Activation of Neuronal Nitric-oxide Synthase by the 5-Methyl Analog of Tetrahydrobiopterin
FUNCTIONAL EVIDENCE AGAINST REDUCTIVE OXYGEN ACTIVATION BY THE PTERIN COFACTOR*

Christoph RiethmüllerDagger , Antonius C. F. GorrenDagger , Eva PittersDagger , Benjamin HemmensDagger , Hans-Jörg HabischDagger , Simon J. R. Heales§, Kurt SchmidtDagger , Ernst R. Werner, and Bernd MayerDagger parallel

From the Dagger  Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria, § Department of Clinical Biochemistry, National Hospital for Neurology and Neurosurgery, Queen Square, London WC 1N 3BG, United Kingdom, and  Institut für Medizinische Chemie und Biochemie, Universität Innsbruck, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria

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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 µM 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.

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Nitric oxide is formed from the guanidino group of L-arginine by nitric oxide synthases (NOSs)1 (EC 1.14.13.39; Refs. 1 and 2). The neuronal (nNOS) and endothelial isoforms are constitutively expressed and require micromolar free Ca2+ for activity, whereas the isoform first described in murine macrophages is cytokine-inducible and Ca2+-independent (inducible NO synthase (type II)). The enzymes consist of an oxygenase domain with a prosthetic heme group and a flavin-containing reductase domain shuttling reducing equivalents from the cosubstrate NADPH to the heme.

Unlike other P450s, NOS requires H4biopterin as a cofactor, but the actions of the pterin that underlie this requirement remain a puzzle (3). It has been suggested to participate as a reactant in the enzymatic hydroxylation of L-arginine to NG-hydroxy-L-arginine and/or the subsequent oxidation of the hydroxylated intermediate to NO and L-citrulline (4-7). However, the allosteric effects of H4biopterin binding are more obvious and have been more accessible to experimentation. They involve changes in the conformation of the protein around the heme and substrate binding pocket (8), facilitate the binding of L-arginine (9), and are coupled to a transition of the heme from a low spin, hexacoordinate state to a high spin, pentacoordinate state (10-12). Also, the binding of H4biopterin promotes dimerization of the enzyme. In inducible NO synthase (type II), it increases the level of dimerization per se (13), and in all NOS isozymes, it induces a form of the dimer that is unusually resistant to dissociation by SDS (14). The time course of conversion from low spin heme to high spin heme correlates closely with enzyme activation (12); also, only dimeric forms of the enzyme have been found to be active (15). Thus, it could be supposed that the allosteric effects of H4biopterin fully explain the pterin dependence of NOS.

However, several pterin derivatives, such as dihydrobiopterin (9, 14, 16) or the 4-amino analog of H4biopterin (17, 18), produce the same allosteric effects as the natural cofactor but do not support NO synthesis. Inhibition of NOS by the oxidation-resistant deaza analog of 6-methyltetrahydropterin (19), as well as by the potent dihydropteridine reductase inhibitor 4-amino-H4biopterin (20), indicated that classical two-electron redox cycling of the pterin cofactor may be essential for NO synthesis. This view was supported by a recent report showing that although both the di- and tetrahydro forms of various pterin derivatives have binding capacity, NOS activation was only observed in the presence of the tetrahydro compounds (16). Finally, a study of reaction intermediates indicated that H4biopterin was necessary for reductive activation of the oxy-ferrous complex of nNOS (21). Taken together, these data provide circumstantial evidence for an essential role of the pterin redox properties in NOS catalysis. In the crystal structure of dimeric inducible NO synthase (type II) oxygenase, the pterin is located proximal to the heme, without apparent access to the distal site where O2 activation and substrate oxidation take place (8). Thus, chemical contributions of the pterin to NOS catalysis are only likely if they occur via the heme.

Another interesting feature of pterin-NOS interactions is the highly anticooperative binding of H4biopterin to two identical binding sites of homodimeric nNOS (12). The biphasic association kinetics of [3H]H4biopterin binding to a pterin-deficient form of the protein pointed to a difference of at least 1,000-fold in the affinities for the binding of the first and the second H4biopterin molecule, respectively. From the rapid association of [3H]H4biopterin, the KD value of the high affinity site was calculated to be less than 1 nM (12, 22). This high binding affinity probably explains why isolated nNOS always contains about 1 equivalent of tightly bound H4biopterin per dimer (23, 24).

