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J Biol Chem, Vol. 274, Issue 35, 24921-24929, August 27, 1999

Tetrahydrobiopterin Inhibits Monomerization and Is Consumed during Catalysis in Neuronal NO Synthase^*

Andreas Reif, Lothar G. Fröhlich^§, Peter Kotsonis^¶, Armin Frey, Heike M. Bömmel, David A. Wink^**, Wolfgang Pfleiderer, and Harald H. H. W. Schmidt

From the Department of Pharmacology and Toxicology, Julius-Maximilians-University, Würzburg, 97078 Germany, the ^** National Cancer Institute, Bethesda, Maryland 20892, and the  Faculty of Chemistry, University of Konstanz, Konstanz, 78434 Germany

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The biosynthesis of nitric oxide (NO) is catalyzed by homodimeric NO synthases (NOS). For unknown reasons, all NOS co-purify with substoichiometric amounts of (6R)-5,6,7,8-tetrahydrobiopterin (H₄Bip) and require additional H₄Bip for maximal activity. We examined the effects of H₄Bip and pterin-derived inhibitors (anti-pterins) on purified neuronal NOS-I quaternary structure and H₄Bip content. During L-arginine turnover, NOS-I dimers time dependently dissociated into inactive monomers, paralleled by a loss of enzyme-associated pterin. Dimer dissociation was inhibited when saturating levels of H₄Bip were added during catalysis. Similar results were obtained with pterin-free NOS-I expressed in Escherichia coli. This stabilizing effect of H₄Bip was mimicked by the anti-pterin 2-amino-4,6-dioxo-3,4,5,6,8,8a,9,10-octahydro-oxazolo[1,2f]-pteridine (PHS-32), which also displaced NOS-associated H₄Bip in a competitive manner. Surprisingly, H₄Bip not only dissociated from NOS during catalysis, but was only partially recovered in the solute (50.0 ± 16.5% of control at 20 min). NOS-associated H₄Bip appeared to react with a NOS catalysis product to a derivative distinct from dihydrobiopterin or biopterin. Under identical conditions, reagent H₄Bip was chemically stable and fully recovered (95.5 ± 3.4% of control). A similar loss of both reagent and enzyme-bound H₄Bip and dimer content was observed by NO generated from spermine NONOate. In conclusion, we propose a role for H₄Bip as a dimer-stabilizing factor of neuronal NOS during catalysis, possibly by interfering with enzyme destabilizing products.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Nitric oxide (NO),¹ a widespread signaling molecule with important physiological as well as pathophysiological functions (1-3), is synthesized from L-arginine at the expense of NADPH and O₂ by a family of NO synthases (NOS; EC 1.14.13.39) comprised of three isoforms (NOS I -III or nNOS, iNOS, and eNOS, respectively; Ref. 4). The native, active enzyme is homodimeric (5) and each subunit contains both a reductase and an oxygenase domain (6). The reductase domain contains binding sites for NADPH, FAD, and FMN and is homologous to the NADPH-dependent cytochrome P450 reductase (7, 8). Binding of calmodulin to NOS promotes electron flow from the reductase to the oxygenase domain (9). In NOS-I and III, this occurs in response to elevated free intracellular calcium concentrations (10), while NOS-II binds calmodulin with high affinity even at the free calcium concentration of resting cells and, therefore, is primarily regulated at the expressional level (9, 11). In the NOS oxygenase domain, which shares spectroscopic but not structural properties with other cytochrome P450-type enzymes (12), a prosthetic heme group (7, 13) forms the catalytic center together with the binding site for L-arginine.

A major difference of all NOS isoforms to other P450 enzymes is their additional requirement for tetrahydrobiopterin (H₄Bip) for maximal activity (14-16). H₄Bip is known to bind in the oxygenase domain close to the heme and L-arginine, although how H₄Bip participates in catalysis is unclear. H₄Bip was first discovered as a cofactor of aromatic amino acid hydroxylases (17) where it acts as the main oxygen acceptor and reacts stoichiometrically to quinoid H₂Bip. In NOS, this function is fulfilled by the prosthetic heme group (18, 19) and any attempts to show redox cycling of H₄Bip have yielded negative results (20-22) making such a role for H₄Bip in NOS catalysis unlikely.

Recent studies on the crystal structure of NOS have yielded controversial results; unlike the initial report by Crane et al. (23), H₄Bip per se does not seem to induce a large conformational shift in the NOS oxygenase domain (24). However, H₄Bip has been reported to stabilize enzyme structure, possibly by keeping essential thiol groups in a reduced state (21, 25-28). Moreover, H₄Bip exerts multiple allosteric effects on NOS by lowering the K_D for the substrate L-arginine (22, 29), facilitating coupled electron transfer between the reductase and oxygenase domains (20, 30), and promoting subunit assembly of NOS-II (31) but not NOS-I (32, 33). Furthermore, H₄Bip stabilizes NOS dimers in the presence of protein denaturants such as 5 M urea (34) or 4% SDS (35). However, the relevance of the above findings in the physiological regulation of NOS is unclear given that the dependence on H₄Bip, at least after several rounds of L-arginine turnover, is absolute.

