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...

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