Activation of neuronal nitric-oxide synthase by the 5-methyl analog of tetrahydrobiopterin. Functional evidence against reductive oxygen activation by the pterin cofactor.

Tetrahydrobiopterin ((6R)-5,6,7,8-tetrahydro-L-biopterin (H4biopterin)) is an essential cofactor of nitric-oxide synthases (NOSs), but its role in enzyme function is not known. Binding of the pterin affects the electronic structure of the prosthetic heme group in the oxygenase domain and results in a pronounced stabilization of the active homodimeric structure of the protein. However, these allosteric effects are also produced by the potent pterin antagonist of NOS, 4-amino-H4biopterin, suggesting that the natural cofactor has an additional, as yet unknown catalytic function. Here we show that the 5-methyl analog of H4biopterin, which does not react with O2, is a functionally active pterin cofactor of neuronal NOS. Activation of the H4biopterin-free enzyme occurred in a biphasic manner with half-maximally effective concentrations of approximately 0.2 microM and 10 mM 5-methyl-H4biopterin. Thus, the affinity of the 5-methyl compound was 3 orders of magnitude lower than that of the natural cofactor, allowing the direct demonstration of the functional anticooperativity of the two pterin binding sites of dimeric NOS. In contrast to H4biopterin, which inactivates nitric oxide (NO) through nonenzymatic superoxide formation, up to 1 mM of the 5-methyl derivative did not consume O2 and had no effect on NO steady-state concentrations measured electrochemically with a Clark-type NO electrode. Therefore, reconstitution with 5-methyl-H4biopterin allowed, for the first time, the detection of enzymatic NO formation in the absence of superoxide or NO scavengers. These results unequivocally identify free NO as a NOS product and indicate that reductive O2 activation by the pterin cofactor is not essential to NO biosynthesis.

Down-Regulation of Porcine Heart Diaphorase Reactivity by Trimanganese Hexakis(3,5-Diisopropylsalicylate), Mn(3)(3,5-DIPS)6, and Down-Regulation of Nitric Oxide Synthase Reactivity by Mn(3)(3,5-DIPS)(6) and Cu(II)(2)(3,5-DIPS)(4).

Purposes of this work were to examine the plausible down-regulation of porcine heart diaphorase (PHD) enzyme reactivity and nitric oxide synthase (NOS) enzyme reactivity by trimanganese hexakis(3,5-diisopropylsalicylate), [Mn(3)(3,5-DIPS)(6)] as well as dicopper tetrakis(3,5- diisopropylsalicylate, [Cu(II)(2)(3,5-DIPS)(4)] as a mechanistic accounting for their pharmacological activities.Porcine heart disease was found to oxidize 114 muM reduced nicotinamide-adenine- dinucleotide-'(3)-phosphate (NADPH) with a corresponding reduction of an equivalent concentration of 2,6-dichlorophenolindophenol (DCPIP). As reported for Cu(II)(2) (3,5-DIPS)(4), addition of Mn(3)(3,5-DIPS)(6) to this reaction mixture decreased the reduction of DCPIP without significantly affecting the oxidation of NADPH. The concentration of Mn(3)(3,5-DIPS)(6) that produced a 50% decrease in DCPIP reduction (IC(50)) was found to be 5muM. Mechanistically, this inhibition of DCPIP reduction with ongoing NADPH oxidation by PHD was found to be due to the ability of Mn(3)(3,5-DIPS)(6) to serve as a catalytic electron acceptor for reduced PHD as had been reported for Cu(II)(2)(3,5-DIPS)(4). This catalytic decrease in reduction of DCPIP by Mn(3)(3,5-DIPS)(6) was enhanced by the presence of a large concentration of DCPIP and decreased by the presence of a large concentration of NADPH, consistent with what had been observed for the activity of Cu(II)(2)(3,5-DIPS)(4)Oxidation of NADPH by PHD in the presence of Mn(3)(3,5-DIPS)(6) and the absence of DCPIP was linearly related to the concentration of added Mn(3)(3,5-DIPS)(6) through the concentration range of 2.4 muM to 38muM with a 50% recovery of NADPH oxidation by PHD at a concentration of 6 muM Mn(3)(3,5-DIPS)(6)Conversion of [(3)H] L-Arginine to [(3)H] L-Citrulline by purified rat brain nitric oxide synthase (NOS) was decreased in a concentrated related fashion with the addition of Mn(3)(3,5-DIPS)(6) as well as Cu(II)(2)(3,5-DIPS)(4) which is an extention of results reported earlier for Cu(II)(2)(3,5-DIPS)(4). The concentration of these two compounds required to produce a 50% decrease in L-Citrulline synthesis by NOS, which may be due to down-regulation of NOS, were 0.1 mM and 8muM respectively, consistent with the relative potencies of these two complexes in preventing the reduction of Cytochrome c by NOS.It is concluded that Mn(3)(3,5-DIPS)(6), as has been reported for Cu(II)(2) (3,5-DIPS)(4) , serves as an electron acceptor in down-regulating PHD and both of these complexes down-regulate rat brain NOS reactivity. A decrease in NO synthesis in animal models of seizure and radiation injury may account for the anticonvulsant, radioprotectant, and radiorecovery activities of Mn(3)(3,5-DIPS)(6) and Cu(II)(2)(3,5-DIPS)(4).

