©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Kinetics and Mechanism of Tetrahydrobiopterin-induced Oxidation of Nitric Oxide (*)

(Received for publication, April 11, 1994; and in revised form, November 10, 1994)

Bernd Mayer (§) Peter Klatt Ernst R. Werner (1) Kurt Schmidt

From the Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, A-8010 Graz, Austria and the Institut für Medizinische Chemie und Biochemie, Universität Innsbruck, A-6020 Innsbruck, Austria

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A Clark-type nitric oxide-sensitive electrode was used for electrochemical determination of NO oxidation kinetics. Reaction with molecular oxygen followed second-order rate law with respect to NO with an overall rate constant of 9.2 ± 0.33 times 10^6M s. Tetrahydrobiopterin, an essential cofactor of NO synthases, was found to induce rapid oxidation of NO in a 1:1 stoichiometry. The reaction required the presence of oxygen, was zero order with respect to NO and first order with respect to tetrahydrobiopterin, completely blocked by 5,000 units/ml superoxide dismutase, and mimicked by a superoxide-generating system. Purified brain NO synthase produced no detectable NO unless high amounts of superoxide dismutase were present. NO synthase-catalyzed citrulline formation was inhibited by superoxide dismutase (5,000 units/ml) in an oxyhemoglobin-sensitive manner, indicating that NO induces feedback inhibition of NO synthase. NO-stimulated soluble guanylyl cyclase was inhibited by tetrahydrobiopterin at half-maximally active concentrations of 2 µM. The present data suggest that NO is inactivated to peroxynitrite by superoxide generated in the course of tetrahydrobiopterin autoxidation.


INTRODUCTION

The biologic effects of NO are almost exclusively determined by its chemical properties. As a free radical, NO reacts rapidly with molecular oxygen, superoxide, and iron-containing proteins. Reaction with oxygen results in the formation of as yet poorly defined reactive intermediates causing oxidative damage of DNA(1, 2) . Reaction with superoxide is extremely fast and generates peroxynitrite, which is rapidly inactivated when protonated to the corresponding peroxynitrous acid(3) . Decomposition of peroxynitrous acid involves formation of cytotoxic intermediates with chemical and biological properties similar to hydroxyl radical(4) . Finally, the activity of a wide variety of iron-containing enzymes is affected by NO. Several non-heme iron proteins, such as cis-aconitase or ribonucleotide reductase are inhibited(5) , whereas heme-containing soluble guanylyl cyclase is stimulated several hundredfold upon binding of NO to its regulatory prosthetic heme group(6) . Microsomal cytochromes P-450, however, which contain a catalytically active heme, are completely inhibited at submicromolar concentrations of NO(7) .

The enzymes involved in biosynthesis of NO from L-arginine, the NO synthases (NOS), (^1)have been identified as cytochrome P-450-like hemeproteins(8, 9, 10, 11) , and it is conceivable, therefore, that NO blocks its own formation. We have recently studied the possible feedback inhibition of purified brain NOS, but observed no appreciable inhibition of citrulline formation upon addition of high concentrations of authentic NO or drugs believed to release NO. Moreover, reaction rates were not enhanced in the presence of oxyhemoglobin, an effective scavenger of NO(12) . However, several reports from different groups demonstrate inhibition of NO synthesis by NO in brain cytosol, endothelial cells, and macrophages (13, 14, 15, 16, 17) . Albeit these studies used fairly high concentrations of NO to produce inhibition, the results clearly conflict with our data.

A major drawback of previous work on feedback inhibition of NOS was that actual NO concentrations have not been measured in the incubation mixtures. In the present study we have used an NO-sensitive Clark-type electrode for NO measurements under various conditions and found that the NOS cofactor H(4)biopterin induced a rapid oxidation of NO. This pronounced effect was apparently due to generation of superoxide in the course of H(4)biopterin autoxidation. Interestingly, purified NOS produced no detectable amounts of NO, unless high concentrations of SOD had been added, regardless whether H(4)biopterin was present or not.


