©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characteristics of the Nitric Oxide Synthase-catalyzed Conversion of Arginine to N-Hydroxyarginine, the First Oxygenation Step in the Enzymic Synthesis of Nitric Oxide (*)

(Received for publication, October 6, 1994; and in revised form, November 18, 1994)

Kenneth L. Campos (§) John Giovanelli (¶) Seymour Kaufman

From the Laboratory of Neurochemistry, National Institute of Mental Health, Bethesda, Maryland 20895

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The nitric oxide synthase-catalyzed conversion of L-arginine to L-citrulline and nitric oxide is known to be the sum of two partial reactions: oxygenation of arginine to N-hydroxyarginine, followed by oxygenation of N-hydroxyarginine to citrulline and nitric oxide. Whereas the conversion of N-hydroxyarginine to citrulline and nitric oxide has been the subject of a number of studies, the oxygenation of arginine to N-hydroxyarginine has received little attention. Here we show that substrate amounts of rat cerebellar nitric oxide synthase, in the absence of added NADPH, catalyze the conversion of arginine to N-hydroxyarginine as the dominant product. The product appears not to be tightly bound to the enzyme. A maximum of 0.16 mol of N-hydroxyarginine/mol of nitric oxide synthase subunit was formed. The reaction requires oxygen and the addition of Ca/calmodulin and is stimulated 3-fold by tetrahydrobiopterin. Upon addition of NADPH, citrulline is formed exclusively. Conversion of N-hydroxyarginine to citrulline, like the first partial reaction, requires Ca/calmodulin and is stimulated by tetrahydrobiopterin but differs from the first partial reaction in being completely dependent upon addition of NADPH. These results indicate that brain nitric oxide synthase contains an endogenous reductant that can support oxygenation of arginine but not of N-hydroxyarginine. The reductant is not NADPH, since the amount of nitric oxide synthase-bound NADPH is appreciably less than the amount required for N-hydroxyarginine synthesis. Possible candidates for this role are discussed in relation to proposed mechanisms of action of nitric oxide synthase.


INTRODUCTION

Nitric oxide synthase (NOS) (^1)catalyzes the oxygenation of arginine in the presence of NADPH to form nitric oxide, citrulline, and NADP. The enzyme is of great interest because nitric oxide participates in a variety of physiological processes including vasodilation, macrophage toxicity, and neurotransmission(1) . There is now convincing evidence that the overall reaction catalyzed by NOS proceeds via two partial reactions,

The intermediary role of L-N^G-hydroxyarginine (NHA) was first suggested by Marletta et al. (2) and detected as a minor product during the conversion of arginine to citrulline by mammalian cells induced for NO synthesis as well as by crude extracts of these cells (3, 4) and by purified macrophage NOS(5, 6) . In the last study, [^3H]NHA formation from [^3H[arginine was detected only under special conditions, namely in the presence of a high concentration of a pool of unlabeled NHA, indicating that this intermediate may not normally be released from the enzyme. The amount of [^3H]NHA accumulating was only 20% of the amount of [^3H]citrulline formed(4, 6) . Recently, Klatt et al. (7) reported the formation of trace amounts of [^3H]NHA from [^3H]arginine catalyzed by purified porcine brain NOS. Here again, the amounts of NHA accumulating were small relative to the amounts of citrulline formed. Under normal reaction conditions, NHA accounted for a maximum of 2% of citrulline formed; in the presence of the redox-active inhibitors of citrulline formation, nitroblue tetrazolium or methylene blue, this value increased up to 20%. The small amounts of NHA relative to citrulline formed under these conditions have hampered attempts to elucidate the characteristics of the first partial reaction.

Here we show that stoichiometric amounts of purified rat brain NOS convert arginine predominantly to NHA in the absence of added electron donor. This experimental system permits examination, for the first time, of the characteristics of the first partial oxygenation reaction. Our findings indicate that the rat brain enzyme contains an endogenous reductant that can support a single cycle oxygenation of arginine to NHA but not of NHA to citrulline. Possible candidates for this role are discussed in relation to the proposed mechanisms of action of nitric oxide synthase.


EXPERIMENTAL PROCEDURES

Materials

L-[2,3,4,5-^3H]Arginine monohydrochloride (Amersham Corp.) was purified by cation exchange HPLC. The natural (6R) isomer of BH(4) was obtained from B. Schircks Laboratories (Jona, Switzerland). Catalase and the enzymes used for NADPH determination (glutamate dehydrogenase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase) were from Boehringer Mannheim; calmodulin and glucose oxidase (Aspergillus niger) were from Calbiochem. NHA was a gift from Dr. Paul L. Feldman, Glaxo Research Institute (Research Triangle Park, NC). [^3H]NHA was prepared by enzymic synthesis. Briefly, [^3H]arginine (1.4 µM, 1.1 times 10^7 dpm) was incubated with 16 µg of NOS and the same concentration of components specified in the standard reaction mixture described under ``Methods.'' The volume of the reaction mixture was 85 µl. The reaction was terminated after 30 min with perchloric acid, followed by addition of 104 pmol of NHA. [^3H]NHA was isolated by HPLC as described under ``Methods.'' The preparation (specific activity 3500 dpm/pmol) was stored at -80 °C and used within 3 months, during which time it remained radiopure. After 5 months of storage, radiopurity had decreased to 80%.

