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J Biol Chem, Vol. 274, Issue 42, 29755-29762, October 15, 1999

Reactivity of Tetrahydrobiopterin Bound to Nitric-oxide Synthase^*

Cor F. B. Witteveen, John Giovanelli, and Seymour Kaufman

From the Laboratory of Neurochemistry, National Institute of Mental Health, Bethesda, Maryland 20892-4096

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Levels of tetrahydrobiopterin (BH₄) bound to nitric-oxide synthase (NOS) were examined during multiple turnovers of the enzyme in the presence of an NADPH-regenerating system. Our findings show that NOS-bound BH₄ does not remain in a static state but undergoes redox reactions. Under these experimental conditions, the redox state of BH₄ was determined by the balance between calcium/calmodulin (Ca²⁺/CaM)-dependent oxidation of BH₄ mediated by the uncoupled formation of superoxide/hydrogen peroxide on the one hand and by reductive regeneration of BH₄ on the other hand. BH₄ oxidation was appreciably increased in the presence of arginine. Levels of NOS-bound BH₄ were also examined under single turnover conditions in the absence of an NADPH-regenerating system and in the presence of added superoxide dismutase and catalase to suppress the accumulation of superoxide and hydrogen peroxide. BH₄ oxidation was again dependent on Ca²⁺/CaM. The insensitivity to superoxide dismutase and catalase suggested that the single turnover oxidation of BH₄ did not proceed through superoxide/peroxide, although the involvement of these oxidants could not be definitively excluded. The amount of BH₄ oxidized was highest in the presence of arginine, and this oxidation significantly exceeded that in the presence of N^G-hydroxy-L-arginine. The findings that single turnover oxidation of BH₄ is stimulated by arginine in the presence of Ca²⁺/CaM and that BH₄ is regenerated are consistent with a role for the pterin as an electron donor in product formation; this role remains to be defined.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Nitric-oxide synthase (NOS)¹ catalyzes the overall conversion of arginine to nitric oxide (NO) and citrulline. The reaction proceeds in two steps; arginine is first oxygenated to the intermediate N^G-hydroxy-L-arginine (NHA), which is then oxygenated to the products NO and citrulline (reviewed in Ref. 1). The enzyme has attracted much interest because of its involvement in a variety of physiological processes such as vasodilation, cytotoxic activity, and neurotransmission (2, 3). NOS is unique as a heme oxygenase in requiring BH₄, and elucidation of the role of BH₄ is essential in understanding how this enzyme functions.

It is well established that BH₄ has pronounced effects on the structure of NOS. These effects include the ability to shift the heme iron to its high spin state, promotion of arginine binding, and stabilization of the active dimeric form of the enzyme (reviewed in Ref. 4), as well as stabilization of the ferrous heme iron coordination structure (5). Furthermore, binding of the first molecule of BH₄ to an NOS dimer has an anticooperative effect on the binding of the second molecule of BH₄ (6). Studies of the effects of pterin analogues on NOS provide graphic evidence that these structural effects cannot fully explain the requirement for BH₄. Whereas the structural effects of BH₄ are mimicked by a series of pterin analogues independent of their oxidation state, pterins must be in the tetrahydro state in order to support NO synthesis (5, 7, 8). This requirement for a tetrahydropterin suggests an additional, redox, role of BH₄.

A number of recent reports support a redox role of BH₄ in which the pterin provides electrons for the oxygenation reactions catalyzed by NOS. Studies with recombinant BH₄-free NOS (9) have implicated BH₄ as acting as a stoichiometric reactant in the first oxygenation step (arginine to NHA) of NO synthesis. The role of BH₄ in this reaction is envisioned by Rusche et al. (9) as being analogous to the well established role of BH₄ in the aromatic amino acid hydroxylase systems (10), except that BH₄ remains tightly bound to NOS throughout the catalytic cycle (11). Further support for this proposal is provided by the finding that non-heme iron, which is an integral component of the aromatic amino acid hydroxylases (10), is a stoichiometric component of NOS and enhances its activity (12). Again by analogy with the hydroxylation of the aromatic amino acids (10), the oxidation of BH₄ by NOS would generate equivalent amounts of NHA and quinonoid dihydrobiopterin (qBH₂), and the latter compound would need to be recycled to BH₄ by a dihydropterin reductase-like activity in order to maintain the catalytic function of BH₄. In these same studies, Rusche et al. (9) further proposed a role for BH₄ in the second oxygenation step (NHA to citrulline and NO). It was suggested (9) that BH₄ may be acting as an electron donor in this step or in an indirect fashion by affecting, for example, the spin equilibrium and reduction potential of the heme.