Herein, we investigate possible structural, non-catalytic roles of H₄Bip for NOS, describe the monomerization of NOS-I during L-arginine turnover, and demonstrate an important role of H₄Bip in inhibiting this process. Additionally, we characterized the separated dimers and monomers. Finally, in order to gain further insight into NOS and pterin interactions, we analyzed the effects of anti-pterins on NOS quaternary structure and measured enzyme-associated and total pterin during catalysis.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- L-[2,3,4,5-³H]Arginine HCl (>2.15 TBq mmol¹) and the ECL Western blotting Kit were purchased from Amersham (Braunschweig, Germany); (6R)-5,6,7,8-tetrahydro-L-biopterin from Dr. Schircks Laboratories (Jona, Switzerland); NADPH from Applichem (Darmstadt, Germany); FAD and GSH from Roche Molecular Biochemicals (Mannheim, Germany); FMN, from Fluka (Buchs, Switzerland); horse heart cytochrome c from Sigma (Deisenhofen, Germany); spermine NONOate from Cayman Chemical Co. (Ann Arbor, MI); HPLC solvents from Roth (Karlsruhe, Germany); anti-NOS-I antibody from Transduction Laboratories (Hamburg, Germany). The anti-pterins (AP) 2-amino-4,6-dioxo-3,4,5,6,8,8a,9,10-octahydro-oxazolo[1,2f]-pteridine (PHS-32) and 4-amino-5,6,7,8-tetrahydrobiopterin (PHS-203) were synthesized as described (22), as well as the NO donor Angeli's salt (Na₂N₂O₃; Ref. 36). Native NOS-I was purified from porcine cerebellum in a two-step chromatography procedure using a DEAE cation-exchange column followed by 2',5'-ADP-Sepharose affinity chromatography. Native enzyme had a specific activity up to 227 nmol of citrulline mg¹ min¹. Recombinant human NOS-I was expressed in a baculovirus/Sf9 cell system, purified by 2',5'-ADP-Sepharose affinity chromatography and subsequent calmodulin-Sepharose affinity chromatography, and had a specific activity of up to 379 nmol of citrulline mg¹ min¹. Pterin-free rat NOS-I was expressed in Escherichia coli, purified to a specific activity of 443 nmol mg¹ min¹ as previously published (33) and kindly provided by Dr. Roman (University of Texas, San Antonio, TX). All other chemicals were of the highest purity available and obtained from either Sigma or Merck AG. Water was deionized to 18 M cm (Milli-Q; Millipore, Eschborn, Germany) and, for some experiments, de-oxygenated with argon.

NOS Incubation-- To determine NOS activity and dimer stability, NOS-I (in the indicated amount) was incubated at 37 °C for up to 30 min in a total assay volume of 100 µl containing 50 mM triethanolamine (TEA) buffer (pH 7.2), 25 µM L-arginine, 1 mM NADPH, 5 µM FAD, 10 µM FMN, 0.5 µM CaM, 1 mM CaCl₂, 7 mM GSH, and 250 µM CHAPSO, in the absence or presence of 2 µM H₄Bip. Given the structural instability of pterin-free NOS-I (33), the concentrations of L-arginine and H₄Bip were increased to 0.75 and 0.1 mM, respectively, to ensure full saturation of the enzyme during turnover. To determine the stability of NOS-associated pterin, NOS was incubated under identical conditions but GSH and H₄Bip were omitted.

Incubation of NOS-I with the NO-donor Spermine NONOate-- We also examined the effects of reagent NO on NOS-I dimer and H₄Bip content by incubating NOS for 2 min in a total assay volume of 100 µl containing 50 mM TEA buffer (pH 7.2), 1 µM CaM, 1 mM CaCl₂ and in the absence or presence of 10 mM spermine NONOate generating a flux of about 91 µM NO min¹ (under these conditions). Spermine NONOate was dissolved freshly prior to the assay in 10 mM NaOH. For control samples, the same amount of the vehicle was used. The samples then were immediately frozen in liquid nitrogen for further analysis.

Citrulline Formation-- To determine L-citrulline formation, NOS-I was incubated (see above) in the presence of 5.55 kBq of L-[2,3,4,5-³H]arginine-HCl. The [³H]citrulline formed by NOS was separated from [³H]arginine by cation-exchange chromatography and measured by liquid scintillation counting as described (37).

Size Exclusion Chromatography-- To determine dimer stability, purified recombinant human NOS-I (17 µg) was incubated (see above) for up to 30 min. The reaction was stopped by adding 10 µl of ice-cold EGTA (5 mM). Samples were immediately frozen in liquid nitrogen and stored at 80 °C for further use. After thawing, samples were centrifuged (10,000 × g, 4 °C, 10 min) and injected onto a Superose 6 HR 10/30 size exclusion chromatography column (Amersham Pharmacia Biotech, Freiburg, Germany), equilibrated with TEA buffer (20 mM, pH 7.5) containing 150 mM NaCl and 5% (v:v) ethylene glycol and connected to a FPLC system (Amersham Pharmacia Biotech, Freiburg, Germany). Proteins were eluted at a flow rate of 0.25 ml min¹ and monitored by their absorbance at 280 nm. NOS-containing fractions (300 µl) were collected to determine citrulline formation, pterin content, and cytochrome c reductase activity. The system used provided a separation range from approximately 5-5000 kDa and was calibrated with a gel filtration calibration kit (Sigma). Calibration curves were obtained by plotting the elution volume (V_e) of the standard proteins (carboanhydratase, albumin, alcohol dehydrogenase, apoferritin, and thyroglobulin) against their known Stokes radii (2.01, 3.55, 4.55, 6.10, and 8.50 nm, respectively). The exclusion volume (V₀) was determined with blue dextran 2000.

Low Temperature SDS-PAGE-- NOS-I was incubated as described above in the absence and presence of 2 µM H₄Bip for the indicated time before low-temperature SDS-PAGE was performed as described previously (35) followed by protein immunoblot.