Methods

Purification of NOS

NOS was purified from frozen rat brain cerebella (8) or from transfected human kidney 293 cells stably transfected with rat brain cDNA NOS(1) . Enzyme from kidney cells was purified by the method of McMillan et al.(9) , except that the 2,5-ADP-Sepharose column was subjected to an additional wash with 10 ml of 0.5 mM NADP in 10 mM TrisbulletHCl, 10% (v/v) glycerol, 0.1 mM EDTA, 0.1 mMp-aminoethylbenzenesulfonyl fluoride, 0.1 mM dithiothreitol, 0.5 µM leupeptin, 0.5 µM pepstatin, pH 7.5, after the wash containing 0.5 M NaCl. NADPH present in the 2,5-ADP-sepharose eluate was decreased to a negligible concentration during the final gel filtration step. For the rat cerebella enzyme, NADPH was decreased by repeated concentration and dilution with 10 mM TrisbulletHCl, 1 mM EDTA, pH 7.4, with the use of a Centricon-30 (Amicon) ultrafilter(8) . Ferricyanide-treated enzyme was prepared by incubation of cloned enzyme (410 µl, 722 µg) with 5 mM potassium ferricyanide in a final volume of 512 µl at 4 °C for 2 min. Ferricyanide was separated from the treated enzyme on a PD-10 column (Pharmacia Biotech Inc.) equilibrated with 50 mM TrisbulletHCl, 10% (v/v) glycerol, 0.1 M NaCl, 0.1 mM EDTA, pH 7.5.

Assay of the First Partial Reaction

For studies of the synthesis of [^3H]NHA, NADPH was omitted from the reaction mixture normally used for assay of the overall reaction of NOS(8) . The standard reaction mixture (14 µl) contained 189 nM [^3H]arginine (2.6 pmol, 2.4 times 10^5 dpm), 250 mM sodium Hepes (pH 6.4), 0.20 µg of calmodulin, 0.83 mM CaCl(2), 60 µM BH(4), 0.6 mM sodium EDTA, 14 µg of bovine serum albumin, 1.5 µg of catalase, and approximately 2 µg of NOS. Changes are noted in the text. Reactions were incubated at 25 °C for the times specified and were routinely conducted under ambient laboratory lighting conditions; in one study, incubations were conducted in the dark by wrapping reaction tubes in aluminum foil. Reactions were terminated by addition of perchloric acid and 150-200 nmol each of authentic NHA and citrulline. The mixture was centrifuged, and the supernatant solution was titrated with KOH to a pH of approximately 2 (thymol blue indicator) when using the HPLC protocol of Chenais et al. (3) or to a pH of approximately 6 (bromcresol purple indicator) for the HPLC protocol described by Klatt et al.(7) . The precipitate of potassium perchlorate was removed by centrifugation, and the supernatant solution was finally adjusted either to pH 2.2 by addition of sodium citrate to a final concentration of 20 mM (Chenais protocol) or to pH 6.5 by addition of sodium acetate to a final concentration of 50 mM (Klatt protocol). Approximately 100 µl of this solution was injected onto a Zorbax 300-SCX column for HPLC. Fractions of 1 ml were assayed for radioactivity; the retention times of NHA and citrulline were determined by amino acid determination of the marker amino acids with fluorescamine(10) .

Assay of the Second Partial Reaction

The complete reaction mixture (85 µl) contained 2.4 nM [^3H]NHA (730 dpm), 250 mM sodium Hepes (pH 6.4), 670 µM NADPH, 1.2 µg of calmodulin, 0.83 mM CaCl(2), 60 µM BH(4), 0.6 mM sodium EDTA, 85 µg of bovine serum albumin, 9 µg of catalase, and 0.7 µg of rat cerebellar NOS. The reaction was terminated after 15 min of incubation at 25 °C by addition of perchloric acid and authentic NHA (100 nmol) and citrulline (200 nmol). The procedure described above for analysis of ^3H in citrulline and NHA was then followed.

HPLC-HPLC was performed by cation exchange chromatography on a Zorbax 300-SCX column (DuPont) (dimensions, 4.6 mm internal diameter times 25 cm) using the solvent system described by Chenais et al. (3) or Klatt et al.(7) . Reverse phase ion pair chromatography was performed on a Waters µ Bondapak C18 column (3.9 mm internal diameter times 30 cm) with the use of a gradient of 0-75% 1-propanol with a stationary phase of 17 mM sodium citrate, pH 2.7, containing 0.5% sodium dodecyl sulfate(11) . Columns were operated at a flow rate of 1 ml/min.