The anticooperative binding of H4biopterin appears to have important functional consequences because the pterin is essential for the coupling of NADPH oxidation to L-arginine metabolism (25). Thus, even in the presence of saturating L-arginine concentrations, activation of pterin-deficient nNOS by Ca2+/calmodulin results in the NADPH-dependent reduction of O2 to Obardot 2 and, eventually, H2O2 (12). Because the two catalytic domains of dimeric NOS operate independently (22, 26, 27), these results suggest that native nNOS containing 1 equivalent of H4biopterin per dimer generates NO and Obardot 2 at the same time, two species known to react with each other very rapidly to yield peroxynitrite (28). Indeed, nNOS was found to produce free NO only in the presence of the Obardot 2 scavenger SOD (29). Inhibition of calmodulin-dependent H2O2 formation in the presence of relatively high, i.e. micromolar, concentrations of free H4biopterin suggests that enzymatic Obardot 2 formation is prevented by saturation of the low affinity pterin binding site (12), but under these conditions, autooxidation of free H4biopterin appears to produce sufficient Obardot 2 to completely inactivate the NO formed by the pterin-saturated enzyme, again rendering SOD essential to detect free NO (29). Because SOD was reported to catalyze the interconversion of NO and nitroxyl anion (NO-) under certain conditions (30), it was proposed that the initial product of the NOS reaction is, in fact, nitroxyl that is converted to free NO by SOD (31). Although we have not observed any significant oxidation of nitroxyl released from Angeli's salt by SOD under our experimental conditions,2 the definitive rebuttal of the nitroxyl hypothesis would require a demonstration of NO formation by nNOS in the absence of SOD or other scavengers, a goal that has not been achieved thus far.

In the present study, we have reconstituted pterin-deficient nNOS with the low affinity binding pterin derivative 5-methyl-H4biopterin. Unlike H4biopterin, this compound proved not to react with O2. This property allowed, for the first time, the detection of the enzymatic formation of free NO in the absence of Obardot 2 or NO scavengers. In addition, the previously calculated 3 orders of magnitude difference in the affinity of the two highly anticooperative pterin binding sites becomes visible in NOS activity assays with 5-methyl-H4biopterin.

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Materials-- Purified rat nNOS containing approximately 1 equivalent of H4biopterin per dimer (H4B(+)nNOS) was obtained from recombinant baculovirus-infected Sf9 cells as described previously (32). Pteridine-deficient nNOS containing <0.1 equivalent of H4biopterin (H4B(-)nNOS) was obtained from Sf9 cells pretreated with 2,4-diamino-6-hydroxypyrimidine as described previously (24). NO solutions were prepared as described previously (33). L-[2,3,4,5-3H]Arginine hydrochloride (57 Ci/mmol) was from American Radiolabeled Chemicals Inc., purchased through Humos Diagnostica GmbH (Maria Enzersdorf, Austria). 3'[3H](6R)-5,6,7,8-Tetrahydro-L-biopterin (14 Ci/mmol) was prepared from [8,5'-3H]GTP as described previously (34). Unlabeled pterins including 5-methyl-H4biopterin were from Dr. B. Schircks Laboratories (Jona, Switzerland). Other chemicals including bovine Cu- and Zn-SOD were from Sigma.

Purity of 5-Methyl-H4biopterin-- The batch of 5-methyl-H4biopterin used in the present study did not contain any detectable contaminating H4biopterin. The detection limit of the applied high performance liquid chromatography method with electrochemical detection (35) was 33 nM H4biopterin in 1 mM solutions of 5-methyl-H4biopterin. Accordingly, the amount of contaminating H4biopterin was <= 0.0033%. Most of the experiments shown here have been repeated with a batch of 5-methyl-H4biopterin containing approximately 0.2 µM H4biopterin in 1 mM solutions with identical results.

Determination of NOS Activity-- NOS activity was determined as the formation of L-[2,3,4,5-3H]citrulline from L-[2,3,4,5-3H]arginine (36). Incubations were for 10 min at 37 °C in 0.1 ml of 50 mM potassium phosphate buffer, pH 7.4, containing 0.3 µg of nNOS, 0.1 mM L-[2,3,4,5-3H]arginine (~60,000 counts/min), 0.2 mM NADPH, 10 µg/ml calmodulin, 0.5 mM CaCl2, 0.2 mM CHAPS, and pteridines as indicated. Of note, standard incubation conditions were modified by omitting exogenous flavins that have been recognized as potential unwanted sources of superoxide (37, 38). H4B(+)nNOS and H4B(-)nNOS exhibited a similar specific activity of approximately 0.4 µmol L-citrulline × mg-1 × min-1 under those conditions.

The stoichiometry of NADPH oxidation and L-citrulline formation was determined with H4B(+)nNOS under slightly modified conditions, i.e. in the presence of 0.2 mM [3H]arginine, SOD (5,000 units/ml), and a limiting concentration of NADPH (10 µM). The amount of [3H]citrulline formed was measured after 3 h of incubation at 37 °C.

Electrochemical Determination of NO and O2-- NO and O2 were measured at 37 °C with commercially available Clark-type electrodes (Iso-NO and ISO2; World Precision Instruments, Berlin, Germany) (29). The electrodes were connected to an Apple Macintosh computer via an analog to digital converter (MacLab; AD Instruments Ltd., Hastings, United Kingdom), and the output current was recorded under constant stirring. The NO electrode was calibrated daily with acidified nitrite according to the recommendations of the manufacturer. Two-point calibration of the O2 electrode was performed with argon and air-saturated distilled water, respectively. O2 consumption was measured at 37 °C in sealed 1.8-ml vials completely filled with 50 mM potassium phosphate buffer, pH 7.4. The observed rates (Kobs) were divided by the O2 and pterin concentrations to obtain the respective second-order rate constants. For the measurement of NO oxidation, a solution of authentic NO was added to 0.5 ml of 50 mM potassium phosphate buffer, pH 7.4, in open glass vials at 37 °C. For the determination of enzymatic NO formation, purified nNOS (5 µg) was incubated in 0.5-ml open glass vials at 37 °C in 50 mM phosphate buffer, pH 7.4, containing 0.1 mM L-arginine, 0.2 mM NADPH, 10 µg/ml calmodulin, 0.5 mM CaCl2, and variable concentrations of H4biopterin or 5-methyl-H4biopterin. Pterins were added as 100-fold concentrated aqueous solutions. Where indicated, SOD was added to give a final concentration of 1,000 units/ml. Experiments were terminated by the addition of hemoglobin (4 µM, final concentration). Specific enzyme activity was calculated from the initial linear rise in the NO concentrations.

H4biopterin Binding-- Saturation binding experiments using [3H] H4biopterin as a high affinity radioligand were performed as described previously with minor modifications (9). H4B(-)nNOS and H4B(+)nNOS (0.5 µg each) were incubated for 10 min at 37 °C with 10 nM [3H]H4biopterin (~14 nCi) and increasing concentrations of 5-methyl-H4biopterin (10-8 to 10-2 M) in 0.1 ml of a 50 mM potassium phosphate buffer, pH 7.4, followed by vacuum filtration of the protein using the MultiScreen Assay System (Millipore) and liquid scintillation counting of bound radioactivity. Data were corrected for nonspecific binding in the presence of 1 mM unlabeled H4biopterin. IC50 values obtained from individual experiments were used to calculate KI values according to the following equation: KI = IC50/(1 + [H4B]f/KD) with [H4B]f corresponding to the concentration of free H4biopterin (10 nM), and KD corresponding to the binding constant for H4biopterin (<= 1 nM) (12, 22).

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Reactivity of 5-Methyl-H4biopterin toward Oxygen-- To test the reactivity of 5-methyl-H4biopterin toward O2, we measured the nonenzymatic consumption of O2 by H4biopterin and its 5-methyl analog. As shown in Fig. 1A, H4biopterin led to a consumption of O2 that was linearly dependent on the concentration of the pterin (0.1-0.5 mM). Assuming a first-order rate law with respect to O2, the calculated second-order rate constant for the reaction was 4.72 ± 0.44 M-1 s-1 (n = 8) at 37 °C. In contrast to H4biopterin, the 5-methyl analog did not trigger a detectable consumption of O2 at concentrations of up to 1 mM. Because the reduction of O2 by H4biopterin results in Obardot 2-mediated oxidation of NO (29), we tested the two pterins for their effects on the rate of NO oxidation measured in O2-containing solutions using a Clark-type NO electrode. Fig. 1B shows that H4biopterin (0.1 mM), but not its 5-methyl analog, led to a rapid, apparently zero-order disappearance of the NO signal. The effect of H4biopterin was completely inhibited by SOD (1,000 units/ml; data not shown). The initial rate of NO consumption by added H4biopterin (0.1 mM, final concentration) was 38.5 ± 2.7 nM s-1 (n = 4) at 37 °C. This rate agrees well with the O2 consumption data showing that the initial rate of Obardot 2 generation by 0.1 mM H4biopterin was about 80 nM s-1 Obardot 2 under identical conditions.


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Fig. 1.   Oxygen consumption (A) and NO oxidation (B) by H4biopterin and its 5-methyl analog. The consumption of O2 (A) and the oxidation of authentic NO (B) were measured electrochemically with O2 and NO electrodes, respectively, at 37 °C as described under "Experimental Procedures." The final pterin concentrations added at the time points shown are indicated by arrows. Results are original traces representative of three experiments.

Activation of NOS by 5-Methyl-H4biopterin-- The interesting chemical properties of 5-methyl-H4biopterin led us to investigate its effect on NOS activity. As shown in Fig. 2, the presence of 5-methyl-H4biopterin resulted in a biphasic stimulation of L-citrulline formation by H4B(-)nNOS. The first phase occurred with an EC50 of approximately 0.2 µM, resulting in an increase of the specific activity from a residual value of about 20 nmol L-citrulline × min-1 × mg-1 to 100 nmol × min-1 × mg-1 in the presence of 10 µM 5-methyl-H4biopterin. The enzyme activity approached the basal activity of H4B(+)nNOS (~150 nmol × min-1 × mg-1) and was not further increased by up to 0.3 mM 5-methyl-H4biopterin. The basal activity of H4B(+)nNOS was not significantly affected by up to 1 mM 5-methyl-H4biopterin. The second phase of enzyme activation occurred with a calculated EC50 of about 10 mM 5-methyl-H4biopterin with both H4B(+)nNOS and H4B(-)nNOS.


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Fig. 2.   Effect of 5-methyl-H4biopterin on nNOS-catalyzed L-citrulline formation. Purified H4B(+)nNOS (open circle ) and H4B(-)nNOS () (0.2 µg each) were incubated for 10 min at 37 °C in 0.1 ml of 50 mM triethanolamine/HCl buffer, pH 7.4, containing 0.1 mM L-[2,3,4,5-3H]arginine (~80,000 counts/min), 0.2 mM NADPH, 0.5 mM CaCl2, 10 µg/ml calmodulin, 2.4 mM 2-mercaptoethanol, and 0.2 mM CHAPS in the presence of the indicated concentrations of 5-methyl-H4biopterin. Data are the mean values ± S.E. of three experiments.

Determination of the stoichiometry of NADP+ and L-citrulline formation by H4B(+)nNOS showed that NADPH oxidation was largely uncoupled from L-arginine oxidation in the absence of 5-methyl-H4biopterin or at low concentrations (up to 1 µM) of 5-methyl-H4biopterin (NADP+/L-citrulline = 2.2 ± 0.2; n = 4). The presence of 0.1 mM 5-methyl-H4biopterin led to an almost complete coupling of the reactions, as indicated by a NADP+/L-citrulline stoichiometry of 1.59 ± 0.07 (n = 4), which is close to the theoretical value of 1.50 (5, 6).