Pterin Determination-- The pterin content (H₄Bip, H₂Bip, and biopterin) in NOS fractions separated by size-exclusion chromatography was analyzed by HPLC (detection limit 0.1-0.2 pmol of pterin) as described (38). To determine pterin stability in NOS-I during L-arginine turnover, native porcine NOS-I (1 µg) was incubated as described above. At the time points indicated, the reaction was stopped by adding ice-cold Tris/HCl (pH 6.7). Samples were immediately centrifuged through 10-kDa cut-off filter caps (Millipore, Eschborn, Germany; 10,000 × g, 4 °C, 20 min). NOS-derived pterins were determined both in the filtrate (i.e. NOS-dissociated pterin) and in the NOS-containing residue (i.e. NOS-associated pterin). Control experiments investigating the chemical stability of reagent H₄Bip (2 µM) were performed under identical conditions but in the absence of enzyme.

Electrochemical NO Detection-- The quantification of reagent NO was performed using an NO electrode as previously published (37). Incubations were carried out in a total volume of 300 µl containing 50 mM MOPS (pH 7.0), 1 mM NADPH, 1 mM CaCl₂, 16.25 µM spermine NONOate and in the absence or presence of 10 µM H₄Bip. The detection limit was 10 nM NO. Calibrations and measurements were performed at room temperature.

Cytochrome c Reduction-- NOS-catalyzed reduction of cytochrome c was measured by incubating 50 µl of each FPLC fraction (see above) in the presence of 0.5 mM NADPH, 0.2 mM cytochrome c, 0.1 µM CaM, 0.1 mM CaCl₂, and TEA buffer (50 mM, pH 7.0) in a final volume of 100 µl for 25 min at 37 °C. Reduced cytochrome c was measured at 550 nm in a SpectraMax 340 microplate reader (Molecular Devices, Sunnyvale, CA). The rate of cytochrome c reduction was calculated using an extinction coefficient of 21 mM¹ cm¹ (39). Blank values were determined in the absence of NOS.

Protein Determination and Immunoblot-- Protein concentrations were determined spectrophotometrically in a microplate assay according to the method of Bradford (40) using bovine serum albumin as a standard. NOS-I was immunodetected by Western blot using a peroxidase enhanced chemiluminescence kit (ECL Western blotting kit; Amersham Pharmacia Biotech) and NOS-I antibody as described (41).

Statistical Analysis-- Data are expressed as mean ± S.E. Statistical analysis was performed using Student's t test for unpaired data. p Values < 0.05 were considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

NOS-I Monomerizes during L-Arginine Turnover-- In order to investigate possible structural changes of NOS-I during catalysis, we measured the dimer and monomer content of recombinant human NOS-I (17 µg) after 0 and 15 min of incubation in the absence of H₄Bip. Samples were then analyzed by size exclusion chromatography as described under "Experimental Procedures." Without incubation (t = 0 min), the enzyme eluted as a single NOS-I immunoreactive protein peak at 12.6 ± 0.05 ml (Fig. 1A, solid line), equivalent to a Stokes radius of 7.89 ± 0.01 nm, which is in the published range for NOS-I dimers (5, 42). Importantly, after 15 min of L-arginine turnover two NOS-I immunoreactive protein peaks were detected (Fig. 1A, broken line): the first coincided with the same elution volume as enzyme without any period of catalysis, the second, at 14.09 ± 0.06 ml, correlating to a Stokes radius of 6.07 ± 0.06 nm, which is in the range of that published for NOS-I monomers (32).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   NOS-I dissociates during L-arginine turnover into inactive and pterin-free monomers. A, recombinant NOS-I (17 µg) was incubated for 0 (solid line) and 15 min (broken line) in the absence of H4Bip as described under "Experimental Procedures." Typical chromatograms are shown and representative of n = 3-7 individual experiments. "D" indicates the dimer and "M" the monomer peak. B, NOS activity in the FPLC eluent fractions was determined as described under "Experimental Procedures" and is reported as absolute citrulline formed during 15 min by 40 µl of eluent. The bars are aligned according to the corresponding elution volume of each fraction; fraction 5 contains the dimer peak, fraction 10, the monomer peak. Open bars indicate the absence, closed bars the presence of 2 µM H4Bip in the citrulline assay mixture. Only fractions containing dimeric NOS-I were found to convert L-arginine to L-citrulline. Addition of H4Bip to the L-citrulline assay mixture markedly enhanced NOS-I activity. C, enzyme-associated H4Bip in the same fractions was determined using HPLC with fluorimetric detection as described under "Experimental Procedures" and is given as the absolute amount of H4Bip per fraction. Only in fractions containing dimeric NOS-I could enzyme-associated pterin be detected. In contrast, in samples containing only monomeric NOS-I, pterin was not detected (fractions 9 to 12; the detection limit was 0.1 to 0.2 pmol pterin per FPLC fraction). The plot represents the mean ± S.E. of three individual experiments, each performed in triplicate.

Only NOS-I Dimers Are Active and Contain H₄Bip-- To characterize NOS dimers and monomers formed during catalysis, we determined the pterin content and enzyme activities (citrulline formation and cytochrome c reductase). In these experiments, the eluent from size exclusion chromatography was fractionated (300 µl). Fractions containing dimeric NOS-I formed L-citrulline (Fig. 1B, open bars) and this enzyme could be stimulated by 10-fold with the addition of 2 µM H₄Bip (total fraction 5, 5.96 ± 0.23 nmol/15 min; Fig. 1B, closed bars), similar to enzyme that was not preincubated. In contrast, fractions containing monomeric enzyme did not convert L-arginine both in the absence and presence of H₄Bip.