Thin Layer Chromatography

Samples for characterization of [^3H]NHA were mixed with authentic unlabeled NHA, arginine, citrulline, and ornithine and subjected to chromatography on Eastman Kodak chromagram cellulose plates developed with n-butanol/acetone/diethylamine/water (70/70/14/35, v/v) or on Whatman silica gel plates (PE SG) developed with acetonitrile/acetic acid/water (4/1/1, v/v). After development, the plates were dipped in a solution of 0.2% (w/v) ninhydrin in acetone to localize the authentic amino acids and then immediately cut into 0.5 cm strips for localization of radioactivity.

Determination of Inhibition by Anaerobiosis and Carbon Monoxide

The reaction mixture (100 µl) for measurement of NHA synthesis under argon contained the components at the concentrations shown for the standard reaction mixture, with the exceptions that arginine was present at 2.5 µM and NOS was omitted. Glucose (4.7 mM) and glucose oxidase (0.7 units) were added as an oxygen scavenger. The reaction mixture, contained in a glass tube, was placed in a Waters vacuum vial and successively evacuated and flushed with argon a total of eight times. The vial was then briefly opened to admit rat cerebella NOS (15 µg) to the mixture, and 0.5 ml of a freshly prepared solution of 0.1 M sodium dithionite in 0.25 M TrisbulletHCl, pH 9.0, was added to the vial. The reaction mixture was again flushed with argon as described and then incubated for 30 min. Measurement of the overall reaction (citrulline synthesis) under argon was similarly determined in a reaction mixture identical to that described except for addition of 667 µM NADPH, 0.15 µM arginine, and 31 ng NOS; [^3H]citrulline was measured by chromatography on Dowex 50 H(8) . Similar procedures were followed for measurements in air except for omission of glucose from reaction mixtures and sodium dithionite from reaction vials.

For determination of the amount of NHA synthesized under carbon monoxide, reaction mixtures were the same as described for the standard reaction mixture, except that the arginine concentration was 11.2 µM. Reaction mixtures (NOS and calmodulin omitted) were pipetted into a Pierce Reactivial (0.3 ml), which was then sealed with a Teflon/silicone disc and flushed for 5 min at room temperature with a mixture of carbon monoxide/oxygen (4/1, v/v). The gas mixture entered the Reactivials through a hypodermic needle and was allowed to impinge on the surface of the reaction mixture, which was shaken frequently to facilitate equilibration. After the initial equilibration, cloned NOS (1.6 µg) was injected with a Hamilton syringe, and equilibration with the gas phase continued for another 5 min. Reactions were started by injection of calmodulin with a Hamilton syringe to a final reaction volume of 14 µl. The final reaction mixture was incubated for 5 or 15 min. For determination of the overall reaction (citrulline synthesis), the above procedure was modified to include 667 µM NADPH and NOS (2.3 ng) in a final volume of 42 µl. Procedures for measurements under air were identical except for substitution of air for the carbon monoxide mixture.

Determination of Enzyme-bound Arginine

Tris buffer, which interferes with the Pico-Tag procedure, was removed from cloned NOS (255 µg) by gel filtration with Sephadex G-25 equilibrated with 50 mM potassium Hepes, 100 mM NaCl, 0.1 mM EDTA, and 0.1 mMp-aminobenzenesulfonyl fluoride, pH 7.4. Aliquots of this preparation were removed for determination of protein and NHA synthesis. The remaining preparation was mixed with marker [U-^14C]arginine (5300 dpm, 7.2 pmol) and trichloroacetic acid to a final concentration of 4.5% (w/v). Denatured protein was removed by centrifugation, and the supernatant solution was extracted with 3.5 volumes of fresh ether; extractions were repeated three times. This material was then subjected to Pico-Tag analysis with the use of the protocol described for the 30-cm Pico-Tag free amino acid analysis column (Waters Manual WM02, revision 1). Fractions from the column were collected and assayed for radioactivity. Ninety percent of the recovered radioactivity comigrated with a retention time (approximately 17 min) characteristic of the phenylthiocarbamyl derivative of authentic arginine. The negligible ultraviolet peak derived from the NOS preparation and corresponding with this radioactive derivative was quantitated by comparison with separate runs of authentic arginine (containing [^14C]arginine) and was corrected for small differences in recovery of [^14C]arginine.