EXPERIMENTAL PROCEDURES

Materials

L-[2,3,4,5-^3H]Arginine hydrochloride (57 Ci/mmol) and [alpha-P]GTP (400 Ci/mmol) were purchased from MedPro (Amersham Corp.), Vienna, Austria. 3-Morpholino-sydnonimine (SIN-1), a generous gift from Cassellla-Riedel, Frankfurt, Germany, was dissolved at pH 5.0 prior to use. NO solutions (2 mM) were prepared as previously described (18) by dissolving NO gas in deoxygenated water. 10 mM stock solutions of GSNO were prepared from GSH and nitrite as described elsewhere(19) . Oxyhemoglobin was prepared by dithionite reduction of hemoglobin and subsequent gel filtration chromatography as previously described(20) . 2,2-Diethyl-1-nitroso-oxyhydrazine sodium salt (DEA/NO) (21) was obtained from NCI Chemical Carcinogen Repository, Kansas City, MO, NADPH from Boehringer Mannheim, Germany, and H(4)biopterin from Dr. B. Schircks Laboratories, Jona, Switzerland. All other chemicals, including bovine erythrocyte CuZn-SOD and milk xanthine oxidase, were from Sigma, Deisenhofen, Germany.

Electrochemical Detection of NO and Oxygen

NO and oxygen were measured with commercially available Clark-type electrodes (Iso-NO and ISO(2), World Precision Instruments, Mauer, Germany). NO and oxygen meters were connected to an Apple Macintosh computer via an analog to digital (A/D) converter (MacLab, World Precision Instruments). Depending on the required resolution in time, data were collected at rates of 0.3-10 Hz. Solutions to be tested (0.5-10 µl) were applied by injection into 1.8-ml glass vials sealed with a septum. To minimize head space volumes, vials were completely filled with 1.8 ml of triethanolamine/HCl buffer (pH 7.0), containing additives as indicated. Measurements of NO and oxygen were performed under constant stirring at ambient temperature. Calibration of the electrodes was performed daily according to the procedure recommended by the manufacturer with air-saturated distilled water (ISO(2)) or acidified nitrite (Iso-NO). The latter was prepared by the addition of KNO(2) to a helium-gassed solution of 0.14 M K(2)SO(4) and 0.1 M KI in 0.1 M H(2)SO(4).

The donor compound DEA/NO (21) was used to generate defined amounts of NO for determination of NO:H(4)biopterin stoichiometry. Following application of DEA/NO (20-120 nmol), NO concentrations rapidly increased to 3-9 µM, followed by a slow decrease of the signals. The areas under the curve were calculated by integration from t(1) = 0 (addition of DEA/NO) and t(2) (defined as the time point at which NO concentrations had decreased to 0.5 µM (30 min)). There was a linear relationship between this area and NO generated from 20 to 120 nmol of DEA/NO. Consumption of NO was measured in the presence of 40-120 nmol of DEA/NO and 10-40 nmol of H(4)biopterin. NO:oxygen stoichiometry was measured by electrochemical determination of oxygen consumption in the presence of 0.1-0.2 mM DEA/NO. Initial oxygen concentrations were 210 ± 5.5 µM (n = 20).

Autoxidation Kinetics of H(4)Biopterin

H(4)Biopterin (1-10 µM) was incubated under constant stirring in 50 mM triethanolamine/HCl buffer (pH 7.0) at ambient temperature. Subsequent to incubation for 10 s up to 10 min, 0.25-ml aliquots were removed and acidified by addition of 30 µl 1 N HCl. Samples were immediately injected onto a 250 times 4-mm C(18) reversed-phase column equipped with a 4 times 4-mm C(18) precolumn (LiChrospher 100 RP-18, 5-µm particle size, Merck) and analyzed for H(4)biopterin by HPLC (LiChroGraph L 6200, Merck); detection was made of ultraviolet absorbance at 266 nm (LiChroGraph L 4200, Merck). Elution was performed at a flow rate of 1.5 ml/min with 20 mM sodium phosphate buffer, pH 3.0, containing 1% (v/v) methanol. The method was calibrated with freshly prepared solutions of H(4)biopterin (0.1-10 µM).

Calculation of Rate Constants

Data obtained from measurements of NO and H(4)biopterin autoxidation were fitted according to the appropriate rate laws using a standard curve fitting program. Assuming first-order kinetics with respect to oxygen uptake (22, 23) , the apparent rate constants (k) for NO and H(4)biopterin oxidation were divided by the initial oxygen concentrations (210 ± 5.5 µM; n = 20) to obtain the respective rate constants. All data are means ± S.E. of n experiments.