To examine whether enzyme activities of NOS-I dimers and monomers and their H₄Bip content were correlated, NOS-associated pterin was determined (Fig. 1C). While dimeric NOS contained 6.13 ± 0.39 pmol of H₄Bip in the peak fraction (fraction 5; Fig. 1C), the pterin content in the monomer-containing fractions (9-12) was below the detection limit. These data suggested that only NOS dimers contained enzyme-associated H₄Bip, as previously reported for rat NOS-I (5).

Since NOS monomers obtained by other procedures usually retain their reductase activity (13), we measured the reduction of cytochrome c in monomers generated under our conditions. Indeed, in contrast to L-citrulline formation, both the dimer and monomer peak fractions (5 and 10, respectively) were able to reduce cytochrome c (8.58 ± 0.28 and 8.6 ± 0.08 nmol of reduced cytochrome c ml¹ min¹, respectively), confirming that the ability of NOS-I to reduce electron acceptors neither requires dimeric enzyme (43) nor the presence of enzyme-associated H₄Bip.²

NOS Monomerization Is Time-dependent in Correlation to Loss of Activity-- To determine the kinetics of NOS-I monomerization during catalysis, NOS-I was incubated for different time points (0-30 min; Fig. 2A, open circles) and then analyzed for dimer content and rate of citrulline formation. Monomerization of NOS-I was found to occur mainly during the first 10 min of L-arginine turnover. Similar results were obtained by low temperature SDS-PAGE under the same incubation conditions, although some monomeric NOS-I was observed also at t = 0 min (not shown). This was probably due to the presence of SDS in this method of analysis. Under the same conditions, the rate of citrulline formation also rapidly declined and then ceased, suggesting that dimer loss and loss in activity may correlate (Fig. 2B, open circles).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   NOS-I monomerizes in a time-dependent manner during catalysis. A, before size exclusion chromatography, recombinant NOS-I (17 µg) was incubated for different time points in the absence (open circles) or presence (closed circles) of 2 µM H4Bip as described under "Experimental Procedures." Monomerization of NOS-I was time-dependent and the addition of H4Bip markedly reduced this, most prominently during the first 15 min. At later time points, mixed effects (i.e. formation of both reactive nitrogen and oxygen species) take place (see Footnote 3) which may explain the destabilization of the dimer even in the presence of H4Bip. Each symbol represents the mean ± S.E. of n = 3-7 individual experiments. B, recombinant human NOS-I (0.6 µg) was incubated for different time points in the absence (open circles) or presence (closed circles) of 2 µM H4Bip. Thereafter, NOS activity was determined as described under "Experimental Procedures." In the presence of H4Bip, the rapid loss of L-citrulline formation was inhibited. C, pterin-free recombinant rat NOS-I (11 µg) was incubated for 15 min in the absence (dashed line) and presence (solid line) of 100 µM H4Bip and subsequently analyzed by size exclusion chromatography as described under "Experimental Procedures." The addition of H4Bip to the reaction mixture markedly inhibited the formation of NOS-I monomers, indicating again a dimer stabilizing function for H4Bip. Each chromatogram is representative of three individual experiments yielding similar results.

H₄Bip Inhibits NOS Monomerization in Correlation with Activity-- To examine the effect of H₄Bip on the time course of NOS-I monomerization, recombinant human NOS-I was incubated as above but in the presence of 2 µM H₄Bip before size exclusion chromatography. At all time points, H₄Bip increased the dimer content (Fig. 2A, closed circles). Similar results were obtained by low temperature SDS-PAGE analysis (not shown). Moreover, in kinetic experiments under the same conditions, the loss of citrulline formation was also greatly inhibited (Fig. 2B, closed circles). The use of higher H₄Bip concentrations to examine whether this would further stabilize the enzyme activity beyond 5 min is complicated, given that high H₄Bip concentrations inhibit NOS-I activity (22). Eventually, NOS-I activity can be abolished with super-saturating H₄Bip concentrations. Thus H₄Bip was added repetitively in lower concentrations in order to retain full catalytic activity as much as possible (see below). The mechanism underlying the inhibitory effect of H₄Bip and a possible antagonism with L-arginine (24) is under investigation. In conclusion, stabilization of the dimers and changes in activity appeared to correlate, although additional mechanisms of inactivation and stabilization of NOS may be involved. Nevertheless, the increase of the dimer/monomer ratio represents a direct, hydrodynamic measurement of an enzyme-stabilizing mechanism of H₄Bip during NOS catalysis.

Pterin-free NOS-I Also Monomerizes in an H₄Bip-inhibitable Manner-- As NOS-I expressed in a baculovirus/Sf9 expression system contains substoichiometric amounts of endogenously bound H₄Bip (63% in the preparation used in this study), we were also interested in analyzing pterin-free NOS-I, i.e. expressed in E. coli (33). Monomerization of pterin-free NOS-I was examined by incubating 11 µg of enzyme for 15 min under catalytic conditions (i.e. all cofactors and 0.75 mM L-arginine) in the absence or presence of 100 µM H₄Bip. Thereafter size exclusion chromatography was performed as described above. E. coli-derived NOS-I that had not been incubated was entirely dimeric. Upon 15 min of L-arginine turnover, a prominent monomer peak appeared (Fig. 2C, dashed line), suggesting that pterin-free NOS monomerizes and inactivates after a few rounds of catalysis similar to Sf9 cell-derived NOS-I. When H₄Bip was included in the incubation mixture, monomerization was markedly reduced (Fig. 2C, solid line) suggesting again a dimer stabilizing role of H₄Bip also for pterin-free NOS-I.