Determination of Enzyme-bound NADPH

NADPH content of NOS preparations was determined by the cycling method of Lowry et al. (12) as modified by Matschinsky(13) . Specificity for NADPH is conferred by preincubation of the sample for 10 min at 60 °C in 0.02 M NaOH, 1.5 mM cysteine to destroy any oxidized pyridine nucleotides followed by cycling in the presence of glucose-6-phosphate dehydrogenase and NADPH-specific glutamate dehydrogenase. A standard curve for NADPH was determined by carrying authentic NADPH through the procedure. Final NADPH content of NOS was corrected for the relatively small fluorescence values measured in the absence of NADPH and in NOS preparations carried through the cycling procedure in the absence of cycling enzymes. NADPH contents of NOS were also corrected for recovery of authentic NADPH (70%) added to the preparation.

Gel Filtration of Products of First Partial Reaction

Cloned NOS (12.8 µg) was incubated with 11 µM [^3H]arginine and the remaining components of the standard reaction mixture for assay of the first partial reaction (final volume, 46 µl). The reaction was incubated for 30 min at 25 °C and then stopped by the addition of 1 µl of 100 mM EDTA and cooled to 4 °C. One sample of 14 µl was mixed with perchloric acid. Another sample was immediately applied to a 200-µl bed of Sephadex G-25 medium (Pharmacia) equilibrated at 4 °C with 50 mM sodium acetate, 2 mM EDTA, pH 6.5. Based on a calibration determined with blue dextran 2000 (Pharmacia), the column was eluted with the same buffer to yield protein and non-protein fractions. Each of the samples was then assayed for [^3H]NHA as described above.

Miscellaneous

Protein was determined with the bicinchoninic acid method (Pierce manual 23225X) with the use of a bovine serum albumin standard.


RESULTS

Formation of NHA and Citrulline from Arginine

Fig. 1illustrates the radioactive products formed during the NOS-catalyzed oxidation of [^3H]arginine. In the absence of added NADPH (Fig. 1, panel A), the major radioactive product (93% of the total) comigrates with authentic NHA marker. The other enzyme-dependent radioactive product comigrates with the authentic citrulline marker. Upon addition of NADPH (Fig. 1, panel B), radioactivity appears exclusively in citrulline. Fig. 1, panel C, in which NOS was omitted, demonstrates the enzyme dependence of the reactions.


Figure 1: Products derived from [^3H]arginine in the absence and presence of NADPH. Panel A, reaction was incubated for 30 min and contained 2 µg of rat cerebellar NOS and the components specified in the standard reaction mixture for assay of the first partial reaction. Panel B, addition of 670 µM NADPH. Panel C, same as panel A except for omission of enzyme. ^3H is shown as histograms. The truncated values for ^3H in arginine (panel A) are 3.58 and 4.80 times 10^4 (37 and 38 min, respectively), and for citrulline (panel B) the values are 4.07 and 4.80 times 10^4 (9 and 10 min, respectively). Positions of carrier citrulline and NHA determined by fluorescamine assay are shown as solid squares. These values have been displaced on the y axes to avoid confusion with the values for ^3H dpm.



The dominant radioactive product formed under the conditions used in Fig. 1, panel A , was characterized as [^3H]NHA by its comigration with authentic NHA during HPLC on cation exchange and reverse phase ion pair columns and thin layer chromatography on cellulose and silica gel. A summary of the chromatographic properties of the radioactive product compared with those of related amino acids is given in Table 1. Further evidence for the identity of the radioactive product with NHA is provided by its conversion predominantly to [^3H]citrulline when incubated with rat cerebellar NOS (see below).



Table 2summarizes the effects of components of the reaction mixture on the synthesis of radioactive products from [^3H]arginine. Omission of Ca/calmodulin or BH(4) resulted in marked decreases in [^3H]NHA synthesis. Omission of catalase increased the proportion of radioactivity in citrulline without affecting the total radioactivity in NHA plus citrulline. In the presence of added NADPH, total ^3H incorporated into product was increased 15-fold, and radioactivity was detected only in citrulline (see also Fig. 1, panel B).



Time Course and Effect of BH(4) on Formation of NHA and Citrulline from Arginine

Fig. 2illustrates the time course of formation of [^3H]NHA and [^3H]citrulline from [^3H]arginine in the absence or presence of BH(4). At all times studied, [^3H]NHA comprised at least 83% of the total radioactive products in the presence of BH(4) and at least 91% in the absence of BH(4). The presence of BH(4) resulted in approximately 3-fold increases in [^3H]NHA formation and even greater increases in [^3H]citrulline formation. Under the conditions of this experiment, the conversion of [^3H]arginine to products was essentially complete after 30 min. Cessation of [^3H]NHA formation was not caused by inactivation of the enzyme; assays of aliquots of the BH(4)-supplemented reaction mixture removed at 30 and 60 min showed NOS activities (measured in the presence of NADPH) of 65% that of an aliquot removed at zero time. In a separate experiment, we examined whether the yield of [^3H]NHA was increased by the addition of a 45-fold excess of unlabeled NHA (8.5 µM) over [^3H]arginine during a 60-min incubation. Under these conditions 98% inhibition of ^3H incorporation into NHA was observed.