Further support for a redox role of BH₄ is provided by the single turnover studies of NOS (13). It was suggested that BH₄ plays a critical role in the first oxygenation step by supplying an electron for reducing the ferric heme superoxide species to the ferric heme peroxo species. Based largely on studies of cytochrome P-450, the latter species, or a closely related form, is believed to carry out the oxygenation of arginine (1, 14). Since only one electron is required for reduction of the ferric heme superoxide species to the ferric heme peroxo species by BH₄, the oxidized pterin product was formally represented as the trihydrobiopterin radical (BH₃^·), although the possibility of qBH₂ being the product could not be excluded. Based on crystallographic studies and the unexpected finding that arginine recognizes the BH₄-binding site, Raman et al. (15) suggest that this site may stabilize the positively charged pterin cation radical, thereby supporting a scheme in which electrons are shuttled between the protonated forms of BH₄ and BH₃^·, rather than between BH₄ and qBH₂. It has recently been reported (16) that the 5-methyl analogue of BH₄ is functionally active with NOS but does not react with oxygen or stimulate phenylalanine hydroxylase. It is suggested (16) on the basis of these studies that the redox chemistry of BH₄ is not the same in NOS as it is in the aromatic amino acid hydroxylases, but it could not be excluded that BH₄ interacts with the heme or another metal group of NOS through an as yet unrecognized redox chemistry.

Since catalytic amounts of BH₄ are sufficient for NOS activity (11), a minimum requirement for any role of BH₄ as a reductant is that the oxidized pterin products be regenerated with each catalytic cycle by a dihydropterin reductase-like activity intrinsic to the enzyme. No recycling of exogenous BH₄ was detected during NOS turnover (11), but this study did not exclude the possibility of recycling of BH₄ tightly bound to the enzyme (17). The reduction of added qBH₂ to BH₄ by NOS, probably by the flavoprotein "diaphorase" activity of the enzyme, has been demonstrated (18), but the significance of this finding to the essential role of BH4 was uncertain. Furthermore, under the standard assay conditions of this latter study, any regenerated BH₄ that remains firmly bound to NOS would not have been detected, since any such regeneration is limited to the number of BH₄-binding sites; these sites are negligible relative to the observed amounts of qBH₂ reduced.

The present work provides experimental support for a redox role of BH₄ by showing that NOS-bound BH₄ can cycle with its oxidized form(s). Although details of the oxidation of BH₄ remain to be clarified, this work is an important step toward defining the mechanistic significance of this reaction in NO formation. The regeneration of BH₄ from its oxidized products is probably important in maintaining the pterin in its active, tetrahydro, state.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chemicals-- The following materials were obtained from the sources shown in parentheses: N^G-nitro-L-arginine (NNA) and L--lysolecithin (Sigma), CaM from bovine brain (Calbiochem), BH₄ (B. Schircks Laboratories, Jona, Switzerland), L-[2,6-³H]phenylalanine, L-[2,3,4,5-³H]arginine, and L-[U-¹⁴C]tyrosine (Amersham Pharmacia Biotech), and NHA (Alexis Corp., San Diego, CA),

Enzymes-- Beef liver catalase was obtained from Roche Molecular Biochemicals. Superoxide dismutase (SOD) from bovine erythrocytes and glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides were obtained from Sigma. Recombinant rat phenylalanine hydroxylase was prepared by minor modification (19) of the procedure of Citron et al. (20) and had a specific activity of approximately 2 µmol/min/mg. Rat brain NOS was purified from transfected human kidney 293 cells (21). NOS isolated in this way showed a single band on an SDS-polyacrylamide gel and typically had a specific activity of 1.0-1.6 µmol/min/mg as determined by citrulline formation over a 10-min incubation at 37 °C with the assay of Bredt and Snyder (22). Molar concentrations of NOS are reported for the 160-kDa subunit. The BH₄ content of the purified NOS was typically 0.5 mol/mol subunit as determined by high performance liquid chromatography (HPLC) analysis of oxidized samples (23). Protein was determined by the bicinchoninic acid method (Pierce manual 23225X) with the use of a bovine serum albumin standard.

BH₄ Levels during Turnover of NOS-- Reaction mixtures used for these studies contained 1 µM NADPH and an NADPH-regenerating system. This low concentration of NADPH was used to prevent significant non-enzymic regeneration of BH₄ that occurs at higher concentrations of NADPH (18, 24). We have previously shown (18) that NOS has normal activity using this low concentration of NADPH. The standard reaction mixture contained 2 µM NOS, 1 µM NADPH, 1.5 mM glucose 6-phosphate, 32 µg/ml glucose-6-phosphate dehydrogenase, 35 mM sodium Bicine (pH 7.6), 0.9 mM CaCl₂, and 0.6 mM EDTA. The following components were also added as indicated: 4 µM CaM, 800 µg/ml SOD, 16 µg/ml catalase, 50 µM arginine or NHA, and 200 µM NNA. In some experiments, reaction mixtures were supplemented with a concentration of BH₄ equal to one-half the concentration of NOS monomer. In this way, native NOS containing approximately 0.5 BH₄/NOS monomer was reconstituted to approach a value of 1/NOS monomer.² Incubations were at 15 °C. Samples of 25 µl were taken to establish the initial levels of BH₄. Immediately thereafter, CaM was added, where appropriate, in order to start the reaction, and samples of 25 µl were taken at fixed times for BH₄ analysis. As illustrated under "Results," negligible oxidation of BH₄ occurred in the absence of CaM.

BH₄ Levels under Single Turnover Conditions-- Two different procedures were followed depending on whether only BH₄ levels were determined or whether BH₄ levels together with NHA were determined.