Effect of Anti-pterins on the NOS-I Structure, Pterin Content, and Activity-- To examine whether anti-pterins mimic the stabilizing effect of H₄Bip on NOS, monomerization of NOS-I during catalysis was investigated in the absence or presence of the anti-pterins PHS-32 and 4-aminotetrahydrobiopterin (PHS-203). These compounds have recently been shown to selectively bind to the H₄Bip-binding site of NOS, displace enzyme-bound H₄Bip, and inhibit H₄Bip-stimulated NOS activity (22, 44). In the presence of PHS-32, monomerization was inhibited at t = 15 min (a typical chromatogram is given in Fig. 3A, solid line), and the H₄Bip content in the dimer fractions was greatly reduced (Fig. 3B). These latter data are consistent with our previous observation that PHS-32, which has a similar K_D as H₄Bip,³ displaces enzyme-associated H₄Bip almost completely (22) and, thus, readily accesses the occupied high affinity (45) pterin-binding site of dimeric NOS-I. This interaction was reversible since the addition of 2 µM H₄Bip after size exclusion chromatography in the subsequent citrulline assay restored NOS activity to 86 ± 2% of control. Interestingly, both anti-pterins (at 100 µM) increased dimer stability during L-arginine turnover analogous to H₄Bip (at 2 µM; Fig. 4, closed bars). PHS-203 was even more effective than the natural cofactor. However, in contrast to H₄Bip, which stimulates NOS activity, the anti-pterins had no effect on NOS activity in the absence of H₄Bip (Fig. 4, open bars). Thus dimer stabilization alone is not sufficient to maintain maximal activity and cannot fully explain the mechanism of action of H₄Bip.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   PHS-32 displaces enzyme-associated H4Bip from dimeric NOS-I. A, a typical chromatogram following preincubation of NOS-I in the absence of H4Bip, but either in the absence (broken line) or presence of the anti-pterin PHS-32 (100 µM; solid line). Typical chromatograms are shown and representative of n = 7 individual experiments. B, H4Bip displacement by PHS-32. Enzyme-associated H4Bip was determined using HPLC with fluorimetric detection as described under "Experimental Procedures" and is given as the absolute amount of H4Bip per fraction. PHS-32 is not detectable with this method and thus does not itself interfere with pterin measurements. Fraction 5 contained the peak height of the dimers, fraction 10 the peak height of the monomers. Open bars indicate the absence, closed bars the presence of 100 µM PHS-32 in the preincubation mixture; in the fractions 9-12, pterin was below the detection limit (see above). H4Bip present in dimeric NOS-I was almost completely displaced upon addition of PHS-32. The plot represents the mean ± S.E. of three individual experiments, performed in triplicate.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Effect of anti-pterins on NOS monomerization and activity. Anti-pterins mimic the stabilizing effect of H4Bip on NOS quaternary structure, but do not activate NOS-I. Recombinant NOS-I was incubated as described under "Experimental Procedures" for 15 min with either 100 µM anti-pterin (PHS-32 and 203) or 2 µM H4Bip. Thereafter, quaternary structure (closed bars) as well as activity (open bars) of NOS-I were determined as described under "Experimental Procedures." Dimer content is expressed as the ratio of dimer versus monomer peak heights. In structural studies, the values represent the mean ± S.E. of n = three to seven individual experiments; in enzyme activity measurements, the values represent the mean ± S.E. of two experiments, performed in triplicates. An asterisk indicates a significant difference from control, whereas + indicates significant difference from incubation with H4Bip. Student's t test was used and p < 0.05 was considered statistically significant.

Resubstitution of NOS with H₄Bip Enhances Subsequent Activity-- To examine whether such hypothetically dimeric and inactive enzyme was due to the source of NOS from a recombinant expression system, we performed kinetic studies with native NOS-I purified from porcine cerebellum. NOS activity of this preparation was found to cease after only 3 min of L-arginine turnover in the absence of exogenous H₄Bip (Fig. 5A, open circles). Interestingly, the earlier H₄Bip was added to the incubation mixture, the more pronounced was the effect on subsequent citrulline formation (Fig. 5B). These data suggest that there is a population of NOS-I which is reversibly inactivated during L-arginine turnover and can be reactivated upon addition of H₄Bip. Consistent with our data using recombinant human NOS-I, the anti-pterin PHS-32 (in the absence of H₄Bip) had no effect on the initial velocity of catalysis under basal conditions and only marginally affected the subsequent loss of porcine NOS-I activity (Fig. 5A). However, in order to maintain maximal NOS activity for more than 15 min, H₄Bip had to be added repetitively every 15 min (Fig. 5C). This suggested that during L-arginine turnover, which generated about 5 µM reactive nitrogen oxide (RNS) species, initially saturating levels of H₄Bip (2 µM) progressively decreased to suboptimal levels. As mentioned above, higher concentrations of H₄Bip (>100 µM) could not be tested.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Early as well as repetitive resubstitution with exogenous H4Bip reduces NOS inactivation. A, in contrast to H4Bip, PHS-32 does not stimulate basal NOS activity. L-Citrulline formation by native NOS-I (3 µg) was determined at the time points indicated in the absence (open circles) or presence of either 2 µM H4Bip (closed circles) or 100 µM PHS-32 (closed triangles). The addition of the anti-pterin did not prevent the complete inactivation of NOS-I observed at 5 min and only slightly increased subsequent L-arginine turnover. B, the delayed addition of H4Bip resulted in reduced recovery of NOS activity. Native porcine NOS-I (3 µg) was incubated in the presence of all cofactors and substrates but without H4Bip. To this mixture, H4Bip (2 µM) and L-[3H]arginine (5.55 kBq) were added at 0, 5, 10, 15, or 20 min after the onset of the reaction and incubated for another 15 min. Thereafter, L-citrulline formation was determined. The later H4Bip was added, the lower was activity that could subsequently be recovered. C, repetitive H4Bip addition preserves Vmax. Native porcine NOS-I (3 µg) was incubated in the presence of all cofactors and substrates including H4Bip (2 µM). At 15, 30, and 45 min, either vehicle (closed circles) or 2 µM H4Bip (squares) were added to the reaction mixture, as indicated by the arrows. L-Citrulline accumulation was determined at 15, 30, 45, and 60 min. When H4Bip was added repetitively, the reaction remained at Vmax for almost 45 min. In contrast, when only vehicle was added, the reaction remained at Vmax for only 15 min. Values are given as mean ± S.E. and represent n = two to seven individual experiments, performed in triplicate.