Figure 2: Time course and effect of BH(4) on products derived from [^3H]arginine. Conditions are those described in Fig. 1(panel A) except for omission of BH(4) where indicated. Values of pmol of [^3H]NHA and [^3H]citrulline were calculated by dividing the amounts of radioactivity in these compounds by the specific activity of [^3H]arginine.



Conversion of NHA to Citrulline

Fig. 3, panel A, illustrates the conversion of [^3H]NHA to [^3H]citrulline in the presence of added NADPH. No conversion took place when NADPH was omitted (Fig. 3, panel B) or when NOS was omitted (Fig. 3, panel C). No detectable activity (leq10% of the complete reaction) was observed when calmodulin was omitted (data not shown). Omission of BH(4) decreased activity to 44% of the control, whereas omission of catalase had little or no effect (data not shown).


Figure 3: Enzymic conversion of [^3H]NHA to [^3H]citrulline. The complete system (described under ``Methods'') is shown in panel A. NADPH is omitted in panel B. Panel C is the same as panel A except for omission of NOS. Symbols are the same as for Fig. 1.



Effect of Enzyme and Arginine Concentration on NHA Synthesis

The amount of [^3H]NHA formed increased with the amount of NOS present (Fig. 4, panel A). NHA formation also increased with argininine concentration to a value of 0.13 NHA/NOS subunit at 20 µM arginine (Fig. 4, panel B). These results indicate that even higher ratios of NHA/NOS might have been obtained at concentrations of arginine greater than 20 uM. The effects of higher concentrations of arginine were not examined because they reduced the sensitivity of the assay to an unacceptable level. It should be noted that the values of NHA synthesis were measured over a 30-min incubation period, during which time the reaction had gone to completion (Fig. 2). This was confirmed in separate experiments that showed that the same relationships depicted in Fig. 4were observed for incubation periods of 60 and 120 min. Each of the values for NHA synthesis shown in Fig. 4, therefore, represent maximum amounts of product formed rather than initial rates of synthesis.


Figure 4: Effect of enzyme and arginine concentration on NHA synthesis. Reactions were as described for the standard reaction mixture except that those of panel A contained 2.6 µM arginine and varying amounts of cloned NOS, whereas those of panel B contained varying concentrations of arginine and 2.8 µg (17 pmol) of rat cerebellar NOS. All reactions were incubated for 30 min.



Synthesized [^3H]NHA Is Not Tightly Bound to the Enzyme

The products of the first partial reaction were subjected to fractionation by a gentle procedure (gel filtration at 4 °C) designed to permit the detection of any [^3H]NHA bound to the enzyme. [^3H]NHA was recovered in the combined non-protein and protein fractions in 98% yield. Of the recovered [^3H]NHA, 97% was recovered in the non-protein fraction, suggesting that, under the conditions used for assay of the first partial reaction, [^3H]NHA does not remain bound to the enzyme.

Effects of Anaerobiosis and Carbon Monoxide Inhibition

The effect of anaerobiosis was determined in experiments in which air was replaced by argon, and the effect of carbon monoxide was determined by replacing air with a mixture of carbon monoxide/oxygen (4/1, v/v). Replacement of air by argon caused a potent inhibition of both NHA (94%) and citrulline (91%) synthesis. The carbon monoxide/oxygen mixture caused 66% inhibition of citrulline synthesis (measured over 15 min). The effect of the carbon monoxide/oxygen mixture on NHA synthesis was determined over two time periods: at 15 min, a time at which the reaction had gone to completion (see Fig. 2) and at 5 min, which gives a value better approximating a true rate. Compared with a 66% inhibition of citrulline synthesis, relatively modest inhibitions of NHA synthesis of 19 and 28%, respectively, were observed.

[^3H]NHA Synthesis Is Not Light-dependent

The possible role of photoreduction of enzyme-bound heme (see ``Discussion'') in the conversion of arginine to NHA was examined by comparing [^3H]NHA synthesis in reactions incubated either under routine assay conditions (ambient light) or in the dark. [^3H]NHA formation was determined after 5, 25, and 60 min of incubation. Identical amounts of [^3H]NHA were formed under the two conditions (data not shown), indicating that neither the rate (approximated by the 5-min value) or the final amount (approximated by the 25- and 60-min values) of NHA synthesis is light-dependent.