The standard reaction mixture in which only BH₄ levels were determined contained 1.7 µM NOS, 0.9 mM CaCl₂, 0.6 mM EDTA, 400 units/ml SOD, 8 µg/ml catalase, and 50 mM sodium Hepes (pH 7.4). Where indicated, NOS was reconstituted with BH₄ as described above to contain approximately 1 BH₄/NOS subunit. Immediately prior to the start of the reaction, NADPH was added to the standard reaction mixture (1 NADPH per NOS subunit), and the mixture preincubated for 2.25 min. This procedure, which is fully described in a separate section below, generated a maximum level of NOS-bound flavin semiquinone. Separate experiments showed that BH₄ was stable during this preincubation period. After the preincubation, samples were taken to establish initial BH₄ levels. Immediately thereafter, the indicated additions of 4 µM CaM, 50 µM arginine, or 50 µM NHA were made, and 25 µl samples for BH₄ analysis removed at 30, 60, 120, and 180 s.

For determination of both BH₄ and NHA, reaction mixtures initially contained 3.4 µM NOS, 15 µM L-arginine, 0.9 mM CaCl₂, 0.6 mM EDTA, 800 units/ml SOD, 16 µg/ml catalase, and 50 mM sodium Hepes (pH 7.4). During the preincubation period with 3.4 µM NADPH (see below), part of the reaction mixture to be used for determination of NHA was transferred to a tube containing L-[³H]arginine, resulting in an increase in the arginine concentration to 18.4 µM. In the other tube containing the remainder of the reaction mixture, unlabeled arginine was added to achieve the same final concentration of 18.4 µM arginine; this tube was used for determination of BH₄ levels. Immediately after samples were taken to establish initial levels of BH₄ and NHA, CaM was added to a final concentration of 4.7 µM to each tube, and 25-µl samples were removed at the intervals shown above for determination of NHA formation and BH₄. All additions were made in very small volumes so as not to significantly change the specified concentrations of reaction components.

All incubations were at 15 °C.

Determination of BH₄ Oxidation-- BH₄ was determined by coupling this pterin to the phenylalanine hydroxylase-catalyzed conversion of an equivalent amount of [³H]phenylalanine to [³H]tyrosine. Reproducibility of the original method (18) was improved by the modifications described below. To the 25-µl samples taken from reaction mixtures, 5 µl of 1.5 M HClO₄ was added immediately to stop the reaction, release BH₄ from NOS, and stabilize the pterin in its reduced form. Within 4 min after adding the acid, 5 µl KHCO₃ was added to partially neutralize the mixture to approximately pH 2.5-3.0. The precipitate of KClO₄ was removed by a 10-s centrifugation, and 30 µl of the supernatant solution was added to 9.6 µl of a solution containing the remaining reaction components. The final reaction mixture contained, in addition to the BH₄ to be assayed, 1 µg of phenylalanine hydroxylase, 4.8 µM L-[³H]phenylalanine (6 µCi), 150 mM potassium phosphate (pH 6.5), 0.1 mM lysolecithin to activate phenylalanine hydroxylase, 0.12 mg/ml catalase, and 333 units/ml SOD. Incubation was for 30 min at 30 °C. The reaction was stopped by adding 25 µl of a solution containing 273 mM HClO₄, 0.73 mM L-phenylalanine, and 0.73 mM L-[¹⁴C] tyrosine (30 nCi), the last component being added to correct for losses during subsequent purification of radioactive tyrosine by HPLC. HPLC was performed on a Beckman Ultrasphere ODS column (C-18, 4.6 mm internal diameter × 150 mm, 5-µm particles) eluted isocratically with 5 mM sodium acetate buffer (pH 4.8). The ratio of [³H] to [¹⁴C] in tyrosine was used to calculate the amount of [³H]tyrosine formed. For each experiment a separate BH₄ calibration curve was established with increasing amounts of NOS either unsupplemented or supplemented with BH₄. The BH₄ content of NOS samples used for calibration was determined independently by the method of Fukushima and Nixon (23).

Single Turnover Oxygenation of Arginine to NHA-- The formation of NHA in the absence of added NADPH was measured by the incorporation of ³H from L-[³H]arginine into NHA (25).