Determination of NOS Activity

NOS was purified from porcine brain as described previously(24) . Enzyme activity was determined as formation of L-[^3H]citrulline from L-[^3H]arginine. Unless otherwise indicated, purified brain NOS (0.2 µg) was incubated at 37 °C for 10 min in 0.1 ml of 50 mM triethanolamine/HCl buffer, pH 7.5, containing 0.1 mML-[^3H]arginine, 0.5 mM CaCl(2), 10 µg/ml calmodulin, 0.2 mM NADPH, 10 µM H(4)biopterin, 5 µM FMN, and 5 µM FAD. Blank values were determined in the absence of enzyme. For electrochemical detection of NO, incubations were performed at ambient temperature in 0.5 ml of 50 mM triethanolamine/HCl buffer, pH 7.0, containing 5 µg of NOS, 1 mML-arginine, 0.5 mM CaCl(2), 5 µg of calmodulin, 0.3 mM NADPH, 5 µM FMN, and 5 µM FAD. H(4)Biopterin concentration-response curves were recorded in a cumulative manner in the absence and presence of 5,000 units/ml SOD by sequential addition of 100-fold concentrated H(4)biopterin stock solutions.

Determination of Soluble Guanylyl Cyclase Activity

Soluble guanylyl cyclase purified from bovine lung (25) was a generous gift from the late Eycke Böhme, Department of Pharmacology, Free University of Berlin, Germany. Formation of [P]cGMP from [alpha-P]GTP was measured by incubation of 0.15 µg of enzyme at 37 °C for 10 min in 0.1 ml of 50 mM triethanolamine/HCl buffer, pH 7.5, containing 0.5 mM [alpha-P]GTP (200,000-300,000 cpm), 1 mM Mg, 1 mM cGMP, and 1 µM GSNO. Reactions were terminated by ZnCO(3) precipitation, and [P]cGMP was isolated as described elsewhere(26) .


RESULTS

We obtained linear responses by calibration of a Clark-type NO-sensitive electrode with NO generated from 0.05 up to 10 µM acidified nitrite. As an example, Fig. 1shows representative traces obtained by calibration of the electrode with 0.4-2 µM nitrite. A sensitivity of 3.85 nM/pA was calculated from the slope of the linear curve fit shown in the inset to Fig. 1. This is in good accordance with a previously reported sensitivity of 3.92 ± 0.23 nM/pA(27) . Since fluctuations of basal currents were usually 2-3 pA, the detection limit of our setup was approximately 20 nM NO. For unknown reasons, basal currents were quite unstable occasionally, but grounding of the operator with a stainless steel wire markedly improved stability in these cases. It is possible, therefore, that taking additional precautions against interferences by surrounding electromagnetical fields could increase sensitivity. The method appears to be specific for NO, since no response of the electrode was observed in the presence of high concentrations of nitrite, nitrate, peroxynitrite, hydrogen peroxide, or a superoxide-generating system (not shown).


Figure 1: Calibration of the NO electrode. At the indicated time points (bullet), 7.2 µl of a 0.1 mM solution of KNO(2) were added to 1.8 ml of a helium-deoxygenated solution of 0.14 M K(2)SO(4) and 0.1 M KI in 0.1 M H(2)SO(4) yielding NO concentrations of 0.4, 0.8, 1.2, 1.6, and 2.0 µM (2 KNO(2) + 2 KI + 2 K(2)SO(4) + 2 H(2)SO(4) 2 NO + I(2) + 2 H(2)O + 4 K(2)SO(4)). Inset, correlation between NO concentration and output current of the electrode (3.85 nM/pA).



Fig. 2shows representative traces obtained with authentic NO. Addition of 4 µl of an NO solution (2 mM) to total volumes of 1.8 ml induced a pronounced response of the electrode corresponding to a maximum of 1.8 µM NO, followed by a decrease of the signal according to second order kinetics (see Fig. 2, inset). Measurement of oxygen uptake in the presence of defined amounts of NO released from the NO donor DEA/NO (21) showed that 1 molecule of oxygen consumed 3.92 ± 0.23 (n = 5) molecules of NO. Assuming a first order rate law with respect to oxygen(22) , we obtained a third order rate constant for NO autoxidation of 9.2 ± 0.33 times 10^6M s (n = 11), which was not changed by 5,000 units/ml SOD (9.5 ± 0.54 times 10^6M s; n = 4).