Effect of Pretreatment of NOS with Ferricyanide

Ferricyanide oxidizes tetrahydropterins (14) and has been used to oxidize the flavin semiquinone of NOS(15) . The effect of pretreatment of NOS with ferricyanide was examined in order to assess the possible role of the flavin semiquinone and BH(4) in NHA synthesis. In these experiments, rates of the overall reaction (NADPH included in the assay) and of the first partial reaction (NADPH omitted from the assay) were determined for the native and ferricyanide-treated enzyme in the absence and presence of BH(4). Table 3shows that, compared with the native enzyme, pretreatment with ferricyanide decreased the overall reaction to 38 and 36% when assayed in the absence or presence of BH(4), respectively. The first partial reaction was more sensitive to ferricyanide treatment of the enzyme. Indeed, ferricyanide treatment actually increases the amount of citrulline formed in the combined absence of NADPH and BH(4). Radioactive accumulation in NHA was decreased to 6-7%, whereas total accumulation in NHA plus citrulline was decreased to 18 and 8%, measured in the absence and presence of BH(4), respectively. For both the overall reaction and the first partial reaction, addition of BH(4) did not restore activity of the ferricyanide-treated enzyme.



When assayed in the absence of NADPH, the relative amount of radioactivity accumulating in citrulline relative to that in NHA was much higher for ferricyanide-treated enzyme. When assayed in the presence of NADPH, no significant accumulation of NHA was detected with the ferricyanide-treated enzyme. These observations are consistent with ferricyanide treatment inhibiting the first partial reaction to a greater extent than the second partial reaction.

Determination of Enzyme-bound Arginine

Spectral evidence of McMillan and Masters (16) indicated that a major portion of NOS as normally isolated from transfected human kidney cells contains tightly bound arginine. As explained below, it was relevant to our studies to determine whether NOS isolated from the same source in our hands also contained bound arginine. Direct determination of arginine in cloned NOS (Table 4) showed an upper limit of 0.06 mol of bound arginine/subunit of NOS. The enzyme preparation, which had been subjected to gel filtration before assay of arginine, synthesized 0.10 mol of NOHA/mol of NOS subunit.



Comparison of NADPH Content of NOS with NHA Synthesis

Table 5compares the NADPH content of two preparations of cloned NOS with the amount of NHA synthesis. NOS contained small amounts of NADPH/subunit, which for each preparation is far less than the amount of NHA formed per subunit.




DISCUSSION

The system described here permits a determination of the characteristics of the first step in nitric oxide synthesis, i.e. the oxygenation of arginine to NHA. One of the most interesting findings is that NHA synthesis proceeds in the absence of added NADPH. This is in sharp contrast to the stringent requirement for added NADPH for further conversion of NHA to citrulline. Synthesis of NHA was stimulated approximately 6-fold by Ca/calmodulin and approximately 3-fold by BH It is well established that BH(4) participates in the second partial reaction(5, 6, 7) , and it has been suggested (17) that the site of action of BH(4) may be restricted to this reaction. Our results clearly demonstrate a role of BH(4) also in the first partial reaction. Omission of catalase did not affect total radioactivity accumulating in NHA plus citrulline but increased radioactivity in citrulline approximately 4-fold at the expense of that in NHA. This effect may result from accumulation, in the absence of added catalase, of hydrogen peroxide, which can then react with NHA in the presence of NOS to form citrulline. Hydrogen peroxide is a major product of NOS activity under conditions of low concentrations of arginine (less than approximately 10 µM) or of BH(4)(18) . Further, hydrogen peroxide in the absence of NADPH can support the conversion of NHA to citrulline catalyzed by murine macrophage NOS(19) .

Ferricyanide treatment of the enzyme appears to preferentially inhibit the first partial reaction. As can be seen in Table 3, the ratio of citrulline to NHA formed increases from 0.02 for the native enzyme to 0.34 for the ferricyanide-treated enzyme. This suggests that ferricyanide treatment of the enzyme may preferentially interfere with its ability to catalyze the conversion of arginine to NHA or with the ability of NHA to dissociate from the enzyme. The observed decrease in the formation of the oxygenated amino acid products (i.e. NHA plus citrulline) by ferricyanide treatment can also be explained by a ferricyanide-mediated inhibition of the conversion of arginine to NHA. Although experimental conditions are not comparable, other redox compounds such as nitroblue tetrazolium and methylene blue have been reported to decrease the ratio of citrulline/NHA formed, suggesting that these compounds may interfere with the conversion of NHA to citrulline(7) .

NOS is believed to belong to the cytochrome P-450 class of enzymes(1) , which show varying susceptibilities to carbon monoxide. This variation has been ascribed to differences in relative affinities of oxygen and carbon monoxide for the ferrous form of heme and differences in the steady-state concentrations of Fe in heme(20) . Inhibition by carbon monoxide/oxygen (4/1) in the range of 60-80% has been reported for the overall conversion of arginine to citrulline (21, 22, 23) and of approximately 30% for conversion of NHA to citrulline (23) . Whereas these combined findings are consistent with a role of heme at least in the second partial reaction, they do not critically address its role in the first partial reaction. In our studies, comparable inhibitions by the carbon monoxide/oxygen mixture of 66% for the overall reaction and 28% for NHA synthesis support a role for heme in the first partial reaction. The requirement of the first partial reaction for calmodulin, which has recently been demonstrated to trigger the transfer of electrons from flavin to heme(24) , provides further support for this suggestion. The apparent role of heme in the first partial reaction argues against the suggestion (25) that this reaction may proceed via a heme-independent mechanism similar to that established for the aromatic amino acid hydroxylases.