Generation of FSQ by Addition of NADPH-- The air-stable NOS-bound flavin semiquinone free radical, FMNH^· (FSQ) was generated immediately before use by preincubation of NOS for 2.25 min in air with NADPH (26). One mol of NADPH (two electron eq) per mol of NOS subunit was required to obtain the maximum level of FSQ (reduced at the level of one electron eq), presumably to compensate for reaction of reduced flavin species with oxygen during the titration. Total reduced flavins were measured by the decrease in absorbance at 458 nm and FSQ by the increase in absorbance at 590 nm (27). All absorbance values were determined with a Hewlett-Packard 8453 diode array spectrophotometer. Addition of NADPH typically resulted in an instantaneous decrease in absorbance at 458 nm, followed by a slow increase in absorbance (first order rate constant of 2.6 min¹) over the next 2 min to approach a constant value approximately midway between the minimal and initial absorbance values. Parallel measurements at 590 nm showed a slow increase in absorbance throughout the incubation period (first order rate constant of 3.4 min¹) to approach a constant value. The combined results are interpreted as reflecting a rapid reduction of the flavins (presumably by formation of FADH₂ and subsequently FMNH₂) followed by formation of a stable FMNH^· species resulting from the partial oxidation of FMNH₂ by oxygen. These spectral data are consistent with values 0.4-0.6 FMNH^· per NOS subunit estimated from the amount of NHA formed per NOS subunit (21). Gachhui et al. (27) similarly observed approximately 0.5 FMNH^· per reductase domain of NOS resulting from titration of this domain with NADPH in air.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Levels of NOS-bound BH₄ were examined under three conditions of continuous turnover. In these experiments, continuous turnover of neuronal NOS (nNOS) was achieved in the presence of a low concentration (1 µM) of NADPH and an NADPH-regenerating system. Clearly, in the absence of arginine, no NO is formed. Under this fully uncoupled condition, electrons are transferred from NADPH to oxygen with the exclusive formation of superoxide and peroxide (6, 28-30). In the presence of arginine, but no added BH₄, nNOS is partially coupled, resulting in the formation of both NO and superoxide/peroxide (28, 30). Maximum coupling of NADPH oxidation to NO formation is approached in the presence of saturating concentrations of both arginine and BH₄ (6, 28). However, complete coupling of electron transfer from NADPH to NO formation in the presence of arginine and BH₄ would not be expected under our conditions. This is because it appears that some superoxide is formed even in the presence of saturating arginine and BH₄ (31), and the addition of BH₄ in our studies resulted in incomplete saturation² of NOS with BH₄.

BH₄ Levels during Fully Uncoupled Turnover of NOS-- Fig. 1 illustrates representative results for the levels of NOS-bound BH₄ in the absence of added BH₄ and arginine. In the absence of CaM, the level of BH₄ remains constant (curve 1). Addition of CaM results in approximately 40% oxidation of BH₄ (curve 2). CaM-dependent oxidation of BH₄ is largely prevented by the additional presence of SOD and catalase (curve 3). Furthermore, the CaM-dependent oxidation of BH₄ proceeding over an 80-s period (curve 4, open circles) is partially reversed by addition of SOD and catalase, in combination with NNA (curve 4, closed circles). NNA is a potent inhibitor of heme-catalyzed reduction of oxygen by neuronal NOS and the consequent formation of superoxide/hydrogen peroxide and nitric oxide (28, 29, 32, 33). Regeneration of BH₄ did not occur in the absence of NNA. These combined findings strongly suggest that BH₄ is oxidized by superoxide and/or peroxide generated by NOS during the uncoupled reduction of oxygen by NADPH and that regeneration of BH₄ (possibly from qBH₂) proceeds when the concentration of superoxide/peroxide is decreased to a low level in the combined presence of SOD, catalase, and NNA.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   BH4 levels during fully uncoupled turnover of NOS. , curve 1, no additions to the standard reaction mixture; , curve 2, addition of CaM; , curve 3, addition of CaM, SOD, and catalase; curve 4, addition of CaM (), followed by further addition of SOD, catalase and NNA after 80 s (). No measurements were taken during the period indicated by the broken line. The standard reaction mixture and concentration of all components are described under "Experimental Procedures." BH4 levels are expressed as a percentage of the initial values.

Effects of Supplementation with Arginine on BH₄ Levels during Turnover of NOS-- Fig. 2 compares the BH₄ levels of NOS under partially coupled conditions (presence of supplemental arginine) with those described above under fully uncoupled conditions (Fig. 1). Curves 1 and 2 of Fig. 2 confirm the requirement of CaM for BH₄ oxidation. BH₄ levels are largely stabilized by addition of NNA at the beginning of the incubation (curve 3). In the presence of CaM, addition of arginine markedly increased the oxidation of BH₄ from 40% (curve 2) to 80-90% (curve 4). Oxidation under the latter conditions (arginine and CaM) was greatly attenuated by addition of SOD plus catalase (curve 5), and partially reversed by addition of SOD, catalase, and NNA (curve 6, closed circles versus open circles), indicating that sufficient superoxide/peroxide is generated even in the presence of supplemental arginine to cause extensive oxidation of BH₄.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Effects of supplemental arginine on BH4 levels during NOS turnover. Conditions are the same as in Fig. 1. , curve 1, no additions to the standard reaction mixture; , curve 2, addition of CaM; , curve 3, addition of CaM and NNA; , curve 4, addition of CaM and arginine; , curve 5, addition of CaM, arginine, SOD, and catalase; curve 6, addition of CaM and arginine (), followed by further addition of SOD, catalase, and NNA after 80 s (). No measurements were taken during the period indicated by the broken line.