Loss of NOS-associated H₄Bip during Catalysis-- To further analyze the observed loss of H₄Bip during L-arginine turnover, we examined chemical interactions between H₄Bip and NOS-derived reactive species. While a role for reactive oxygen species (ROS) has already been suggested,³ we here focused on the possible involvement of RNS. These results were compared with the non-enzymatic decomposition of reagent H₄Bip (in the absence of NOS). Under NOS assay conditions, reagent H₄Bip was found to be surprisingly stable (Fig. 6A, open circles). In contrast, during the first minutes of L-arginine turnover, NOS-associated H₄Bip not only rapidly dissociated from the enzyme into solution but the total recovery of H₄Bip, i.e. enzyme-associated plus dissociated, was increasingly incomplete and decreased to a much greater extent (Fig. 6B) than expected from the chemical stability of H₄Bip (see Fig. 6A). The observed dissociation of H₄Bip from NOS-I appeared to coincide with the loss in dimer (see Fig. 2A). It is unlikely that this loss in total H₄Bip was due to autoxidation because neither concomitant dihydrobiopterin nor biopterin generation, which would have been expected in the case of oxidative breakdown, was observed. Rather, these data suggested that activated NOS plays a direct role in the decomposition of enzyme-associated H₄Bip to a product distinct from H₂Bip and biopterin.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Chemical stability and loss of NOS-associated pterin during catalysis. A, chemical decomposition of H4Bip. H4Bip (2 µM) was incubated in the absence of NOS but otherwise under NOS assay conditions (i.e. 50 µM L-arginine, 1 mM NADPH, 5 µM FAD, 10 µM FMN, 500 nM CaM, 1 mM CaCl2, 250 µM CHAPSO) either in the absence (open circles) or presence of 100 µM NO (closed circles) or 150 µM NO (squares). NO was applied by using Angeli's salt; the source of NO was a NO-saturated solution. At the time points indicated, total pterin was measured as described. NO as well as NO reduced total pterin recovery in a time-dependent manner whereas H4Bip remained stable in the control experiments. At all incubation time points, neither H2Bip nor biopterin could be detected. In contrast, neither H2Bip nor biopterin reacted with either NO or NO (not shown). Data points represent mean ± S.E. of four independent experiments, each performed in triplicate. B, total recovery of NOS-associated pterin was lowered during L-arginine turnover. Native porcine NOS-I (1 µg) was incubated as described under "Experimental Procedures" for different time points and thereafter the amount of enzyme-bound (open bars) and free (closed bars) pterin was determined. Again, neither H2Bip nor biopterin could be detected. During catalysis, enzyme-bound H4Bip was rapidly lost from NOS. Furthermore, total pterin recovery, bound and dissociated, was lowered in a time-dependent manner. The plot represents mean ± S.E. of three experiments, each performed in triplicate. C, reagent NO is rapidly scavenged by H4Bip. The NO signal, generated from spermine NONOate (16.25 µM), was monitored electrochemically as described under "Experimental Procedures." When a steady state flux was reached, 10 µM H4Bip was added to the reaction mixture (arrow). The NO signal then immediately ceased correlating well with the disappearance of H4Bip upon exposure to NO (A).

NO and H₄Bip Interactions-- To examine whether the apparent loss of total pterin during L-arginine turnover could be explained in part by a reaction of H₄Bip with NO, we measured the recovery of reagent H₄Bip when incubated with NO in amounts that were similar to those which could be formed during L-arginine turnover. Indeed, in the presence of NO, H₄Bip decreased to 59 ± 4.5% of control (Fig. 6A, closed circles). Similarly, when reagent H₄Bip was incubated in the same manner with NO derived from Angeli's salt (a model compound for alternative arginine-derived products from NOS; Ref. 37), the H₄Bip loss was even more pronounced (Fig. 6A, closed squares). To further confirm our hypothesis, we electrochemically monitored the release of NO from the NO donor compound spermine NONOate (37). When a steady state NO signal was reached, H₄Bip was added (Fig. 6C, arrow at t = 0 min) upon which the NO signal was abolished. With respect to the mechanism of interference between NO and H₄Bip, the formation of superoxide during H₄Bip autoxidation and the subsequent diffusion-limited reaction of NO with superoxide to peroxynitrite (46) has been suggested. However, recently published EPR data arguing against superoxide formation from H₄Bip (47) make this mechanism unlikely. Thus, the mechanism of interaction remains open and may in fact involve a direct interaction between NO and H₄Bip.