Figure 2: Autoxidation of NO. Experiments were performed at ambient temperature in the absence (-) and presence of 5,000 units/ml SOD (bulletbulletbulletbulletbullet). At time point zero, 4 µl of a saturated NO solution (2 mM) were added to 1.8 ml of 50 mM triethanolamine/HCl buffer, pH 7.0, and changes in NO concentrations were monitored over 13 min (350 data points). Inset, replot of the original data (2.5 to 13 min; 230 points) according to second-order rate law. The original tracing and the plot shown in the inset are representative of 11 (Control) or four (SOD) similar experiments.



Rates of NO oxidation were markedly increased by addition of H(4)biopterin. As can be seen from the representative experiment shown in Fig. 3, oxidation kinetics was changed from second to zero order with respect to NO, suggesting that NO itself was not involved in the rate-limiting reaction step that induced its rapid oxidation. The reaction was first order with respect to H(4)biopterin, and gassing the buffer with helium for 15 min prior to the experiments completely blocked the effect of H(4)biopterin (not shown), indicating that NO oxidation was triggered by a reaction of molecular oxygen with H(4)biopterin. Experiments in which DEA/NO was used for release of defined amounts of NO revealed that 1 mol of H(4)biopterin consumed 0.98 ± 0.10 (n = 23) mol of NO. However, the pteridine became substoichiometrically active in the presence of reducing compounds. Pure NADPH (0.1 mM), for instance, did not affect autoxidation of NO but markedly decreased H(4)biopterin:NO stoichiometry to at least 1:10.


Figure 3: Effect of H(4)biopterin on NO oxidation. Experiments were performed at ambient temperature in the absence (-) and presence of 5,000 units/ml SOD (bulletbulletbulletbulletbullet). At time point zero, 4 µl of a saturated NO solution (2 mM) were added to 1.8 ml of 50 mM triethanolamine/HCl buffer, pH 7.0. At the maximum of the NO peak, a solution of H(4)biopterin (30 µM final concentration) was added. Inset, replot of the data (2.5 to 15 min; 270 points) obtained in the presence of SOD according to second-order rate law. The original tracing and the plot shown in the inset are representative of 11 (Control) or 4 (SOD) similar experiments.



Since SOD (5,000 units/ml) completely prevented H(4)biopterin-induced oxidation of NO (Fig. 3), we speculated that the effect was mediated by superoxide generated via H(4)biopterin autoxidation. Two sets of experiments were performed to substantiate this hypothesis. First, we determined rates of H(4)biopterin autoxidation directly by quantitative HPLC analysis. Autoxidation was first order with respect to H(4)biopterin (k = 3.03 ± 0.37 times 10 s; n = 5). Assuming first order kinetics of oxygen consumption(23) , we obtained a rate constant for H(4)biopterin autoxidation of 1.44 ± 0.18 M s (n = 5), which was comparable with the respective constant obtained by determination of NO oxidation in the presence of H(4)biopterin (1.88 ± 0.15 M s; n = 11). Second, we tested whether generation of superoxide by hypoxanthine/xanthine oxidase mimicked H(4)biopterin-induced oxidation of NO. As shown in Fig. 4, addition of xanthine oxidase (1.67 milliunits) to an NO solution containing 1 mM hypoxanthine induced an SOD-inhibitable switch of NO oxidation from second to zero order kinetics, which was very similar to that observed in the presence of H(4)biopterin. Consumption of NO was 10.3 ± 0.73 nM s (n = 4) under these conditions, demonstrating that rates of zero order NO oxidation were a good measure for superoxide production by the hypoxanthine/xanthine oxidase system. These data suggest that H(4)biopterin-induced generation of superoxide accounts for the rapid oxidation of NO in the presence of the NOS cofactor H(4)biopterin.