Effects of Supplementation with Arginine and BH₄ on BH4 Levels during Turnover of NOS-- Since maximum coupling is approached in the combined presence of arginine and BH₄ (6, 28), it was therefore of interest to determine the effect of adding BH₄ to the arginine-supplemented reaction mix used in the experiment described in Fig. 2. The results shown in Fig. 3 confirm that little or no oxidation of NOS-bound BH₄ occurs in the absence of CaM (curve 1) and further show that similar stability of BH₄ is observed in the presence of supplemental BH₄ (curve 2). CaM-dependent oxidation is increased marginally by supplementary BH₄ (curve 4 versus curve 3). In the presence of arginine, supplementary BH₄ initially protects BH₄ against oxidation (curve 6 versus curve 5), but this protective effect is lost as the incubation progresses. These results show that even in the presence of arginine and BH₄, conditions that favor the coupled formation of NO, BH₄ is oxidized ultimately to the same low level as observed with arginine alone.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Effects of supplemental BH4 and arginine on BH4 levels during NOS turnover. Native NOS containing 0.5 BH4/NOS subunit was reconstituted with supplemental BH4 to a value approaching 1 BH4/NOS subunit (see "Experimental Procedures"). BH4 levels are expressed as percentages of the initial total BH4 content. Thus, although the amount of initial total BH4 in the BH4-supplemented reaction mixtures is twice that of the unsupplemented reaction mixtures, there is also almost twice as much NOS containing bound BH4. This representation allows direct approximation of relative BH4 levels based on the corresponding amount of BH4-bound NOS. Conditions are the same as those described in Fig. 1 except for the absence or presence, respectively, of supplemental BH4:  and  curves 1 and 2, no additions;  and , curves 3 and 4, plus CaM;  and , curves 5 and 6, plus CaM and arginine.

BH₄ Levels during Single Turnover of NOS-- In the studies reported above, reaction mixtures contained an NADPH-regenerating system that provides a source of electrons for both the superoxide/peroxide-mediated oxidation of BH₄ and, as demonstrated in the presence of SOD plus catalase plus NNA, the regeneration of BH₄ from its oxidized products. The effects noted above therefore represent steady-state changes determined by the amount of oxidation of BH₄ on the one hand and regeneration of BH₄ on the other. Clearly, in order to detect any oxidation of BH₄ that is stoichiometrically coupled with the overall oxygenation of arginine to NO, it is necessary to prevent regeneration of BH₄ from its oxidized products. We therefore examined BH₄ levels of NOS during the single turnover of arginine to NHA and of NHA to citrulline in the absence of an NADPH-regenerating system; SOD/catalase were included in the reaction mixtures to minimize any oxidation of BH₄ by superoxide/peroxide.

Fig. 4 illustrates results of experiments to determine the effects of arginine and NHA on BH₄ oxidation. The possible trace amount of BH₄ oxidation in the absence of CaM (curve 1) could reflect the inherent instability of BH₄ under these conditions. Addition of CaM (curve 2) or CaM plus NHA (curve 3) caused a small increase in the oxidation. Maximal BH₄ oxidation was observed in the presence of CaM plus arginine (curve 4). Higher levels of SOD/catalase added to the reaction mixture of curve 2 resulted in similar results (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   BH4 levels under single turnover conditions. NOS was preincubated with NADPH to maximize the level of FSQ, and reaction mixtures were supplemented with BH4. Details are given under "Experimental Procedures." , curve 1, no additions; , curve 2, addition of CaM; , curve 3, addition of CaM and NHA; , curve 4, addition of CaM and arginine. Error bars indicate standard deviation of at least three experiments.

The relationship between BH₄ oxidation and oxygenation of arginine to NHA under single turnover conditions is illustrated in Fig. 5. NHA, the immediate product of arginine oxidation, was the dominant product; citrulline formation was negligible (data not shown). The ratio of BH₄ oxidized to NHA formed was not constant. It may be estimated from the results of Fig. 5 (inset) that this ratio increased with time from 1.0 at 30 s to 1.7 at 180 s. In this experiment, NOS was not supplemented with BH₄. Variable ratios were also observed in a similar experiment in which NOS was supplemented with 1 mol of BH₄ per mol of NOS subunit. Under this condition the ratio of BH₄ oxidized to NHA formed was 3.1 at 30 s and progressively decreased to 1.8 at 180 s (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 5.   BH4 levels and NHA formation under single turnover conditions. NOS was preincubated with NADPH to maximize the level of FSQ. Reaction mixtures, which were not supplemented with BH4, are described under "Experimental Procedures." , values for BH4; , values for NHA.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

BH₄ Oxidation during Continuous Turnover-- Under continuous turnover conditions in the presence of an NADPH-regenerating system (absence of arginine and BH₄), oxidation of NOS-bound BH₄ is dependent upon the presence of Ca²⁺/CaM and is prevented by SOD/catalase and by NNA. Furthermore, BH₄ can be regenerated upon addition of SOD/catalase and NNA. These observations are consistent with the oxidation of NOS-bound BH₄ being mediated by superoxide/peroxide, formed during the uncoupled reduction of oxygen by heme-mediated transfer of electrons from NADPH (6, 28-30). The oxidation state of NOS-bound BH₄ appears to be determined by a balance between BH₄ oxidation by superoxide/peroxide on the one hand, and reductive regeneration of BH₄ on the other.

For nNOS, added arginine and BH₄ are known to increase the coupling of oxygen reduction to NO formation at the expense of superoxide/peroxide formation (28, 30). We therefore expected that the presence of arginine, especially in the presence of added BH₄, would decrease the amount of BH₄ oxidation. Instead, arginine appreciably increased BH₄ oxidation (Fig. 2, curve 4 versus curve 2; Fig. 3 curve 5 versus curve 3). The combined presence of arginine and added BH₄ initially delayed BH₄ oxidation (Fig. 3, curve 6), but ultimately BH₄ was oxidized to the same low level as observed in the presence of arginine alone. A likely interpretation of these effects is that arginine facilitates the reduction of heme iron (34). Bearing in mind that some superoxide is produced by nNOS even at saturating concentrations of arginine and BH₄ (31) and that NOS was not completely saturated² with added BH₄, an increase in heme reduction can lead not only to an increase in (coupled) NO formation but also to an increase in (uncoupled) formation of superoxide/peroxide and resultant increase in BH₄ oxidation. Peroxynitrite can also be formed by reaction of superoxide and NO (35). In the combined presence of arginine and BH₄ (Fig. 3, curve 6), it is envisioned that the oxygenation at early times is largely coupled to NO formation, thereby limiting the formation of superoxide and peroxynitrite required for BH₄ oxidation. As the incubation proceeds, formation of superoxide/peroxynitrite and oxidation of BH₄ resume, as the level of BH₄ is decreased to a level that is no longer effective in coupling.

It is not entirely clear from these studies whether the increased oxidation of BH₄ in the presence of arginine reflects a donation of electrons by BH₄ for arginine oxygenation. The conditions of continuous turnover of NOS in this experiment, in which BH₄ is in a dynamic redox state, are not optimal for assessing such a role for BH₄; this role of BH₄ is examined below in a more rigorous manner under single turnover conditions. It is emphasized again that the arginine-stimulated oxidation of BH₄ under continuous turnover conditions is sensitive to SOD/catalase. If SOD/catalase-sensitive oxidation of BH₄ is an essential step in arginine oxygenation,³ product formation by NOS would be expected to show a similar sensitivity. No such inhibition of NOS by SOD has been observed (36-41).⁴ Limited reports in the literature on the effects of catalase on NOS are conflicting. A preliminary abstract (42) claimed that NO formation (measured by the conversion of oxyhemoglobin to methemoglobin) catalyzed by NOS purified from porcine brain was catalase-dependent. On the other hand, Mittal (38) reported that NO formation (detected by activation of guanylate cyclase) catalyzed by crude extracts of rat brain was abolished by catalase. In neither of these studies did catalase affect citrulline formation, in agreement with reports by other workers (11, 41, 43).

Other explanations for the effect of arginine during continuous turnover of NOS are considered as follows.

(i) It is possible that the presence of arginine increases BH₄ oxidation by increasing the formation of peroxynitrite at the expense of superoxide. As explained above, whereas superoxide is the major product of oxygen reduction in the absence of arginine, the presence of arginine allows both NO and superoxide to be formed. The latter two compounds can react rapidly with each other to form peroxynitrite (35). Peroxynitrite is generally considered to be a more potent oxidant under physiological conditions than is superoxide or NO (35). However, preliminary studies⁵ on the oxidation of 1 mM BH₄ in phosphate buffer (pH 7.6) showed comparable potencies for superoxide and peroxynitrite; hydrogen peroxide was relatively inactive, exhibiting an oxidation rate barely exceeding that of autoxidation by oxygen. These observations argue against an increased formation of peroxynitrite in the presence of arginine causing the increased oxidation of NOS-bound BH₄. It should be noted, however, that the rank order of potency determined in free solution may not be identical to that determined for NOS-bound BH₄.

(ii) It is possible that the increased net oxidation of BH₄ in the presence of arginine can be explained by the nature of the BH₄ oxidation products and their recycling to BH₄. The nature of the product (BH₃^· or qBH₂) could be affected by whether BH₄ is oxidized by the obligate one electron oxidant, superoxide (44), or by peroxynitrite, which can catalyze one or two electron oxidations (45). Possible ways in which each of these oxidized pterin products could be reduced to BH₄ are discussed below.

BH₄ Oxidation under Single Turnover Conditions-- In the single turnover system employed in the current work, it was previously shown that FSQ can provide all the electrons required for oxygenation of both arginine and NHA (21). Our NOS preparations contain only one FSQ per NOS dimer, thereby raising the problem of explaining the mechanism of delivery of the second electron required for arginine oxygenation (21). These considerations suggested, and our preliminary findings did not exclude, the possibility that compounds such as BH₄ might provide the second electron for arginine oxygenation by reduction of the ferric superoxide heme species to the ferric peroxo heme species (21). A similar proposal has recently been made by Bec et al. (13) based on the results of single turnover studies of the oxygenation of arginine to NHA supported by the ferrous oxygen complex of nNOS.

It should be noted that the current single turnover experiments were carried out under conditions that optimized the ability to detect any coupling of the oxidation of BH₄ to arginine oxygenation. Thus, any regeneration of BH₄ from qBH₂ was suppressed by omission of an NADPH-regenerating system from the reaction mixture. Furthermore, SOD and catalase were included in the reaction mixture to minimize any oxidation of BH₄ arising from the accumulation of ROS (superoxide and hydrogen peroxide). SOD and catalase were considered the best scavengers of ROS to be employed in our single turnover studies, for the following reasons. (i) It is generally believed that superoxide is a critical component of ROS and that SOD provides the primary defense mechanism, with catalase and the glutathione peroxidase/glutathione system sharing the task of converting hydrogen peroxide to water and oxygen (46-48). As explained below, it was not feasible to include both catalase and the glutathione peroxidase/glutathione in our single turnover studies. (ii) SOD and catalase are quite specific for superoxide and peroxides, respectively, and are not known to catalyze reactions unrelated to their intended use. (iii) SOD and catalase resulted in efficient, although possibly not complete, scavenging ROS in our studies, even under the extreme conditions that favor the formation of superoxide and other ROS (Figs. 1 and 2). (iv) As previously explained in the discussion of BH₄ oxidation during continuous turnover, available evidence indicates that neither SOD nor catalase inhibits arginine oxygenation. (v) SOD and catalase do not interfere with the assay of BH₄ since they are removed from the reaction mixture by precipitation with perchloric acid. Furthermore, SOD and catalase do not interfere with any oxidation of BH₄ coupled to arginine oxygenation. This is not so with a number of other widely used antioxidants. For example, the antioxidants ascorbate and thiols such as glutathione regenerate BH₄ from qBH₂ (24, 49, 50) and could thus lead to an underestimation of the amount of oxidation of BH₄ under single turnover conditions. Similarly, ferricytochrome c could not be used as a scavenger of superoxide, because its oxidation of BH₄ (51) under our experimental conditions could not be excluded; such a reaction would lead to an overestimation of any oxidation of BH₄ coupled to arginine oxygenation. Various nitroxides act as mimics of SOD, and their excellent cell permeability has led to their extensive use as superoxide scavengers in biological systems. These compounds are not an appropriate substitute for SOD in our studies because the rate constants for reaction of superoxide with these nitroxides are generally smaller by 3-5 orders of magnitude than that with SOD (52).

The significantly greater oxidation of NOS-bound BH₄ under single turnover conditions in the presence of arginine (Fig. 4, curve 4) relative to NHA (Fig. 4, curve 3) is indeed consistent with a scheme in which BH₄ provides an electron for arginine oxygenation but is not required for the one electron-requiring oxygenation of NHA (53). A molar ratio for BH₄ oxidized/NHA formed of 0.5 is expected if the two-electron oxidation of BH₄ to qBH₂ supplied only the second electron for NHA formation, and a ratio of 1.0 if BH₄ provided both electrons for NHA formation. The observed molar ratio was not constant but increased from 1.0 to 1.7 as the reaction progressed (Fig. 5). This indicates that NOS-bound BH₄ is able to provide all the electrons for oxygenation of arginine to NHA. However, other pathways for BH₄ oxidation must operate in order to explain the high molar ratios of BH₄ oxidized per NHA formed at later incubation times and the significant oxidation of BH₄ in the absence of arginine (Fig. 4, curve 2). Despite the efficiency of SOD and catalase in scavenging ROS, it is important to note that a contribution of ROS in the single turnover oxidation BH₄ cannot be definitively excluded.

Implications of the Redox Reactions of NOS-bound BH₄-- The cycling between BH₄ and its oxidized product(s) reported here is of major significance in that it provides experimental support for a redox role of NOS-bound BH₄ and provides important insights toward defining the mechanistic details of this redox role of BH₄. The possible significance of the oxidation of NOS-bound BH₄ and regeneration of the oxidized pterin products to BH₄ is discussed separately below.

Our combined results are consistent with, but do not differentiate between, the two mechanistic roles recently proposed for BH₄ oxidation: as a reductant of oxygen (9), analogous to its role in the aromatic amino acid hydroxylases (10), and as an electron donor for reducing the ferric heme superoxide species to the ferric heme peroxo species in the oxygenation of arginine to NHA (13). Two additional roles of BH₄ oxidation that are consistent with our findings should be mentioned. The first is based on the report that the basal activity of NOS (measured in the absence of added BH₄) is positively correlated with the amount of bound BH₄ (6, 54). Oxidation of NOS-bound BH₄ may therefore act as a switch in shutting off the formation of toxic levels of peroxynitrite, formed by reaction of NO with superoxide, that results in a variety of pathophysiological conditions (55, 56). Whereas superoxide could continue to be formed by the BH₄-depleted enzyme (6, 28, 57), studies of the injury that occurs during postischemic myocardial reperfusion, for example, indicate that peroxynitrite was the oxidant species responsible (56). The second suggested role for NOS-bound BH₄ oxidation is that the reaction is coupled to the conversion of some group(s) on NOS to the reduced form required for activity (11). A precedent for this model is the reduction by BH₄ of the inactive ferric form of phenylalanine hydroxylase to the active ferrous form (58, 59). Our current findings could reflect an analogous Ca²⁺/CaM  dependent reduction by BH₄ of some group(s) on NOS that must be in the reduced form for catalytic activity.

Regardless of the specific role of electron donation by BH₄, regeneration of BH₄ from its oxidized product(s) probably plays a critical role in maintaining the pterin in its active, reduced, form and provides experimental support for the suggestion that BH₄ recycling is involved in the higher and more linear rates of NO formation in the presence of a BH₄-regenerating system such as dihydropteridine reductase or reducing thiols (60-62). Unless qBH₂ is effectively reduced to BH₄, it rapidly rearranges to 7,8-dihydrobiopterin (63, 64). The latter compound is not reduced by NOS to BH₄ (18), resulting in a net loss of BH₄ from the enzyme.

