©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Neuronal Nitric Oxide Synthase
EXPRESSION IN ESCHERICHIA COLI, IRREVERSIBLE INHIBITION BY PHENYLDIAZENE, AND ACTIVE SITE TOPOLOGY (*)

(Received for publication, March 14, 1995; and in revised form, April 21, 1995)

Nancy Counts Gerber Paul R. Ortiz de Montellano (§)

From the Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, California 94143-0446

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A gene coding for rat neuronal nitric oxide synthase (nNOS) has been cloned into pCWori and the vector has been expressed in Escherichia coli. The expressed enzyme has been purified with a final yield of purified protein of approximately 1 mg/g of wet cells. The recombinant protein reconstituted with calmodulin and Ca exhibits spectroscopic and catalytic properties identical to those reported in the literature for nNOS. Reaction of recombinant nNOS with phenyldiazene produces a phenyl-iron (FePh) complex with a maximum at 470 nm. Formation of this complex is paralleled by inactivation of the enzyme and is inhibited by arginine, the natural substrate of the enzyme. Phenyl-iron complex formation does not alter the rate of electron transfer from the flavin domain to cytochrome c. Addition of ferricyanide triggers migration of the phenyl residue from the iron to the porphyrin nitrogens. The N-phenylprotoporphyrin isomers with the phenyl on the nitrogens of pyrrole rings B, A, C, and D are formed in, respectively, approximately a 14:20:21:45 ratio. The regioisomer pattern indicates that the active site of NOS is open to some extent above all four pyrrole rings but more so above pyrrole ring D. Arylhydrazines are thus not only a new class of inhibitors of nNOS but provide useful information on the active site topology of the enzyme.


INTRODUCTION

Nitric oxide (NO) functions physiologically as a neurotransmitter and vasodilator and pathologically as a cytotoxic agent. Nitric oxide is produced by nitric oxide synthases (EC 1.14.13.39, NOS)()that catalyze the conversion of L-arginine to nitric oxide and citrulline (Fig. 1)(1, 2, 3, 4, 5) . Two general categories of NOS are known: (a) constitutive enzymes that are regulated by Ca and calmodulin, and (b) inducible enzymes with a tightly bound calmodulin unit that are not regulated by Ca. Both types of NOS consist of a heme and a two-flavin domain linked by a polypeptide that serves as the binding site for calmodulin.


Figure 1: Steps in the NOS-catalyzed oxidation of arginine to nitric oxide.



Neuronal nitric oxide synthase (nNOS) is one of the constitutive, calmodulin-regulated forms of the enzyme. In the absence of calmodulin, limited proteolysis cleaves the protein into heme and flavin domains that retain partial structural and functional integrity(6) . The flavoprotein domain contains one FAD and one FMN as prosthetic groups and has extensive sequence identity with cytochrome P450 reductase(7) . The absorption and resonance Raman spectra of the heme domain(8, 9, 10) indicate that the heme group is coordinated, as in cytochrome P450, to a cysteine thiolate(8, 9, 10, 11) . Site-specific mutagenesis of the cysteine residues proposed to be the iron ligands in endothelial (Cys->Ala) and neuronal (Cys->His) NOS produces inactive proteins(12, 13) . Sequence comparisons with the known cytochrome P450 enzymes, however, reveal no identifiable sequence identity with the exception of 4 or 5 residues within a decapeptide surrounding the proposed cysteine iron ligand (F/W-X-X-A/G-X-R-X-C-hydrophobic aliphatic-G)(14) . The flavin domain is thus closely related to cytochrome P450 reductase, but the heme domain is virtually unrelated to cytochrome P450. Nevertheless, the catalytic chemistry of NOS, involving hydroxylation of L-arginine to N-hydroxyl-L-arginine followed by oxidation of this intermediate to nitric oxide and citrulline, is reminiscent of P450-catalyzed transformations.

Despite the differences in their sequences, the presence of a thiolate iron ligand and the analogy between the catalytic chemistries of cytochrome P450 and NOS suggest that approaches used to characterize the P450 monooxygenases may be useful in defining the structure and function of NOS. Recent work from this laboratory has established that myoglobin, catalase, and cytochrome P450 react with phenyldiazene (PhN=NH), or its precursor phenylhydrazine (PhNHNH), to give stable -bonded phenyl-iron complexes (Fig. 2) (15, 16, 17, 18) . Formation of these complexes results in loss of hemoprotein function. The structures of the phenyl-iron complexes obtained with myoglobin and cytochrome P450 have been confirmed by x-ray crystallography(16, 17) . Oxidation of the P450 phenyl-iron complexes with ferricyanide results in migration of the phenyl group from the iron to the heme porphyrin nitrogens within the intact protein active site(19, 20, 21) . The four nitrogen atoms of the porphyrin are distinguishable due to the substitution pattern at the heme periphery (Fig. 2). The extent to which the phenyl group migrates toward each of the pyrrole nitrogens can therefore be determined by HPLC analysis of the N-phenylporphyrins derived from the N-phenyl hemes(22) . As the shift occurs within the intact active site, the N-phenylporphyrin ratio provides information on the location, with respect to a grid defined by the heme group, of active site residues that interfere with migration of the phenyl group. Although phenyl-iron complexes are obtained with various hemoproteins, migration of the phenyl group within the intact active site has only been observed with thiolate-ligated hemoproteins (i.e. cytochrome P450 and chloroperoxidase)(15, 18, 19, 20, 21, 23) .