The protein inhibitor of neuronal nitric oxide synthase (PIN): characterization of its action on pure nitric oxide synthases.

Neuronal NO synthase (nNOS) was discovered recently to interact specifically with the protein PIN (protein inhibitor of nNOS) [Jaffrey, S.R. and Snyder, S.H. (1996) Science 274, 774-777]. We have studied the effects on pure NOS enzymes of the same GST-tagged PIN used in the original paper. Unexpectedly, all NOS isoenzymes were inhibited. The IC50 for nNOS was 18 +/- 6 microM GST-PIN with 63 nM nNOS after 30 min at 37 degrees C. Uncoupled NADPH oxidation was inhibited similarly, whereas cytochrome c reductase activity, the K(M) for L-arginine, and dimerization were unaffected. We reconsider the physiological role of PIN in the light of these results.

Characterization of the inducible nitric oxide synthase oxygenase domain identifies a 49 amino acid segment required for subunit dimerization and tetrahydrobiopterin interaction.

The oxygenase domain of inducible NO synthase (residues 1-498, iNOSox) is the enzyme's catalytic center. Its active form is a homodimer that contains heme and tetrahydrobiopterin (H4biopterin) and binds l-arginine [Ghosh, D. K., & Stuehr, D. J. (1995) Biochemistry 34, 801]. To help identify protein residues involved in prosthetic group and dimeric interaction, we expressed H4biopterin-free iNOSox in Escherichia coli. The iNOSox was 80% dimeric but contained a low-spin heme iron that bound DTT as a sixth ligand. The iNOSox bound H4biopterin or L-arginine with high affinity, which displaced DTT from the heme and caused spectral changes consistent with a closing up of the heme pocket. The H4biopterin-replete iNOSox could catalyze conversion of Nomega-hydroxyarginine to citrulline and NO in a H2O2-supported reaction. Limited trypsinolysis of the H4biopterin-free iNOSox dimer cut the protein at a single site in its N-terminal region (K117). H4biopterin protected against the cleavage whereas l-arginine did not. The resulting 40 kDa protein contained thiol-ligated low-spin heme, was monomeric, catalytically inactive, showed no capacity to bind H4biopterin or l-arginine, and did not dimerize when provided with these molecules, indicating that residues 1-117 were important for iNOSox dimerization and H4biopterin/l-arginine interaction. A deletion mutant missing residues 1-114 was partially dimeric but otherwise identical to the 40 kDa protein regarding its spectral and catalytic properties and inability to respond to l-arginine and H4biopterin, whereas a deletion mutant missing residues 1-65 was equivalent to wild-type iNOSox, narrowing the region of importance to amino acids 66-114. Mutation of a conserved cysteine in this region (C109A) decreased H4biopterin affinity without compromising iNOSox dimeric structure, L-arginine binding, or catalytic function. These results suggest that residues 66-114 of iNOSox are involved in productive H4biopterin interaction and subunit dimerization. H4biopterin binding appears to stabilize the protein structure in this region, and through doing so activates iNOS for NO synthesis.