The amount of NHA formed was always less than the amount of enzyme subunits added. With low concentrations of arginine such as 189 nM used in the experiments of Fig. 2, a maximum value of 0.02 mol NHA/mol NOS subunit was obtained. This ratio increased with increasing concentrations of arginine (Fig. 4, panel B) to a value of 0.13 at 20 µM arginine. The maximum value of NHA/subunit observed was 0.16 (Table 5). The possibility was considered that ratios of less than unity may reflect the presence of L-arginine bound to the enzyme, as suggested by spectral studies(16) . The presence of nonradioactive enzyme-bound arginine in equilibrium with a small amount of radioactive arginine would lower the specific radioactivity of arginine and its products and lead to the underestimation of the amount of NHA formed. Reference to the results of Table 4shows this not to be the case. Assuming equilibrium between [^3H]arginine (157 pmol) and a maximum of 0.72 pmol (0.06 times 12) of nonradioactive enzyme-bound arginine, the ratio of NHA/NOS calculated in Table 4(0.10) would be underestimated by less than 1%. Even with the extreme assumption that all enzyme-bound arginine is converted to nonradioactive NHA (i.e. with no equilibration between enzyme-bound arginine and added radioactive arginine), the total amount of NHA formed would be increased only from 1.2 pmol to 1.92 (1.2 plus 0.72) pmol, corresponding to an increase in NHA/subunit from 0.10 to 0.16. The reason for the apparent discrepancy between the negligible amounts of bound arginine determined in this work with the values of greater than 0.8 mol of arginine/mol of NOS subunit suggested by spectral studies (16) is not clear. Kinetic studies by Matsuoke et al. (26) also indicate that the purified native cloned enzyme contains little, if any, bound arginine. The differing amounts of enzyme-bound arginine may reflect differences in conditions used to culture the cells or in the procedure used to isolate the enzyme.

Two reasons can be suggested to explain why values of less than unity were observed for the ratio of NHA/NOS subunit. First, NOS may be heterogeneous in the sense that not all species contain the putative enzyme-bound factor (see below) required for NHA synthesis. Second, formation of NHA may not proceed to completion because this compound competes with arginine for binding to the enzyme. This reason seems less likely based on the observation that the product NHA is recovered in the non-protein fraction obtained by gel filtration at 4 °C. However, the possibility cannot be excluded that NHA remains enzyme-bound during the assay but is dissociated during the gel filtration step. Indeed, spectral studies of the cloned enzyme (16) indicate that NHA may be able to displace bound arginine, a notion that is consistent with our observation that a pool of unlabeled NHA essentially abolished the incorporation of ^3H from arginine into NHA. Based on the evidence presented, it is proposed that NOS contains one or more endogenous enzyme-bound factors that substitute for NADPH in the formation of NHA. It is generally believed the path of electron flow in NOS resembles that proposed for NADPH-cytochrome P-450 reductase(15, 27) , with which it shares close sequence homology(1) .

In analogy with current ideas about the mechanism of oxygenation catalyzed by cytochrome P-450(28, 29) , it has been suggested(7, 23) that ferrous heme formed in this electron chain complexes with oxygen. Further addition of an electron to this ferrous-oxygen complex, followed by electron rearrangement, converts it to a ferric peroxide complex. This complex or its fission product, perferryl oxygen, is believed to be the oxygenation species.

A priori, the endogenous enzyme-bound factor could be either a reductant that generates an oxygenation species, or an oxygenation species itself. The oxygen dependence for NHA synthesis is consistent with the endogenous factor being a reductant rather than an oxygenation species. It is therefore suggested that synthesis of NHA in the system described here proceeds as follows.

NHA is shown here as dissociated from the enzyme, based the finding that NHA appears in the non-protein fraction following gel filtration.

Possible candidates for this reductant are discussed below.

Light

Many heme-containing proteins, including cytochrome P-450, are easily photoreduced(30, 31, 32, 33) . Since NHA synthesis was routinely studied under ambient lighting conditions, we examined the possibility that photoreduction was involved in this process. The finding that neither the rate nor final amount of NHA synthesis was affected by incubation in the dark clearly excludes a role of photoreduction.