Exogenous NO Decreases Enzyme-associated H₄Bip and NOS Dimer Content-- To test our hypothesis that, in addition to ROS, RNS could also contribute to NOS dimer destabilization, the effects of reagent NO, generated by the NO donor compound spermine NONOate, on both NOS-associated H₄Bip and dimer content were examined. Recombinant human NOS-I (17 µg, that would generate a flux of 64 µM NO min¹ under catalytic conditions) was exposed for 2 min to 10 mM spermine NONOate (generating, under our conditions, a flux of approximately 91 µM NO min¹) under non-catalytic conditions (i.e. in the absence of NADPH and L-arginine) and assayed for pterin content (see "Experimental Procedures"). In control experiments, NOS-I was incubated under identical conditions but in the absence of spermine NONOate. In the presence of spermine NONOate, the amount of enzyme-associated H₄Bip decreased to 58 ± 10% of control (Fig. 7, open bars). A further 10 ± 3% of total H₄Bip dissociated from NOS-I and were recovered in the solute (Fig. 7, hatched bar). Thus the total recovery of H₄Bip (enzyme-associate and free) was lowered to 68% of control. This suggests that NO rapidly diffuses in the catalytic center of NOS-I and then reacts with bound H₄Bip to a yet unknown derivative, which thereafter dissociates from the enzyme.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Reagent NO decreases both enzyme-associated H4Bip and NOS-I dimer content. Recombinant human NOS-I (17 µg) was incubated for 2 min at 37 °C in a total volume of 100 µl of 50 mM TEA buffer (pH 7.4) containing 1 µM CaM, 1 mM CaCl2 and in the absence (control) or presence (NO) of 10 mM spermine NONOate. The NO flux rate, under these conditions, was approximately 91 µM NO min1 which is similar to the NO flux that would be generated during l-arginine turnover by the amount of NOS-I used (64 µM NO min1). Thereafter, the samples were analyzed for pterin content and dimer content as described under "Experimental Procedures." Open bars indicate the amount of enzyme-associated H4Bip, hatched bars H4Bip in the solute, and closed bars the loss of NOS-I dimers expressed in % of control. The addition of spermine NONOate markedly reduced enzyme-bound pterin; furthermore, NOS-I dimers were decreased indicating a corresponding destabilization of the enzyme's quaternary structure. Values represent mean ± S.E. of three individual experiments. The asterisk indicates a significant difference from control value (p < 0.05).

Additionally, the effects of reagent NO on the quaternary structure of NOS-I were investigated. Similar to the dimer loss that was induced in NOS-I during L-arginine turnover (21 ± 2% after 2.5 min; see Fig. 2A), exposure to reagent NO also significantly decreased the dimer content of quiescent NOS-I by 16 ± 3% (after 2 min; Fig. 7, closed bar). Almost identical results were observed when the effects of NO on pterin-free rat NOS-I (11 µg) were studied. Here, the dimer content was lowered by 12 ± 3%. Collectively, these data suggest that pterin loss and destabilization of the quaternary structure are mechanistically linked.

	DISCUSSION

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The finding that dimeric NOS-I time dependently monomerizes during catalysis (Scheme I) is novel and may be important in the regulation of NO, RNS, and ROS synthesis. To date, purified native NOS-I has only been observed in its dimeric state (5, 35) and monomerization was shown to occur in the presence of a 10-kDa protein inhibitor of neuronal NOS, PIN (48), although recent evidence questions this finding (49). NOS also monomerizes in the presence of protein denaturants. For example, treatment of NOS-II with 2 M urea causes unfolding of the NOS reductase domain and flavin release; a higher concentration of urea (5 M) is required to cause subunit dissociation (50, 51). Moreover, SDS induces NOS-I monomerization associated with a dramatic increase in -helical structure (35). In contrast to these studies, the present analysis using native NOS describes the appearance and detection of inactive, monomeric enzyme during L-arginine turnover in the absence of PIN and protein denaturants. In agreement with our findings, Stuehr's (31) laboratory found that only dimeric NOS-II is active with respect to L-citrulline formation.

View larger version (18K): [in this window] [in a new window]   Scheme I.

Monomerization and inactivation of NOS-I was paralleled by a loss of enzyme-associated H₄Bip. While the addition of H₄Bip largely inhibited monomerization, also of pterin-free NOS-I, we were not able to achieve reactivation of monomerized NOS-I. In contrast, NOS-II monomers generated by exposure to 5 M urea reassemble upon preincubation with L-arginine and H₄Bip to form active enzyme (51). Thus, the underlying mechanism by which H₄Bip stabilizes the NOS-I dimer complex is unclear and appears to be isoform-specific. H₄Bip may act as a molecular clip to prevent NOS-I subunit dissociation, as Crane et al. (23) recently suggested that H₄Bip might induce a large conformational change upon binding to the NOS-II oxygenase domain. However, recent structural data argue against such a mechanism as the H₄Bip-free and containing structures of the NOS-III oxygenase domain show no major differences (24). Thus, H₄Bip may alternatively prevent uncoupled catalysis and the formation of ROS (30) which otherwise destabilize NOS-I dimers.³ Nevertheless, the present findings are consistent with the stabilizing effect of H₄Bip on SDS-denaturated NOS-I (35) and therefore provide a means to examine structural interactions between H₄Bip and native NOS-I during L-arginine turnover.

It is still controversial whether NOS-II (13, 31, 51, 52) and NOS-III (53-57) require H₄Bip for the initial subunit assembly. However, this can be excluded for NOS-I (32, 33, 58). In contrast, our data suggest that the intracellular pterin content may regulate NOS-I dimer dissociation during catalysis after the initial, H₄Bip-independent subunit assembly during protein synthesis.