Figure 4: Effect of hypoxanthine/xanthine oxidase on NO oxidation. Experiments were performed at ambient temperature in the absence (-) and presence of 5,000 units/ml SOD (bulletbulletbulletbulletbullet). At time point zero, 4 µl of a saturated NO solution (2 mM) were added to 1.8 ml of 50 mM triethanolamine/HCl buffer, pH 7.0, containing 1 mM hypoxanthine. After reaching the maximal NO concentration, 1.67 milliunits of xanthine oxidase were added. Inset, replot of the data (2.5-15 min; 270 points) obtained in the presence of SOD according to second-order rate law. The original tracing and the plot shown are representative of four.



Since these findings appeared to be of potential relevance to the biology of NO, we investigated whether the biological activity of NO was affected by H(4)biopterin. Fig. 5shows that H(4)biopterin produced a concentration-dependent inhibition of purified soluble guanylyl cyclase, which had been stimulated with 1 µM GSNO. Half-maximal effects were observed at 2 µM H(4)biopterin, and inhibition was complete at 0.1 mM.


Figure 5: Effect of H(4)biopterin on NO-induced cGMP formation by purified soluble guanylyl cyclase. Soluble guanylyl cyclase purified from bovine lung (0.15 µg) was incubated at 37 °C for 10 min in 50 mM triethanolamine/HCl buffer, pH 7.5 in the presence of 1 µM GSNO and increasing concentrations of H(4)biopterin as described under ``Experimental Procedures.'' Data are means ± S.E. of three experiments performed in triplicate.



Taking into account the role of H(4)biopterin as essential cofactor of NOS, we measured enzymatically produced NO with the NO-sensitive electrode. As shown in Fig. 6(upper panel), we observed no detectable signal by incubation of purified NOS with L-arginine and requisite cofactors in the absence and presence (0.1-1 µM) of exogenously added H(4)biopterin. However, subsequent addition of 5,000 units/ml SOD resulted in the generation of NO at initial rates of approximately 20 nM s, yielding steady state concentrations of 0.3 µM NO after about 1 min. The lower panel in Fig. 6shows a representative experiment performed in the presence of SOD. No response of the electrode was observed in the absence of calmodulin, addition of calmodulin produced a signal corresponding to 0.1 µM NO, and this was further increased to 0.3 µM in a concentration-dependent manner by added H(4)biopterin. The half-maximally active concentration of H(4)biopterin was approximately 0.1 µM and thus similar to the potency of the pteridine determined previously with other methods(24, 28) .


Figure 6: Electrochemical detection of enzymatically formed NO. Experiments were performed at ambient temperature in 0.5 ml of 50 mM triethanolamine/HCl buffer, pH 7.0, containing 0.5 mM CaCl(2), 1 mML-arginine, 0.3 mM NADPH, 5 µM FAD, 5 µM FMN, and 5 µg of purified brain NOS. Original tracings of experiments performed in the absence (upper panel) and presence of 5,000 units/ml SOD (lower panel) are shown. Where indicated, calmodulin (10 µg/ml), H(4)biopterin (0.1-100 µM), or SOD (5,000 units/ml) were added (final concentrations in parentheses). The tracings shown are representative of three.



These results demonstrated that NOS produced detectable amounts of NO only when considerably high concentrations of SOD were present. We speculated that this may explain the controversy regarding feedback inhibition of NOS by NO and measured formation of citrulline by the purified enzyme in the absence and presence of 5,000 units/ml SOD. As shown in Fig. 7, the enzyme was inhibited to 51.2% of controls in the presence of SOD, and this effect was completely prevented by 20 µM oxyhemoglobin. SIN-1 (1 mM) had a slight, oxyhemoglobin-insensitive inhibitory effect in the absence of SOD (75.5% of controls), but rates of citrulline formation were reduced down to 26.6% of untreated controls when both SIN-1 and SOD were present. Again, the inhibitory effect of SOD was not observed in the presence of 20 µM oxyhemoglobin.


Figure 7: Effects of oxyhemoglobin on SOD-induced inhibition of NOS. Purified brain NOS (0.2 µg) was incubated at 37 °C for 10 min in 50 mM triethanolamine/HCl buffer, pH 7.5, in the absence and presence of SOD (5,000 units/ml), oxyhemoglobin (20 µM) or SIN-1 (1 mM) as described under ``Experimental Procedures.'' Data are means ± S.E. of five experiments performed in duplicates. OxyHb, oxyhemoglobin.




