Phe1395 stacks parallel to the FAD isoalloxazine ring in neuronal nitric-oxide synthase (nNOS) and is representative of conserved aromatic amino acids found in structurally related flavoproteins. This laboratory previously showed that Phe 1395 was required to obtain the electron transfer properties and calmodulin (CaM) response normally observed in wild-type nNOS. Here we characterized the F1395S mutant of the nNOS flavoprotein domain (nNOSr) regarding its physical properties, NADP ؉ binding characteristics, flavin reduction kinetics, steady-state and pre-steady-state cytochrome c reduction kinetics, and ability to shield its FMN cofactor in response to CaM or NADP(H) binding. F1395S nNOSr bound NADP ؉ with 65% more of the nicotinamide ring in a productive conformation with FAD for hydride transfer and had an 8-fold slower rate of NADP ؉ dissociation. CaM stimulated the rates of NADPH-dependent flavin reduction in wild-type nNOSr but not in the F1395S mutant, which had flavin reduction kinetics similar to those of CaM-free wild-type nNOSr. CaM-free F1395S nNOSr lacked repression of cytochrome c reductase activity that is typically observed in nNOSr. The combined results from pre-steady-state and EPR experiments revealed that this was associated with a lesser degree of FMN shielding in the NADP ؉ -bound state as compared with wild type. We conclude that Phe 1395 regulates nNOSr catalysis in two ways. It facilitates NADP ؉ release to prevent this step from being rate-limiting, and it enables NADP(H) to properly regulate a conformational equilibrium involving the FMN subdomain that controls reactivity of the FMN cofactor in electron transfer. Nitric-oxide synthases (NOS)1 are homodimeric enzymes that synthesize NO via oxidation of L-Arg and participate in various physiological and pathological settings (1-6). In the NOS polypeptide, an N-terminal oxygenase domain is linked to a C-terminal reductase domain by a calmodulin (CaM)-binding sequence. The NOS oxygenase domain contains binding sites for iron protoporphyrin IX (heme), (6R)-5, 6, 7, 8-tetrahydro-Lbiopterin (H 4 B), and L-Arg and is the site where oxidative catalysis takes place. The NOS reductase domain (NOSr) contains binding sites for FMN, FAD, and NADPH and functions to transfer reducing equivalents from NADPH to the oxygenase domain.NOSr belongs to a small family of structurally related dualflavin reductases that also includes cytochrome P450 reductase (CYPR) (7, 8), methionine synthase reductase (9), and novel reductase-1 (10). These proteins are comprised of separate FMN and FAD/NADPH modules attached by a flexible hinge region (11,12). It is believed that these reductases are the product of gene fusion because their FMN and FAD/NADPH subdomains show a high similarity to flavodoxins (13) and ferredoxin NADP ϩ reductases (FNR) (14), respectively. In NOSr and related flavoproteins, the FAD receives electrons from NADPH via hydride transfer and then sequentially passes the electrons to the FMN cofactor. Ultimately, it is the 2-electron reduced FMN hydroqu...

Nitric oxide (NO) is a physiological mediator synthesized by NO synthases (NOS).Despite their structural similarity, endothelial NOS (eNOS) has a 6-fold lower NO synthesis activity and 6 -16-fold lower cytochrome c reductase activity than neuronal NOS (nNOS), implying significantly different electron transfer capacities. We utilized purified reductase domain constructs of either enzyme (bovine eNOSr and rat nNOSr) to investigate the following three mechanisms that may control their electron transfer: (i) the set point and control of a two-state conformational equilibrium of their FMN subdomains; (ii) the flavin midpoint reduction potentials; and (iii) the kinetics of NOSr-NADP ؉ interactions. Although eNOSr and nNOSr differed in their NADP(H) interaction and flavin thermodynamics, the differences were minor and unlikely to explain their distinct electron transfer activities. In contrast, calmodulin (CaM)-free eNOSr favored the FMN-shielded (electron-accepting) conformation over the FMN-deshielded (electron-donating) conformation to a much greater extent than did CaM-free nNOSr when the bound FMN cofactor was poised in each of its three possible oxidation states. NADPH binding only stabilized the FMN-shielded conformation of nNOSr, whereas CaM shifted both enzymes toward the FMN-deshielded conformation. Analysis of cytochrome c reduction rates measured within the first catalytic turnover revealed that the rate of conformational change to the FMN-deshielded state differed between eNOSr and nNOSr and was rate-limiting for either CaM-free enzyme. We conclude that the set point and regulation of the FMN conformational equilibrium differ markedly in eNOSr and nNOSr and can explain the lower electron transfer activity of eNOSr.