The stabilizing effect of H₄Bip was mimicked by anti-pterins. Given that these compounds are effective inhibitors of NOS, this hints to a partial dissociation of dimer stability and activity. We therefore suggest that there are distinct conformations of the NOS-I dimer (Scheme I, modified from Ref. 13): active (green) and inactive (red) with respect to L-arginine turnover. As we have previously shown, NOS is able to convert L-arginine even in the absence of added H₄Bip. This basally active enzyme was termed NOS* (Ref. 22; II in Scheme I). Interestingly, this enzyme is not affected by the type of anti-pterins used in the present study despite the fact that these compounds effectively displace enzyme-associated pterin. However, II rapidly inactivates during catalysis, possibly due to autoinactivation by NOS-derived NO or ROS.³ The resulting species, NOS° (III in Scheme I), is at first reversibly inactive and can be re-activated by adding exogenous H₄Bip to obtain a maximally active state of NOS, I. Otherwise, III converts to an irreversibly inactive dimer (IV), which subsequently monomerizes (V). Even the time point of H₄Bip addition to basally active enzyme during L-arginine turnover appears to be important in determining subsequent enzyme activity. The later the addition took place, the lower was the subsequently recoverable activity suggesting that increasing amounts of III had converted to irreversibly inactive NOS species, i.e. IV or V.

As depicted in Scheme I, I may also be obtained from II by adding exogenous H₄Bip, resulting not only in an increase in V_max, but also in stability. At present, we attribute this stabilizing effect of H₄Bip to its capability to scavenge autoinhibitory products from NOS, i.e. ROS³ and RNS. The mechanism of this inactivation of NOS remains to be established; however, since NOS contains essential protein thiols within the catalytic center (12, 25), thiol oxidation is one candidate target. During enzyme isolation, unbound pterin is removed so that the amount of NOS* (II) in purified NOS-I could be a "reminder" of cellular H₄Bip levels. This would also explain our recent finding that basal activity of purified NOS-I, native or recombinant, does not correlate with protein-associated pterin (22) when compared between different studies. Anti-pterins compete with exogenous H₄Bip to prevent the formation of I. However, they do not affect L-arginine turnover by II and apparently inhibit the monomerization, but not the inactivation process. Thus, in the presence of anti-pterins NOS-I is locked in stage II-IV.

During L-arginine turnover, we observed a progressive loss of enzyme-associated pterin as detected by our HPLC method. Moreover, reagent as well as enzyme-bound H₄Bip was incompletely recovered when incubated with NO or NO. Furthermore, reagent NO was scavenged when incubated with H₄Bip. This raises the possibility that NOS-associated H₄Bip reacts with NOS catalysis products in a similar manner, which may therefore account for at least some of the loss of H₄Bip during catalysis. Further spectroscopic studies aimed at the identification of the product of H₄Bip with RNS/ROS and the H₄Bip metabolite generated in NOS during L-arginine turnover are presently under way.

The present findings may have important implications for the regulation of NOS-I depending on intracellular H₄Bip concentrations. In situations where both NO and H₄Bip synthesis are high, e.g. stimulated macrophages (59), NOS is likely to be replenished with H₄Bip. However, at H₄Bip levels of resting cells, constitutive NOS may become H₄Bip deficient, especially if turnover is occurring in neighboring NOS molecules. Interestingly, H₄Bip has been reported to prevent NOS from an autoinactivation process by NO (27) as well as causing oxidation of NO to ONOO in the presence of superoxide (46). Monomerization of NOS-I then could contribute to the prevention of harmful peroxynitrite production, which occurs at subsaturating H₄Bip levels (60).

	ACKNOWLEDGEMENT

We are indebted to Linda Roman for the generous gift of pterin-free recombinant rat NOS-I expressed in E. coli. We thank M. Bernhardt, B. Thur, and M. Weeger for expert technical assistance, as well as C. Alvarez, F. Bossle, M. La, Z. Shutenko, R. Wölfel, and U. Zabel for helpful discussions.

	FOOTNOTES

^* This work was supported in part by the Bundesministerium für Bildung und Forchung, Fritz-Thyssen Stiftung, and Deutsche Forschungsgemeinschaft Grant SFB 355/C7 (to H. H. H. W. S.).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.

Submitted to the Faculty of Medicine of the Julius-Maximilians-University, Würzburg, Germany, in partial fulfillment of a Ph.D. thesis. To whom correspondence should be addressed: Dept. of Pharmacology and Toxicology, Julius-Maximilians University, Versbacher Str. 9, D-97078 Würzburg, Germany. Tel.: 49-931-201-3854; Fax: 49-931-201-3539; E-mail: a.reif@gmx.net.

^§ Submitted to the Faculty of Chemistry and Pharmacy of the Julius-Maximilians University, Würzburg, Germany, in partial fulfillment of a thesis.

^¶ Recipient of a C. J. Martin fellowship (Australian National Health & Medical Research Council).

Present address: Biocenter, Dept. of Physiological Chemistry, Julius-Maximilians University, Würzburg, Germany.

² M. Pantke and H. H. H. W. Schmidt, unpublished observation.

³ Kotsonis, P., Frey, A., Fröhlich, L., Hofmann, H., Reif, A., Wink, D., Feelisch, M., and Schmidt, H. (1999) Biochem. J. 340, 745-752.

	ABBREVIATIONS

The abbreviations used are: NO, nitric oxide; AP, anti-pterin; CaM, calmodulin; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate; FPLC, fast performance liquid chromatography; H₂Bip, (6R)-7,8-dihydro-L-biopterin; H₄Bip, (6R)-5,6,7,8-tetrahydro-L-biopterin; HPLC, high performance liquid chromatography; MOPS, 3-(N-morpholino)propanesulfonic acid; NOS, nitric oxide synthase; PHS, anti-pterin code number; PIN, protein inhibitor of nNOS; RNS, reactive nitrogen species; ROS, reactive oxygen species; TEA, triethanolamine hydrochloride; PAGE, polyacrylamide gel electrophoresis.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES