Neuronal nitric-oxide synthase (nNOS) is activated by the Ca 2؉ -dependent binding of calmodulin (CaM) to a characteristic polypeptide linker connecting the oxygenase and reductase domains. Calmodulin binding also activates the reductase domain of the enzyme, increasing the rate of reduction of external electron acceptors such as cytochrome c. Several unusual structural features appear to control this activation mechanism, including an autoinhibitory loop, a C-terminal extension, and kinase-dependent phosphorylation sites. Presteady state reduction and oxidation time courses for the nNOS reductase domain indicate that CaM binding triggers NADP ؉ release, which may exert control over steady-state turnover. In addition, the second order rate constant for cytochrome c reduction in the absence of CaM was found to be highly dependent on the presence of NADPH. It appears that NADPH induces a conformational change in the nNOS reductase domain, restricting access to the FMN by external electron acceptors. CaM binding reverses this effect, causing a 30-fold increase in the second order rate constant. The results show a startling interplay between the two ligands, which both exert control over the conformation of the domain to influence its electron transfer properties. In the fulllength enzyme, NADPH binding will probably close the conformational lock in vivo, preventing electron transfer to the oxygenase domain and the resultant stimulation of nitric oxide synthesis.Mammalian nitric-oxide synthases (NOS) 1 are responsible for generating NO in a wide range of cell types during the immune system response and as part of numerous intercellular signaling mechanisms (1-4). They are homodimeric and consist of a reductase domain, which binds FAD and FMN stoichiometrically, and an oxygenase domain, which contains a P450-like Cys-ligated heme and a tetrahydrobiopterin molecule. The oxygenase domain forms the main dimer interface, and tetrahydrobiopterin is an integral part of this. Crystal structures are available for several NOS oxygenase domain dimers (5, 6) and for the FAD binding subdomain of neuronal NOS (nNOS) (7). The reductase domain closely resembles mammalian cytochrome P450 reductase (7-11) and similarly catalyzes NADPH dehydrogenation at the FAD site and electron transfer to the FMN. The oxygenase domain of one subunit accepts electrons from the reductase domain of the other subunit (12-14) and generates NO from L-arginine via a unique two-step monooxygenation reaction (1-4, 15). The two domains are linked by a functional peptide of 20 -25 amino acids which binds calmodulin (CaM) reversibly at elevated Ca 2ϩ concentrations in the nNOS and endothelial NOS (eNOS) isoforms but irreversibly in the inducible isoform (iNOS). CaM binding activates nNOS and eNOS, providing them with a rapid response mechanism during their participation in signaling cascades. The inducible isoform, on the other hand, is regulated at the transcriptional level. CaM binding has been shown to control NO synthesis by activating electron transfer th...

The reaction of pentaerythritol tetranitrate reductase with reducing and oxidizing substrates has been studied by stopped-flow spectrophotometry, redox potentiometry, and X-ray crystallography. We show in the reductive half-reaction of pentaerythritol tetranitrate (PETN) reductase that NADPH binds to form an enzyme-NADPH charge transfer intermediate prior to hydride transfer from the nicotinamide coenzyme to FMN. In the oxidative half-reaction, the two-electron-reduced enzyme reacts with several substrates including nitroester explosives (glycerol trinitrate and PETN), nitroaromatic explosives (trinitrotoluene (TNT) and picric acid), and ␣,␤-unsaturated carbonyl compounds (2-cyclohexenone). Oxidation of the flavin by the nitroaromatic substrate TNT is kinetically indistinguishable from formation of its hydride-Meisenheimer complex, consistent with a mechanism involving direct nucleophilic attack by hydride from the flavin N5 atom at the electrondeficient aromatic nucleus of the substrate. The crystal structures of complexes of the oxidized enzyme bound to picric acid and TNT are consistent with direct hydride transfer from the reduced flavin to nitroaromatic substrates. The mode of binding the inhibitor 2,4-dinitrophenol (2,4-DNP) is similar to that observed with picric acid and TNT. In this position, however, the aromatic nucleus is not activated for hydride transfer from the flavin N5 atom, thus accounting for the lack of reactivity with 2,4-DNP. Our work with PETN reductase establishes further a close relationship to the Old Yellow Enzyme family of proteins but at the same time highlights important differences compared with the reactivity of Old Yellow Enzyme. Our studies provide a structural and mechanistic rationale for the ability of PETN reductase to react with the nitroaromatic explosive compounds TNT and picric acid and for the inhibition of enzyme activity with 2,4-DNP.

The reaction of morphinone reductase (MR) with the physiological reductant NADH and the oxidizing substrate codeinone has been studied by multiple and single wavelength stopped-flow spectroscopy. Reduction of the enzyme with NADH proceeds in two kinetically resolvable steps. In the first step, the oxidized enzyme forms a charge-transfer intermediate with NADH. The charge-transfer complex is characterized by an increase in absorbance at long wavelength (540 to 650 nm), and its rate of formation is dependent on substrate concentration and is controlled by a second-order rate constant of 4. 8 x 10(5) M-1 s-1 at pH 7.0 and 5 degrees C. In the second step, the enzyme-bound flavin is reduced to the dihydroflavin form. The rate of flavin reduction (23.4 s-1 at pH 7.0 and 5 degrees C) is independent of substrate concentration and is observed as a monophasic decrease in absorbance at 462 nm. The oxidative half-reaction proceeds in three kinetically resolvable steps. The first is due to the formation of a reduced enzyme-codeinone charge-transfer complex and is observed at long wavelength (about 650 nm). The rate of charge-transfer complex formation is dependent on codeinone concentration and is controlled by a second-order rate constant of 11.5 x 10(3) M-1 s-1 at pH 7.0 and 5 degrees C. The second step represents flavin reoxidation and is observed at 462 (absorption increase) and 650 nm (absorption decrease) and progresses with a rate (about 45 s-1) which is independent of codeinone concentration. The third step is observed as a further small increase in absorbance at 462 nm and proceeds with a rate of about 2.5 s-1. This step most likely represents hydrocodone release from the oxidized enzyme. Analysis of the temperature dependence of the reductive half-reaction has enabled calculation of the entropic and enthalpic contributions for charge-transfer formation, charge-transfer decay (yielding free enzyme and substrate), and electron transfer to the enzyme-bound FMN, and the construction of a partial energy profile for the reaction catalyzed by MR. The reaction scheme and redox properties of MR are compared with those described previously for the closely related flavoprotein, old yellow enzyme. Although common features are identified, there are notable differences in the kinetic and redox properties of the two enzymes.

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