NADPH

Any free NADPH present in the eluate from the 2`,5`-ADP column would have been decreased to a negligible value by gel filtration (cloned enzyme) or by serial dilutions (enzyme from rat brain). It was estimated that the five serial dilutions routinely employed would have decreased any free NADPH to less than one-thousandth of the amount of NOS on a molar basis. Furthermore, direct assays showed enzyme-bound NADPH to be appreciably less than the amount required for NHA synthesis (Table 5). These findings, therefore, argue against NADPH playing a major role as the endogenous reductant.

Flavins

NOS contains 1 mol each of tightly bound FAD and FMN per NOS subunit(1) , and flavin semiquinone has been detected in resting preparations of NOS isolated from macrophage and brain(15) . Studies of cytochrome P-450 reductase indicate that the air-stable semiquinone form, (FMNH) (FAD) is itself not a reductant but requires initial reduction by NADPH to form the species (FMNH) (FADH(2)), which is then converted to the actual reductant, (FMNH(2)) (FADH)(27, 34, 35) . The flavin species involved in the electron flow catalyzed by NOS have not been elucidated. A role of the flavin semiquinone as a source of endogenous electrons remains a possibility in light of our finding (Table 3) that the first partial reaction is more sensitive to ferricyanide pretreatment on NOS than is the overall reaction.

Heme

NOS contains 1 equivalent of cytochrome P-450-type iron protoporphyrin IX per subunit(1) . This prosthetic group itself is unlikely to act as a reductant since any ferrous heme, after complexing with O(2), requires further input of one electron to form an oxygenation species (see and associated text). Moreover, the cloned enzyme, as purified, is mainly a ferric species(16) .

BH(4)

Whereas BH(4) is known to be involved in NOS activity(1) , a reductive role for this cofactor has not been reported. The BH(4) content of NOS preparations used in these studies ranged between 0.3 and 0.4 mol/mol of NOS subunit, more than adequate for the amounts of NHA formed. Although the decrease in NHA synthesis associated with the ferricyanide-treated enzyme (Table 3) is consistent with a role of BH(4), activity was not restored by addition of BH(4). This finding should be interpreted with caution, however, since added BH(4) may not displace any enzyme-bound BH(2) formed by ferricyanide oxidation of enzyme-bound BH(4).

In summary, of the redox factors believed to be involved in NOS function, neither NADPH, flavins, nor heme appears to account for the endogenous reductant supporting NHA synthesis. Photoreduction was also excluded as a source of electrons. This work does not exclude a possible role of BH(4). Using a sensitive method developed for specific assay of BH(4) in the presence of the quinonoid form of dihydrobiopterin, we are currently examining the possible role of enzyme-BH(4) in the oxygenation of arginine. (^2)

A question with important mechanistic implications posed by these findings is why the putative endogenous reductant is available for oxygenation of arginine but not of NHA. One explanation proposed is that arginine has a higher affinity for NOS-reductant than does NHA. Another interesting possibility is that electrons ultimately donated from NADPH may proceed in different domains, each one subserving only one of the partial reactions. It is interesting to note that recent sequence and structural homology studies (^3)indicate the presence of a second NADPH binding site, distinct from the NADPH binding site associated with FAD on NOS. Further, resonance Raman scattering studies (36) of NOS have been interpreted to suggest that either there are two different heme-protein conformations or that the two protein subunits bind heme differently.


FOOTNOTES

*
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.

§
Present address: Dept. of Health Services, San Diego County Psychiatric Hospital, 3851 Rosecrans St., San Diego, CA 92110.

To whom correspondence should be addressed: NIHMH, DIRP, Laboratory of Neurochemistry, Bldg. 36, Rm. 3D30, 36 Convent Dr., MSC 4096, Bethesda, MD 20892-4096. Tel.: 301-402-4896; Fax: 301-480-9284.

(^1)
The abbreviations used are: NOS, nitric oxide synthase; BH 6-(L-erythro-1,2-dihydropropyl)-5,6,7,8-tetrahydropterin; HPLC, high performance liquid chromatography; NHA, N^G-hydroxyarginine.

(^2)
C. F. B. Witteveen, J. Giovanelli, and S. Kaufman, unpublished results.

(^3)
J. C. Salerno, personal communication.


ACKNOWLEDGEMENTS

We thank Dr. Sheldon Milstien of this laboratory for determinations of the amount of BH(4) in various preparations of purified NOS; Dr. Peter Backlund, Jr. (Laboratory of General and Comparative Biochemistry) for determination of arginine by Pico-Tag analysis; Kun Park of this laboratory for determination of amino acids with fluorescamine; Drs. Bettie Sue Siler Masters and Kirk McMillan (University of Texas Health Science Center, San Antonio, TX) for an inoculum of transfected human kidney 293 cells and advice on their culture; and Dr. C. W. Witteveen of this laboratory for preparations of the cloned enzyme and for helpful discussions and critical reading of the manuscript.


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