The present work opens up a number of promising lines of research in clarifying the redox role of BH₄. For example, neither the products of BH₄ oxidation nor the mechanism of regeneration of BH₄ from these products is understood. qBH₂ is the usual product of enzymic oxidation of BH₄, and regeneration of BH₄ from qBH₂ is catalyzed by dihydropteridine reductase (65). Regeneration of BH₄ by purified NOS proceeds in the absence of any added reductase, and must therefore be catalyzed by an inherent reductase activity of NOS. NOS does indeed catalyze the reduction of qBH₂ (18)_. However, a number of observations cast doubt on the physiological significance of this reduction. For example, very high concentrations of qBH₂ were required to achieve rates that are comparable to those of NO synthesis, and the quinonoid form of 6-methyldihydropterin was more active than qBH₂, whereas the product of this reduction, 6-methyltetrahydropterin, shows relatively little activation of NOS (18). An alternate proposal is that BH₄ cycles with the free radical, BH₃^· (13, 15). There is no precedent for the enzymic reduction of BH₃^·. Possible mechanisms include direct reduction of BH₃^· to BH₄ (see Equation 1) (15) or the conversion of BH₃^· to qBH₂ and BH₄ by dismutation (see Equation 2) (66). Alternatively, BH₃^· can react with oxygen to form qBH₂ and hydrogen peroxide (H₂O₂) (Equation 5, the sum of Equations 3 and 4) (67). Quinonoid BH₂ formed in Equations 2-5 could be further reduced to BH₄ by an inherent dihydropterin reductase activity of NOS.

(Eq. 1)

(Eq. 2)

(Eq. 3)

(Eq. 4)

(Eq. 5)

It is noteworthy that, at least under our particular continuous turnover conditions in the presence of arginine, NOS appears to have a limited capacity for regeneration of BH₄ in relation to BH₄ oxidation. Only under conditions that severely inhibit superoxide formation (Fig. 2, curve 6) does BH₄ regeneration become the dominant reaction. However, our current work did not allow quantitation of the relative rates of oxidation of NOS-bound BH₄ and regeneration of BH₄ from its oxidized products. Thus, BH₄ regeneration observed on blocking the oxidation reaction was essentially complete by the time the first sample was taken (Fig. 1, curve 4, and Fig. 2, curve 6), and the observed oxidation of BH₄ in Figs. 1-3 is a composite of both oxidation and reduction reactions. Such quantitations will be invaluable in viewing the significance of the redox reactions of BH₄ in relation to overall NOS activity and to the reduction of qBH₂ by the "diaphorase activity" of NOS (18). A better understanding of these redox reactions might also help clarify why the uncoupled reduction of molecular oxygen is much more prominent with nNOS than with the other isozymes of NOS (reviewed in Ref. 4).

	ACKNOWLEDGEMENTS

We thank Drs. Bettie Sue Siler Masters and Kirk McMillan for an inoculum of transfected human kidney 293 cells and advice on their culture. We also thank Anna-Maria Vasquez for outstanding tissue culture work.

	FOOTNOTES

^* 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.

To whom correspondence should be addressed: Laboratory of Neurochemistry, National Institute of Mental Health, Bldg. 36, Rm. 3D30, 36 Convent Dr. MSC 4096, Bethesda, MD 20892-4096. Tel.: 301-402-4896; Fax: 301-480-9284; E-mail: giovanel@codon.nih.gov.

² It was calculated that supplementation with BH₄ resulted in 90% (presence of arginine) and 75% (absence of arginine) of NOS subunits containing BH₄. These calculations were based on K_D values of 37 nM (presence of arginine) and 0.25 µM (absence of arginine) determined for the dimer containing two bound BH₄ molecules dissociating to the dimer containing a single bound BH₄ molecule (68). Supplementation of reaction mixtures with higher concentrations of BH₄ was precluded because of the resultant decreased sensitivity in the assay of BH₄ oxidation.

³ Because the observed oxidation of BH₄ is the net result of the oxidation of BH₄ and regeneration of its oxidized products, we cannot strictly exclude the ad hoc proposal that an SOD-insensitive component of BH₄ oxidation is also present and that formation of the oxidized product of BH₄ by this component is balanced by reduction of this product to regenerate BH₄.

⁴ The presence of SOD can inhibit citrulline formation up to 60% (36, 37, 39), but this effect was explained by SOD allowing the accumulation of the feedback inhibitor NO, rather than by a direct effect of SOD on the reactions of NO formation.

⁵ J. Giovanelli and S. Kaufman, unpublished data.

	ABBREVIATIONS

The abbreviations used are: NOS, nitric-oxide synthase; NO, nitric oxide; BH₄, 6-(L-erythro-1,2-dihydroxypropyl)-5,6,7,8-tetrahydropterin; qBH₂, quinonoid dihydrobiopterin; BH₃^·, trihydrobiopterin radical; CaM, calmodulin; FSQ, the air-stable NOS-bound flavin semiquinone free radical (FMNH^·); HPLC, high performance liquid chromatography; SOD, superoxide dismutase; NHA, N^G-hydroxy-L-arginine; NNA, N^G-nitro-L-arginine; nNOS, neuronal NOS (NOS-I); ROS, reactive oxygen species; Bicine, N,N-bis(2-hydroxyethyl)glycine.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES