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 FMN module of nitric-oxide synthase (NOS) plays a pivotal role by transferring NADPH-derived electrons to the enzyme heme for use in oxygen activation. The process may involve a swinging mechanism in which the same face of the FMN module accepts and provides electrons during catalysis. Crystal structure shows that this face of the FMN module is electronegative, whereas the complementary interacting surface is electropositive, implying that charge interactions enable function. We used site-directed mutagenesis to investigate the roles of six electronegative surface residues of the FMN module in electron transfer and catalysis in neuronal NOS. Results are interpreted in light of crystal structures of NOS and related flavoproteins. Neutralizing or reversing the negative charge of each residue altered the NO synthesis, NADPH oxidase, and cytochrome c reductase activities of neuronal NOS and also altered heme reduction. The largest effects occurred at the NOS-specific charged residue Glu 762. Together, the results suggest that electrostatic interactions of the FMN module help to regulate electron transfer and to minimize flavin autoxidation and the generation of reactive oxygen species during NOS catalysis.

SYNOPSIS The Nitric Oxide Synthases (NOS; EC 1.14.13.39) contain a C-terminal flavoprotein domain (NOSred) that binds FAD and FMN and an N-terminal oxygenase domain that binds heme. Evidence suggests that the FMN-binding domain undergoes large conformational motions to shuttle electrons between the NADPH/FAD-binding domain (FNR) and the oxygenase domain. previously we showed that three residues on the FMN domain (Glu762, Glu816 and Glu819) that make charge-pairing interactions with the FNR help to slow electron flux through nNOSred. In this study, we show that charge neutralization or reversal at each of these residues alters the setpoint (KeqA) of the NOSred conformational equilibrium to favor of the open (FMN-deshielded) conformational state. Moreover, computer simulations of the kinetic traces of cytochrome c reduction by the mutants suggest that they have relatively larger effects on the conformational transition rates (from 1.5 to 4x faster) and the rate of interflavin electron transfer (from 1.5 to 2x faster) relative to wild type nNOSred. We conclude that the three charge-pairing residues on the FMN domain govern electron flux through nNOSred by stabilizing its closed (FMN-shielded) conformational state and by retarding the rate of conformational switching between its open and closed conformations.

Calmodulin (CaM) activates the nitric-oxide synthases (NOS) by a mechanism that is not completely understood. A recent crystal structure showed that bound CaM engages in a bridging interaction with the NOS FMN subdomain. We investigated its importance in neuronal NOS (nNOS) by mutating the two residues that primarily create the bridging interaction (Arg 752 in the FMN subdomain and Glu 47 in CaM). Mutations designed to completely destroy the bridging interaction prevented bound CaM from increasing electron flux through the FMN subdomain and diminished the FMN-to-heme electron transfer by 90%, whereas mutations that partly preserve the interaction had intermediate effects. The bridging interaction appeared to control FMN subdomain interactions with both its electron donor (NADPH-FAD subdomain) and electron acceptor (heme domain) partner subdomains in nNOS. We conclude that the Arg 752 -Glu 47 bridging interaction is the main feature that enables CaM to activate nNOS. The mechanism is bi-modal and links a single structural aspect of CaM binding to specific changes in nNOS protein conformational and electron transfer properties that are essential for catalysis. Nitric oxide (NO)4 is an essential signal and effector molecule in biology (1). NO is produced in animals from L-Arg by the NO synthases (NOS, EC 1.14.13.39) (2). Three types of NOS are expressed in mammals: inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS) (3-5). The three NOS are structurally homologous and are active as homodimers (6, 7). Each NOS monomer is comprised of two domains: an N-terminal oxygenase domain (NOSoxy) that contains cofactors protoporphyrin IX (heme) and (6R)-5,6,7,8-tetrahydro-L-biopterin (H 4 B) and binds the substrate L-Arg, and a C-terminal reductase domain that contains FAD, FMN, and binds NADPH. The NOS reductase domain is homologous to cytochrome P-450 reductase and related dual-flavin enzymes (5, 8), but also contains up to three regulatory inserts that are unique to the NOS enzymes (3, 5). Significantly, a calmodulin (CaM) binding site is located in the connecting sequence between the NOSoxy and reductase domains (3-5). CaM binding to this site activates NO synthesis by triggering electron transfer to the heme in NOS enzymes (9). The ability of CaM to activate a redox enzyme like NOS is novel and the mechanism is a topic of current interest.Much of the actions of CaM impinge on the NOS FMN subdomain, which is thought to undergo large conformational motions to transfer electrons during catalysis (10 -17). This may be a common feature in the dual-flavin reductase enzyme family (18 -20). Fig. 1 illustrates a model for FMN conformational switching during electron transfer between the FMN and heme within a NOS homodimer. The FMN subdomain must first interact with the NADPH-FAD subdomain (FNR) in a "FMN-shielded" conformation to receive electrons, according to equilibrium A. Once the FMN hydroquinone forms (FMNH 2 ), it must swing away to a "FMN-deshielded" conformation, and then must interact with the NOSoxy do...

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