbinding sites. FNR-like modules in related flavoproteins have three conserved residues that regulate electron transfer between bound NAD(P)H and FAD. To investigate the function of one of these residues in neuronal NOS (nNOS), we generated and characterized mutants that had Val, Glu, or Asn substituted for the conserved Asp-1393. All three mutants exhibited normal composition, spectral properties, and binding of cofactors, substrates, and CaM. All had slower NADPH-dependent cytochrome c and ferricyanide reductase activities, which were associated with proportionally slower rates of NADPH-dependent flavin reduction in the CaM-free and CaM-bound states. Rates of NO synthesis were also proportionally slower in the mutants and were associated with slower rates of CaMdependent ferric heme reduction. However, a D1393V mutant whose flavins had been prereduced with NADPH had a normal rate of heme reduction. This indicated that the kinetic defect was restricted to flavin reduction step(s) in the mutants and suggested that this limited their catalytic activities. Together, our results show the following. 1) The presence and positioning of the Asp-1393 carboxylate side chain are critical to enable NADPHdependent reduction of the nNOS flavoprotein. 2) Control of flavin reduction is important because it ensures that the rate of heme reduction is sufficiently fast to enable NO synthesis by nNOS.
Nitric oxide (NO)1 is a widespread signal and effector molecule in biology (1-3). Animals generate NO by virtue of the nitric-oxide synthases (NOSs), which catalyze a stepwise, NADPH-dependent oxidation of L-Arg to NO and L-citrulline, with N -hydroxy-L-arginine (NOHA) being formed as an intermediate. Each NOS is composed of an N-terminal oxygenase domain that binds iron protoporphyrin IX (heme), (6R)-tetrahydrobiopterin (H 4 B), and substrate, L-Arg, and a C-terminal flavoprotein domain that binds FAD, FMN, and NADPH. A calmodulin (CaM)-binding sequence connects the oxygenase and flavoprotein domains. During catalysis, electrons from NADPH transfer into the FAD and FMN groups of the NOS flavoprotein domain, and then transfer one at a time from the FMN hydroquinone (FMNH 2 ) to the ferric heme (4 -8) (Scheme 1).Heme reduction enables the enzyme to bind O 2 and initiate an H 4 B-dependent oxygen activation that is required for catalysis (9, 10). In all NOSs studied so far, the rates of NADPHdependent flavin reduction are fast relative to the rates of electron transfer from FMNH 2 to the ferric heme in the CaMbound enzymes (11)(12)(13)(14). This makes heme reduction rate-limiting for NO synthesis and implies that the NOS flavins predominantly exist in their reduced forms during steady-state NO synthesis.NOSs are part of a small enzyme family whose members have covalently linked flavoprotein and heme domain...