DISCUSSION

Electrochemical detection with a Clark-type NO-sensitive electrode showed that reaction of NO with molecular oxygen followed second order kinetics with respect to NO. For the overall reaction we obtained a third order rate constant of 9.2 times 10^6M s, which is in good accordance to kinetic data published previously(22, 29, 30) . Determination of an NO:oxygen stoichiometry close to 4:1 confirms that NO autoxidation may follow a reaction sequence suggested recently(31) :

The major finding of the present study is that fairly low concentrations of the NOS cofactor H(4)biopterin induce rapid oxidation of NO. Mechanism of H(4)biopterin-mediated NO oxidation is apparently different from that of NO autoxidation, because it followed zero order instead of second-order kinetics with respect to NO. Thus, the rate-limiting reaction is independent of the actual NO concentrations and appears to solely involve H(4)biopterin and oxygen. Autoxidation of H(4)biopterin is thought to generate superoxide(32) , which again rapidly reacts with NO to form peroxynitrite(3) . Several observations led us to suggest that the effect of H(4)biopterin is mediated by superoxide anions. First, SOD completely blocked H(4)biopterin-induced oxidation of NO. Second, rate constants for NO oxidation in the presence of the pteridine correlated well with rates of H(4)biopterin autoxidation, and, finally, a superoxide generating system was found to mimic the effects of H(4)biopterin on NO oxidation kinetics (see Fig. 4).

Accordingly, superoxide generated during autoxidation of H(4)biopterin seems to react with NO to peroxynitrite, explaining the rapid, apparently zero order decrease in NO concentrations induced by H(4)biopterin. The fact that the H(4)biopterin-induced inactivation of NO was not apparent when either NOS-catalyzed NO production (28) or authentic NO (this study; not shown) were measured as loss of oxyhemoglobin may be explained by recent findings demonstrating NO-like reaction of peroxynitrite with oxyhemoglobin(33) . Thus, it is likely that peroxynitrite is produced from NO in the presence of H(4)biopterin, but direct demonstration of this was precluded by interference of the reduced biopterin with several photometrical assays for peroxynitrite (34) (not shown).

Our data suggest that H(4)biopterin reacts with oxygen to yield 5,6-H(2)biopterin (q-H(2)biopterin) and superoxide. If not efficiently removed by SOD, superoxide may combine rapidly with NO to peroxynitrite. In the presence of reducing compounds such as thiols or NADPH, q-H(2)biopterin is non-enzymatically reduced back to H(4)biopterin,^2 presumably explaining the apparent substoichiometrical activity of the pteridine on NO oxidation under these conditions. Since it is likely that generated peroxynitrite reacts with H(4)biopterin or one of its metabolites, the system consisting of NO, oxygen, and H(4)biopterin may be highly complex, and the pertinent reactions deserve detailed investigation.

The experiments with purified soluble guanylyl cyclase demonstrate that the products of H(4)biopterin-induced NO oxidation do not stimulate cGMP formation. In fact, H(4)biopterin proved to be a potent inhibitor of the NO-stimulated enzyme. Inhibition was observed between 0.1 and 10 µM H(4)biopterin, which is in the range of biopterin concentrations determined in tissue extracts (35) , suggesting that intact cells may express protective mechanisms preventing NO oxidation and/or convert peroxynitrite to a biologically active product. In support of latter hypothesis, evidence has been provided recently that peroxynitrite may act as vasodilator and inhibit platelet aggregation(36, 37) .

We have previously demonstrated that purified brain NOS, not saturated with H(4)biopterin, generates hydrogen peroxide(38, 39) , a reaction that involves production of superoxide anions as intermediates (40) . Since addition of exogenous H(4)biopterin also leads to superoxide generation, pteridine-deficient NOS may produce sufficient superoxide for NO inactivation both in the absence and presence of added H(4)biopterin, and this is supported by the experiments shown in Fig. 6. Thus, at low concentrations of H(4)biopterin (leq1 µM), NOS generates superoxide due to uncoupled oxygen activation(38) , whereas saturating concentrations of the pteridine, which prevent the enzymatic oxygen reduction, produce superoxide non-enzymatically. In each case, the amounts of generated superoxide are apparently sufficient for complete inactivation of NO, although we cannot exclude at this stage that another, as yet unidentified, source of oxygen radicals has contributed to NO inactivation under the experimental conditions used for this study.