A synthetic peptide corresponding to the putative dihydrofolate reductase domain of nitric oxide synthase inhibits uncoupled NADPH oxidation.

A stretch of about 150 amino acids located between the heme and the calmodulin recognition sequence of nitric oxide synthase (NOS) has been strongly conserved within isoforms and was proposed to participate in pteridine binding because of sequence similarities to the folate binding site of dihydrofolate reductase (DHFR). In the present study we tested four synthetic peptides corresponding to sequences located within the putative DHFR domain of rat neuronal NOS for their effects on catalytic and binding activities of the recombinant enzyme purified from baculovirus-infected insect cells. Three of the selected peptides had no effects at concentrations of up to 0.1 mM, but one peptide, corresponding to amino acid residues 564-582 of neuronal NOS, led to a concentration-dependent inhibition of L-citrulline formation. The potency of the peptide decreased with increasing assay concentrations of NOS, pointing to a competitive interaction with a specific structure of the enzyme. The peptide was not competitive with L-arginine and H4biopterin, did not antagonize binding of radiolabeled NG-nitro-L-arginine or H4biopterin, and had no effect on Ca2+/calmodulin-dependent reduction of cytochrome c. However, the presence of the peptide led to a pronounced inhibition of NADPH oxidation in the absence of L-arginine and prevented stimulation of this reaction by the amino acid substrate. These results indicate that sequence 564-582 of neuronal NOS does not contribute to L-arginine or H4biopterin binding but is critically involved in the electron transfer from the reductase domain to the heme.

Allosteric modulation of rat brain nitric oxide synthase by the pterin-site enzyme inhibitor 4-aminotetrahydrobiopterin.

We investigated the functional and allosteric effects of the 4-amino analogue of tetrahydrobiopterin, (6R)-2,4-diamino- 5,6,7,8-tetrahydro-6-(L-erythro-1,2-dihydroxypropyl) pteridine (4-amino-H4biopterin) on pteridine-free rat neuronal nitric oxide synthase. In the presence of added (6R)-5,6,7,8-tetrahydro-L-erythrobiopterin (H4biopterin; 10 microM), 4-amino-H4biopterin completely inhibited the conversion of both L-arginine and NG-hydroxy-L-arginine with half-maximally effective concentrations of 1.1+/-0.09 and 1.3+/-0.09 microM, respectively. Inhibition was reversible, as shown by a time-dependent restoration of citrulline formation upon dilution of the inhibitor-treated enzyme (t1/2=3.0 min). Binding of 4-amino-H4biopterin led to a complete conversion of the haem from low-spin to high-spin state, and to the formation of stable homodimers which partially survived electrophoresis under denaturating conditions. These results show that oxidation of both L-arginine and NG-hydroxy-L-arginine is pteridine-dependent, and that the allosteric effects of H4biopterin do not fully explain the essential role of the pteridine cofactor in nitric oxide biosynthesis.

Tetrahydrobiopterin-free neuronal nitric oxide synthase: evidence for two identical highly anticooperative pteridine binding sites.

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.

Identification of the 4-amino analogue of tetrahydrobiopterin as a dihydropteridine reductase inhibitor and a potent pteridine antagonist of rat neuronal nitric oxide synthase.

The binding of tetrahydropteridines with 6-di- and trihydroxypropyl side chains to recombinant rat neuronal nitric oxide (NO) synthase (EC 1.14.13.39) was determined by competition with 6R-[3'-3H]-5,6,7,8-tetrahydro-L-erythro-biopterin (6R-[3'-3H]H4biopterin). Although all but one of the derivatives exhibited only poor affinities (Ki 50 microM), the 4-amino analogue of 6R-H4 biopterin was a potent antagonist of 6R-H4 biopterin binding (Ki 13.2 nM). The 4-amino analogue of 6R-H4 biopterin inhibited NO synthase stimulation by the natural cofactor 6R-H4 biopterin with an IC50 of 1 microM without affecting the basal activity observed in the absence of added 6R-H4 biopterin. Because the 4-amino analogue of 6R-H4biopterin also inhibited dihydropteridine reductase (EC 1.6.99.7; IC50 20 microM), our results support the hypothesis that redox cycling of H4 biopterin might be required for the NO synthase reaction.