The neuronal nitric-oxide synthase (nNOS) flavoprotein domain (nNOSr) contains regulatory elements that repress its electron flux in the absence of bound calmodulin (CaM). The repression also requires bound NADP(H), but the mechanism is unclear. The crystal structure of a CaM-free nNOSr revealed an ionic interaction between Arg 1400 in the C-terminal tail regulatory element and the 2-phosphate group of bound NADP(H). We tested the role of this interaction by substituting Ser and Glu for Arg 1400 in nNOSr and in the full-length nNOS enzyme. The CaM-free nNOSr mutants had cytochrome c reductase activities that were less repressed than in wild-type, and this effect could be mimicked in wild-type by using NADH instead of NADPH. The nNOSr mutants also had faster flavin reduction rates, greater apparent K m for NADPH, and greater rates of flavin auto-oxidation. Single-turnover cytochrome c reduction data linked these properties to an inability of NADP(H) to cause shielding of the FMN module in the CaM-free nNOSr mutants. The full-length nNOS mutants had no NO synthesis in the CaM-free state and had lower steady-state NO synthesis activities in the CaM-bound state compared with wild-type. However, the mutants had faster rates of ferric heme reduction and ferrous heme-NO complex formation. Slowing down heme reduction in R1400E nNOS with CaM analogues brought its NO synthesis activity back up to normal level. Our studies indicate that the Arg 1400 -2-phosphate interaction is a means by which bound NADP(H) represses electron transfer into and out of CaM-free nNOSr. This interaction enables the C-terminal tail to regulate a conformational equilibrium of the FMN module that controls its electron transfer reactions in both the CaM-free and CaM-bound forms of nNOS. Nitric oxide (NO)2 has diverse biological functions and is generated in mammals by the NO synthase (NOS) enzymes (EC 1.14.13.39) (1, 2). Three NOS isozymes (inducible NOS or iNOS, neuronal NOS or nNOS, and endothelial NOS or eNOS) have evolved to function in health and disease (3-7). All are homodimeric enzymes that catalyze an NADPH-and O 2 -dependent oxidation of L-arginine (Arg) to 9). Each NOS is composed of an N-terminal oxygenase domain that contains binding sites for iron protoporphyrin IX (heme), 6R-tetrahydrobiopterin, and Arg, and a C-terminal flavoprotein domain (NOSr) that contains binding sites for FAD, FMN, and NADPH (10, 11). The oxygenase and NOSr domains are connected by a calmodulin (CaM)-binding polypeptide (12, 13). CaM plays a critical role in activating NO synthesis, because its Ca 2ϩ -dependent binding triggers electron transfer from the FMN hydroquinone to the ferric heme (14 -18). This enables the heme to bind O 2 and initiate its reductive activation as required for NO synthesis (19,20).NOSr contains separate ferredoxin-NADP ϩ -reductase (FNR) and FMN modules (21,22) and in this way is similar to a number of NADPH-utilizing dual flavin oxidoreductases (23-27). In NOSr, a hydride transfer occurs from NADPH to FAD within the FNR module, foll...

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...

Nitric oxide synthases (NOS) are flavoheme enzymes with important roles in biology. The reductase domain of neuronal NOS (nNOSr) contains a widely conserved acidic residue (Asp(1393)) that is thought to facilitate hydride transfer between NADPH and FAD. Previously we found that the D1393V and D1393N mutations lowered the NO synthesis activity and the rates of heme and flavin reduction in full-length nNOS. To examine the mechanisms for these results in greater detail, we incorporated D1393V and D1393N substitutions into nNOSr along with a truncated NADPH-FAD domain construct (FNR) and characterized the mutants. D1393V nNOSr had markedly lower (