Effect of 5-Methyl-H4biopterin on [3H]H4biopterin Binding to nNOS-- Radioligand binding experiments using [3H]H4biopterin as a high affinity ligand (9) showed that 5-methyl-H4biopterin bound specifically to the pterin site of nNOS. As illustrated in Fig. 3, the 5-methyl analog displaced [3H]H4biopterin from H4B(-)nNOS and H4B(+)nNOS with IC50 values of 78 ± 7.1 and 62 ± 1.3 µM (means ± S.E.; n = 3), respectively. Because the KD of the low affinity site is too high to be observed in radioligand binding experiments, 5-methyl-H4biopterin most likely displaced H4biopterin bound with high affinity to H4B(+)nNOS. Based on the KD of <= 1 nM for high affinity binding of H4biopterin (12, 22), the maximal KI values for 5-methyl-H4biopterin binding to H4B(-)nNOS and H4B(+)nNOS were calculated to be 7.2 and 5.5 µM, respectively.


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Fig. 3.   Effect of 5-methyl-H4biopterin on [3H]H4biopterin binding. Purified H4B(+)nNOS (open circle ) and H4B(-)nNOS () (0.5 µg each) were incubated for 30 min at 25 °C with 10 nM [3H]H4biopterin (~14 nCi) in 0.1 ml of a 50 mM triethanolamine/HCl buffer, pH 7.4, in the presence of increasing concentrations of 5-methyl-H4biopterin (10-8 to 10-2 M) followed by liquid scintillation counting of protein-bound radioactivity. Data (means ± S.E.; n = 3) were corrected for nonspecific binding in the presence of 1 mM unlabeled H4biopterin.

Effect of 5-Methyl-H4biopterin on NO Formation by nNOS-- The inactivity of 5-methyl-H4biopterin toward O2 suggested that incubation of nNOS with an excess of this synthetic pterin should not lead to the inactivation of enzymatically produced NO, as observed with the natural cofactor. Indeed, as shown in Fig. 4, significant NO formation was detectable when H4B(-)nNOS was incubated with 5-methyl-H4biopterin (1 mM; Fig. 4A) instead of the natural cofactor (0.1 mM; Fig. 4B). The specific enzyme activity calculated from the initial linear increase in the NO concentration was 52 ± 3 nmol × mg-1 × min-1, giving a steady-state concentration of 0.20 ± 0.05 µM NO (means ± S.E.; n = 3). The addition of SOD (1,000 units/ml) further increased this level to 0.31 ± 0.09 µM NO. The response of the electrode was completely abolished by the addition of the NO scavenger hemoglobin (4 µM, final concentration). As shown in Fig. 4B, the enzyme did not produce any detectable NO in the presence of 0.1 mM H4biopterin without SOD. The addition of SOD (1,000 units/ml) led to the formation of NO with an apparent specific activity of 45 ± 12 nmol NO × mg-1 × min-1 and a steady-state NO concentration of 0.29 ± 0.05 µM (means ± S.E.; n = 3). Again, the signal returned to baseline after the addition of hemoglobin (4 µM, final concentration). Similar results were obtained with H4B(+)nNOS (data not shown).


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Fig. 4.   Effects of 5-methyl-H4biopterin and SOD on the formation of NO by nNOS. Purified H4B(-)nNOS (10 µg) was incubated at 37 °C in the presence of 1 mM 5-methyl-H4biopterin (A) or 0.1 mM H4biopterin (B). NOS, SOD, and hemoglobin (Hb) were added at the time points indicated by arrows to give the indicated final concentrations. NO was measured electrochemically with a Clark-type electrode as described under "Experimental Procedures." Results are original traces representative of six similar experiments.

The concentration-dependent effects of 5-methyl-H4biopterin on NO formation by H4B(-)nNOS and H4B(+)nNOS are summarized in Fig. 5. With both preparations, half-maximal enzyme activation was observed at 5-10 µM 5-methyl-H4biopterin. The maximal specific activities of H4B(-)nNOS and H4B(+)nNOS calculated from the initial rates of NO formation at 1 mM of 5-methyl-H4biopterin were 52 ± 3 and 88 ± 4 nmol NO mg-1 × min-1 (means ± S.E.; n = 3) respectively, values that are in good agreement with the rate of NO formation obtained with H4biopterin in the same assay.


