The properties of neuronal nitric oxide synthase containing one tetrahydrobiopterin (BH4) per dimer [nNOS(BH4+)] were compared to those of the BH4-free enzyme [nNOS(BH4-)]. The stimulation by BH4 of the formation of L-citrulline at the expense of H2O2 production unambiguously demonstrated that BH4 is essential in coupling reductive oxygen activation to Arg oxidation. The clear difference between the Stokes radii of nNOS(BH4-) and nNOS(BH4+) indicates that the introduction of one BH4 per dimer significantly changes the enzyme structure. Whereas the heme in nNOS(BH4+) was primarily high-spin, nNOS(BH4-) contained mainly low-spin heme. This was slowly converted into the high-spin form with Arg and/or BH4, with a rate that was independent of the concentration of either compound. Dithiothreitol inhibited the Arg/BH4-induced spin conversion by stabilizing low-spin heme. Formation of high-spin heme, with rates varying from 0.04 to 0.4 min-1, always correlated to an equally fast increase in activity. Radioligand binding studies showed the rapid association (within 20 s) of BH4 to nNOS(BH4-), but not to nNOS(BH4+), after preincubation with Arg. Complete and monophasic dissociation of radioligand occurred in the presence of excess unlabeled BH4, demonstrating the exchangeability of high-affinity bound BH4. Studies of the association of NG-nitro-L-arginine (L-NNA) to nNOS(BH4+) revealed that excess BH4 increased the amount of bound L-NNA 2-fold. Most of the binding data are explained by a model in which nNOS dimers accommodate two identical BH4- and Arg/L-NNA-binding sites, with cooperativity between Arg- and BH4-binding and anticooperativity between the BH4-binding sites.

Nitric oxide (NO), a physiologically important activator of soluble guanylyl cyclase (sGC), is synthesized from Larginine and O 2 in a reaction catalyzed by NO synthases (NOS). Previous studies with purified NOS failed to detect formation of free NO, presumably due to a fast inactivation of NO by simultaneously produced superoxide (O 2 . ). To characterize the products involved in NOS-induced sGC activation, we measured the formation of cyclic 3,5-guanosine monophosphate (cGMP) by purified sGC incubated in the absence and presence of GSH (1 mM) with drugs releasing different NO-related species or with purified neuronal NOS. Basal sGC activity was 0.04 ؎ 0.01 and 0.19 ؎ 0.06 mol of cGMP ؋ mg ؊1 ؋ min ؊1 without and with 1 mM GSH, respectively. The NO donor DEA/NO activated sGC in a GSH-independent manner. Peroxynitrite had no effect in the absence of GSH but significantly stimulated the enzyme in the presence of the thiol (3.45 ؎ 0.60 mol of cGMP ؋ mg ؊1 ؋ min The NO/cGMP pathway involving NO-mediated activation of soluble guanylyl cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2.; sGC 1 ) is essential to signal transduction in several biological systems (1). In the vasculature, NO/cGMP signaling is important for the regulation of blood pressure and platelet function (2); in the brain, this pathway controls the release of neurotransmitters such as glutamate and acetylcholine (3). Biosynthesis of NO is triggered by autacoids increasing the intracellular concentration of free Ca 2ϩ , resulting in activation of Ca 2ϩ /calmodulin-dependent NOS (EC 1.14.13.39), complex homodimeric enzymes that catalyze the synthesis of NO from the guanidino moiety of the amino acid L-arginine (4 -7). The oxidation of L-arginine is catalyzed by a cytochrome P 450 -type heme iron in the oxygenase domain of NOS with O 2 serving as a cosubstrate. The electrons required for reduction of O 2 are shuttled from the cofactor NADPH to the heme via a flavincontaining cytochrome P 450 reductase that forms the C-terminal half of the NOS protein. This electron transport chain only operates when Ca 2ϩ /calmodulin is bound to the enzyme, which then effects the Ca 2ϩ regulation of endothelial and neuronal NO synthesis.At low concentrations of L-arginine or in its absence, the enzymatic reduction of O 2 uncouples from substrate oxidation and results in the generation of superoxide anions and H 2 O 2 (8 -12). The effective coupling of the reaction requires not only saturation with L-arginine but also the pteridine cofactor H 4 biopterin (13). Since the two subunits of neuronal NOS bind H 4 biopterin in a highly anticooperative manner, the purified enzyme always contains Յ1 molecule of H 4 biopterin/dimer, i.e. it consists of a H 4 biopterin-containing and a H 4 biopterin-free subunit (14). In this state, the enzyme can form L-citrulline and is stimulated about 2-fold upon binding of H 4 biopterin to the low affinity site of the pteridine-free subunit. Together with our recent findings that the two NOS subunits function independently (15), this...

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Peroxynitrite (ONOO-) is widely recognized as mediator of NO toxicity, but recent studies have indicated that this compound may also have physiological activity and induce vascular relaxation as well as inhibition of platelet aggregation. We found that ONOO- induced a pronounced increase in endothelial cyclic GMP levels, and that this effect was significantly attenuated by pretreatment of the cells with GSH-depleting agents. In the presence of 2 mM GSH, ONOO- stimulated purified soluble guanylyl cyclase with a half-maximally effective concentration of about 20 microM. In contrast to the NO donor 2,2-Diethyl-1-nitroso-oxyhydrazine sodium salt (DEA/NO), ONOO- was completely inactive in the absence of GSH, indicating that thiol-mediated bioactivation of ONOO- is involved in enzyme stimulation. Studies on the reaction between ONOO- and GSH revealed that about 1% of ONOO- was non-enzymatically converted to S-nitrosoglutathione. The authentic nitrosothiol was found to be stable in solution, but slowly decomposed in the presence of GSH. GSH-induced decomposition of S-nitrosoglutathione was apparently catalyzed by trace metals and was accompanied by a sustained release of NO and a 40-100-fold increase in its potency to stimulate purified soluble guanylyl cyclase. Our data suggest that the biologic activity of ONOO- involves S-nitrosation of cellular thiols resulting in NO-mediated cyclic GMP accumulation.