Charge-pairing interactions control the conformational setpoint and motions of the FMN domain in neuronal nitric oxide synthase.

The NOS (nitric oxide synthase; EC 1.14.13.39) enzymes contain a C-terminal flavoprotein domain [NOSred (reductase domain of NOS)] that binds FAD and FMN, and an N-terminal oxygenase domain that binds haem. Evidence suggests that the FMN-binding domain undergoes large conformational motions to shuttle electrons between the NADPH/FAD-binding domain [FNR (ferredoxin NADP-reductase)] and the oxygenase domain. Previously we have shown 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 (neuronal NOSred). In the present study, we show that charge neutralization or reversal at each of these residues alters the setpoint [Keq(A)] of the NOSred conformational equilibrium to favour the open (FMN-deshielded) conformational state. Moreover, computer simulations of the kinetic traces of cytochrome c reduction by the mutants suggest that they have higher conformational transition rates (1.5-4-fold) and rates of interflavin electron transfer (1.5-2-fold) 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.

Control of electron transfer and catalysis in neuronal nitric-oxide synthase (nNOS) by a hinge connecting its FMN and FAD-NADPH domains.

In nitric-oxide synthases (NOSs), two flexible hinges connect the FMN domain to the rest of the enzyme and may guide its interactions with partner domains for electron transfer and catalysis. We investigated the role of the FMN-FAD/NADPH hinge in rat neuronal NOS (nNOS) by constructing mutants that either shortened or lengthened this hinge by 2, 4, and 6 residues. Shortening the hinge progressively inhibited electron flux through the calmodulin (CaM)-free and CaM-bound nNOS to cytochrome c, whereas hinge lengthening relieved repression of electron flux in CaM-free nNOS and had no impact or slowed electron flux through CaM-bound nNOS to cytochrome c. How hinge length influenced heme reduction depended on whether enzyme flavins were pre-reduced with NADPH prior to triggering heme reduction. Without pre-reduction, changing the hinge length was deleterious; with pre-reduction, the hinge shortening was deleterious, and hinge lengthening increased heme reduction rates beyond wild type. Flavin fluorescence and stopped-flow kinetic studies on CaM-bound enzymes suggested hinge lengthening slowed the domain-domain interaction needed for FMN reduction. All hinge length changes lowered NO synthesis activity and increased uncoupled NADPH consumption. We conclude that several aspects of catalysis are sensitive to FMN-FAD/NADPH hinge length and that the native hinge allows a best compromise among the FMN domain interactions and associated electron transfer events to maximize NO synthesis and minimize uncoupled NADPH consumption.

A bridging interaction allows calmodulin to activate NO synthase through a bi-modal mechanism.

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.

Differences in a conformational equilibrium distinguish catalysis by the endothelial and neuronal nitric-oxide synthase flavoproteins.

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.

Surface charge interactions of the FMN module govern catalysis by nitric-oxide synthase.

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.

The three nitric-oxide synthases differ in their kinetics of tetrahydrobiopterin radical formation, heme-dioxy reduction, and arginine hydroxylation.

The nitric-oxide synthases (NOSs) make nitric oxide and citrulline from l-arginine. How the bound cofactor (6R)-tetrahydrobiopterin (H4B) participates in Arg hydroxylation is a topic of interest. We demonstrated previously that H4B radical formation in the inducible NOS oxygenase domain (iNOSoxy) is kinetically coupled to the disappearance of a heme-dioxy intermediate and to Arg hydroxylation. Here we report single turnover studies that determine and compare the kinetics of these transitions in Arg hydroxylation reactions catalyzed by the oxygenase domains of endothelial and neuronal NOSs (eNOSoxy and nNOSoxy). There was a buildup of a heme-dioxy intermediate in eNOSoxy and nNOSoxy followed by a monophasic transition to ferric enzyme during the reaction. The rate of heme-dioxy decay matched the rates of H4B radical formation and Arg hydroxylation in both enzymes. The rates of H4B radical formation differed such that nNOSoxy (18 s(-1)) > iNOSoxy (11 s(-1)) > eNOSoxy (6 s(-1)), whereas the lifetimes of the resulting H4B radical followed an opposite rank order. 5MeH4B supported a three-fold faster radical formation and greater radical stability relative to H4B in both eNOSoxy and nNOSoxy. Our results indicate the following: (i) the three NOSs share a common mechanism, whereby H4B transfers an electron to the heme-dioxy intermediate. This step enables Arg hydroxylation and is rate-limiting for all subsequent steps in the hydroxylation reaction. (ii) A direct correlation exists between pterin radical stability and the speed of its formation in the three NOSs. (iii) Uncoupled NO synthesis often seen for eNOS at low H4B concentrations may be caused by the slow formation and poor stability of its H4B radical.