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Fig. 5.   Concentration-dependent effect of 5-methyl-H4biopterin on NO formation by nNOS. Purified H4B(+)nNOS (open circle ) and H4B(-)nNOS () (10 µg each) were incubated in the presence of the indicated concentrations of 5-methyl-H4biopterin. NO was measured electrochemically with a Clark-type electrode as described under "Experimental Procedures." Data are the mean values ± S.E. of three experiments.


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Compared with the parent compound H4biopterin, the 5-methyl analog exhibited markedly reduced activity toward O2, which may be due to steric hindrance by the 5-methyl group, preventing the formation of the presumed hydroperoxide intermediate at the C4a position of the pterin ring. Nevertheless, 5-methyl-H4biopterin stimulated pterin-deficient nNOS in a biphasic manner, an effect that most likely reflects binding of the pterin to the high and low affinity sites of the dimeric protein. The KD for binding the natural cofactor, H4biopterin, to the high affinity site is too low to be determined in binding studies, but from the kinetics of [3H]H4biopterin association to H4B(-)nNOS, we have calculated that the binding constant is <= 1 nM (12, 22). Because this value is about 1 order of magnitude lower than the typical NOS assay concentrations (10-30 nM), biphasic pterin binding is not detectable in functional studies using H4biopterin as a cofactor. However, the 3 orders of magnitude lower affinity of the 5-methyl derivative apparently allowed, for the first time, monitoring of the saturation of the high affinity site in the arginine-to-citrulline conversion assay. The 50,000-fold difference in the affinities of the two binding sites (EC50 values of approximately 0.2 µM and 10 mM) agrees well with our earlier estimate that the KD values for high and low affinity binding of H4biopterin to dimeric nNOS differ by at least 3 orders of magnitude (12, 22). Because the second phase of enzyme activation required relatively high concentrations of 5-methyl-H4biopterin, it was conceivable that this effect was mediated by contaminating H4biopterin. However, this is unlikely because (i) the batch of 5-methyl-H4biopterin used throughout this study did not contain any detectable H4biopterin (<= 0.0033%), and (ii) virtually identical data were obtained with a batch containing an at least 10-fold higher amount of H4biopterin (0.03%).

Because the precise KD value for high affinity H4biopterin binding is unknown, it was not possible to convert the IC50 values for inhibition of [3H]H4biopterin binding by the 5-methyl analog into reliable KI values. However, based on the solid evidence that the H4biopterin binding constant is <= 1 nM (12, 22), we have calculated that the upper limit of the KI value for the binding of 5-methyl-H4biopterin is approximately 5 µM. This calculated binding constant is about 20-fold higher than the EC50 value obtained in the arginine-to-citrulline conversion assay, indicating that the KD for high affinity H4biopterin binding to nNOS may be as low as 50 pM.

There are numerous reports demonstrating NO release from intact cells, but it has proven difficult in the past to identify NO as a product of the NOS reaction. Thus far, NO formation by isolated NOS has only been detected in the presence of scavengers of either NO or superoxide. Thus, enzymatic NO formation was apparent when NOS activity was assayed as a hemoglobin-to-methemoglobin conversion (7) or in spin trapping electron paramagnetic resonance spectroscopy experiments performed with excess Fe2+-N-methyl-D-glucamine dithiocarbamate as a NO trap (37). Besides NO scavengers, the presence of the Obardot 2 scavenger SOD was also shown to switch the outcome of the NOS reaction toward detectable free NO (29, 31). Even though those results strongly favor the view that NOS does indeed form NO, it has remained unclear whether or not NOS would also make NO in the absence of scavengers which, with the exception of SOD, are not present in cells. Our results demonstrate for the first time that nNOS forms free NO in the absence of scavengers when the enzyme is reconstituted with the autooxidation-resistant 5-methyl analog of H4biopterin instead of the natural cofactor. The results unequivocally identify free NO as the initial product of the NOS reaction and provide further evidence for our proposal that the failure to detect NO formation by purified nNOS is a consequence of NO inactivation by Obardot 2 formed during autooxidation of exogenously added H4biopterin (29). Unlike L-citrulline formation, NO production was not stimulated in a biphasic manner but gradually increased when the concentration of 5-methyl-H4biopterin was increased from 5 µM to 1 mM. This concentration range is clearly between the concentrations required for high affinity and low affinity activation of L-citrulline formation (see Fig. 2). Interestingly, the stoichiometry of NADP+ and L-citrulline formation showed that the presence of 0.1 mM 5-methyl-H4biopterin was sufficient for virtually complete coupling of NADPH and L-arginine oxidation. These results may indicate that saturation of one pterin site of dimeric nNOS with 5-methyl-H4biopterin leads to inhibition of heme reduction and associated superoxide formation by the pterin-free subunit. Although this hypothesis appears to provide a reliable explanation for the formation of free NO by the enzyme subsaturated with 5-methyl-H4biopterin, additional studies are needed to settle the complex relationship between biphasic 5-methyl-H4biopterin binding and NOS activity.