Using NO-chemiluminescence and co-incubation with purified soluble guanylyl cylcase for detection of NO we have previously observed NO formation by purified brain NOS even in the absence (guanylyl cylcase assay) or presence of as little as 30 units/ml SOD (chemiluminescence) (24) . Of note, in this previous study H(4)biopterin concentration-response curves showed sharp maxima at sub-saturating concentrations of added H(4)biopterin (0.3-1 µM) in the chemiluminescence and cGMP assays but not in the citrulline assay. Accordingly, purified NOS may generate small amounts of free NO at low concentrations of added H(4)biopterin, and NO chemiluminescence as well as bioassays may be considerably more sensitive for NO than Clark-type electrodes.

Our data explain the conflicting reports on feedback inhibition of NOS (12, 13, 14, 15, 16, 17) , if the crude preparations used by others contained predominantly the H(4)biopterin-saturated holoenzyme and low concentrations of the free pteridine. When we blocked H(4)biopterin-induced oxidation of NO by addition of high concentrations of SOD, NOS-catalyzed citrulline formation was inhibited by 50%, and further increasing the NO concentrations by SIN-1 produced inhibition down to about 25% of controls. Lack of effect of SOD in the presence of the NO scavenger oxyhemoglobin indicates that the effect of SOD was indeed mediated by NO.

According to these data, NO could inhibit its own synthesis via interaction with the prosthetic heme group of NOS, but then it seems hard to understand how the enzyme works. For several reasons we think that the key in understanding NOS function is understanding the role of H(4)biopterin. NOS is unique as a pteridine-dependent cytochrome P-450. There must have been compelling reasons for evolution of H(4)biopterin as essential cofactor of NOS, especially in the light of the present data showing that the pteridine and NO hardly co-exist in the presence of oxygen. Recently, we compared binding of various pteridine derivatives with their effects on NOS activity and found that only tetrahydrobiopterins supported catalytic activity(41) . Thus, the role of H(4)biopterin may not be confined to an allosteric effect on NOS(42, 43) , but additionally involve a redox reaction. Considering the odd electron chemistry requisite for formation of the NO radical, it is tempting to speculate that the heme-catalyzed reaction rather yields NO or NO(44) , and that H(4)biopterin or q-H(2)biopterin are required for the eventual formation and release of NO. Such a function of H(4)biopterin would be in accordance with all experimental data available so far, including previous observations that the pteridine is not involved in NOS-catalyzed activation of molecular oxygen(38, 39) .

While this manuscript was under consideration, it has been reported that H(4)biopterin markedly diminished inhibition of rat cerebellar NOS by NO(45) . Griscavage et al.(45) claim that the effects of H(4)biopterin they have observed are not due to a direct chemical interaction between the pteridine and NO. Nevertheless, H(4)biopterin-induced oxidation of NO as described in the present study provides a reliable explanation for virtually all of their data.


FOOTNOTES

*
This work was supported by grants P 10098 (to B. M.), P 9685 (to E. R. W.), and P 9601 (NO and oxygen electrodes) 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. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This work is dedicated to Prof. Dr. Eycke Böhme(1943-1993).

§
To whom correspondence should be addressed: Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria. Tel.: 43-316-380-5567; Fax: 43-316-323-54-14.

(^1)
The abbreviations used are: NOS, NO synthase; DEA/NO, 2,2-diethyl-1-nitroso-oxyhydrazine sodium salt; GSNO, S-nitrosoglutathione; H(4)biopterin, (6R)-5,6,7,8-tetrahydro-L-biopterin; H(2)biopterin, 7,8-dihydro-L-biopterin; q-H(2)biopterin, 5,6-dihydro-L-biopterin; SIN-1, 3-morpholino-sydnonimine; HPLC, high performance liquid chromatography; SOD, superoxide dismutase.

(^2)
E. R. Werner, unpublished observation.


ACKNOWLEDGEMENTS

We wish to thank Eva Leopold for excellent technical assistance.


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