A great deal of published circumstantial evidence supports our early hypothesis that one step of the NOS reaction resembles the hydroxylation of aromatic amino acids, an enzymatic reaction in which H4biopterin functions as a donor of electrons required for reductive O2 activation (5). Thus, stimulation of NOS activity is only observed with fully reduced, i.e. tetrahydro, pterins, whereas dihydro compounds are inhibitory (9, 16). Furthermore, the redox-inactive N5-deaza analog of 6-methyl-tetrahydropterin was found to inhibit H4biopterin-supported NOS activity, although the parent compound was an active cofactor (19). Similarly, the 4-amino analog of H4biopterin, an inhibitor of dihydropteridine reductase-catalyzed pterin redox cycling (20), potently inhibited both steps of the NOS reaction, although it produced allosteric effects identical to those of H4biopterin (17, 18). Finally, a recent study demonstrated that the rate of the NOS reaction is increased by binding of non-heme iron, indicating that an additional metal center might cooperate with H4biopterin in enzyme catalysis (39). Nevertheless, compared with the finding that 5-methyl-H4biopterin does not stimulate phenylalanine hydroxylase,3 the present data unequivocally show that the same redox chemistry of the pterin is not required in the NOS reaction. This constitutes conclusive functional evidence that the pterin in NOS is not the site of O2 activation. Although the redox potential of 5-methyl-H4biopterin is different from that of H4biopterin,4 the 5-methyl compound can still undergo certain redox reactions that are currently being investigated in our laboratory. Thus, at the present time, we cannot rule out that the pterin cofactor, in addition to its allosteric functions, interacts with the heme or another metal center of NOS through an as yet unrecognized redox chemistry.

    FOOTNOTES

* This work was supported by Grants 11478 and 13013 (to B. M.), Grant 12191 (to K. S.), and Grant 11301 (to E. R. W.) of the Fonds zur Förderung der Wissenschaftlichen Forschung in Österreich.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel To whom correspondence should be addressed. Tel.: 43-316-380-5567; Fax: 43-316-380-9890; E-mail: mayer{at}kfunigraz.ac.at.

2 K. Schmidt and B. Mayer, unpublished observation.

3 E. R. Werner, H.-J. Habisch, A. C. F. Gorren, L. Canevari, K. Schmidt, G. Werner-Felmayer, and B. Mayer, manuscript in preparation.

4 A. C. F. Gorren, A. J. Kungl, K. Schmidt, E. R. Werner, and B. Mayer, submitted for publication.

    ABBREVIATIONS

The abbreviations used are: NOS, nitric-oxide synthase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; H4biopterin, (6R)-5,6,7,8-tetrahydro-L-biopterin; 5-methyl-H4biopterin, N5-methyl-(6R)-5,6,7,8-tetrahydro-L-biopterin; NO, nitric oxide; nNOS, neuronal nitric-oxide synthase (type I); H4B(+)nNOS, neuronal nitric-oxide synthase containing one molecule of bound tetrahydrobiopterin per dimer; H4B(-)nNOS, neuronal nitric-oxide synthase containing <= 0.1 equivalent of tetrahydrobiopterin; SOD, superoxide dismutase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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