Figure 2: Scheme illustrating the reaction of phenyldiazene with the prosthetic group of a hemoprotein to give a phenyl-iron complex from which the phenyl migrates to the porphyrin nitrogen atoms. The reagent for stepa is PhN=NH and for step b is KFe(CN). The structure of the N regioisomer of N-phenylprotoporphyrin IX obtained by removal of the iron from the corresponding N-phenylheme is shown (V = CH=CH, p = CHCHCOH). The N, N, and N regioisomers bear the phenyl group on the corresponding pyrrole nitrogen atoms.



We report here the first successful expression of catalytically active nNOS in Escherichia coli. The recombinant enzyme has been used to demonstrate that nNOS is inactivated by reaction with phenyldiazene in a reaction that produces a phenyl-iron complex. We have also established that ferricyanide promotes migration of the phenyl group within the intact active site and have used this reaction to explore the active site topology of nNOS.


EXPERIMENTAL PROCEDURES

Materials

L-Arginine and L-citrulline were obtained from Aldrich. 2`,5`-ADP-Sepharose, calmodulin-Sepharose 4B, and Phast System products were from Pharmacia LKB Biotechnol. Calmodulin, (6R)-5,6,7,8-tetrahydrobiopterin and N-hydroxy-L-arginine were from Alexis Biochemicals (San Diego, CA). Bradford protein assay kits, Chelex-100, and Dowex 50W-X8 were from Bio-Rad. BioScint scintillation mixture was from Fisher. Restriction enzymes, Bacto yeast extract, IPTG, and DH5 E. coli cells were purchased from Life Technologies, Inc.. L-[2,3,4,5-H]Arginine was purchased from Amersham Corp. Methylphenyldiazenecarboxylate azo ester was purchased from Research Organics, Inc. (Cleveland, OH). All other reagents and materials were from Sigma. The cDNA clone for rat brain NOS was a gift from Solomon Snyder (Johns Hopkins University) (6) . The pCWori vector was a gift from Robert Fletterick (University of California, San Francisco)(24) .

Absorption spectra were recorded on an Aminco DW2000 spectrophotometer and were transferred to a Macintosh computer for analysis and display. High pressure liquid chromatography was performed on a Hewlett Packard 1090 HPLC system.

Cloning

The polymerase chain reaction was used to amplify a region of the nNOS gene between the initiator methionine and the unique ApaI site. This allowed the removal of 348-base pairs of non-coding sequence and introduction of a unique NdeI site for cloning purposes. This product was ligated into the vector pCRII (Invitrogen, San Diego), then into the vector Bluescript (SK) containing the nNOS gene using KpnI and ApaI restriction sites. The gene was cloned into pCWori using the NdeI and XbaI sites to give pCWnNOS that was used to transform competent E. coli DH5 cells. The subcloned fragment was sequenced by the method of Sanger et al.(25) .

Expression

An overnight culture of pCWnNOS was used to inoculate 2 liters of 2xYT media in a 2.8-liter Fernbach flask. Casein N-Z plus hydrolyzate (Sigma) was used in place of tryptone in the media. The cultures were grown to an OD of 0.6-0.8 and induced with 1 mM IPTG. The cultures were then grown at 22 °C at a rotation rate of 190 revolutions/min for 16-20 h before they were harvested by centrifugation. Expression of nNOS was checked by SDS-PAGE and Western blotting using an antibody raised against a section of the heme-domain of nNOS.()

Purification

The purification sequence was typically carried out with the protein from 4 to 8 liters of culture. Due to the instability of nNOS, the purification was carried out in the presence of 10% glycerol within a period of 1 day, and the resultant protein was quickly frozen in liquid nitrogen and stored at -70 °C. The protein was purified using a modification of a published protocol(26, 27) . The cells were lysed in Buffer A (50 mM Tris-Cl (pH 8.0) buffer containing 0.5 mML-arginine, 10 µM BH, 5 mM 2-mercaptoethanol, 10% glycerol, and protease inhibitors (1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 1 µM leupeptin, 1 µM pepstatin, 1 µg/ml antipain), 2 mg/ml lysozyme, 32 units/ml DNase I, and 3 units/ml RNase A). The cells were sonicated briefly then centrifuged to sediment the cell debris. The supernatant was brought to 2 mM CaCl and the protein loaded onto a 15-ml calmodulin-Sepharose column (1.8 2.5 cm) equilibrated with Buffer A containing 2 mM CaCl. The column was washed with 100 ml of Buffer B (50 mM HEPES (pH 7.5) containing the same protease inhibitors as Buffer A, 2 mM CaCl, and 0.3 M NaCl) and eluted with Buffer B supplemented with 0.3 M NaCl and 5 mM EGTA. The protein was then loaded onto a 5-ml 2`,5`-ADP-Sepharose column equilibrated with Buffer B supplemented with 0.3 M NaCl. The column was first washed with 50 ml of Buffer B containing 0.5 M NaCl and then with 50 ml of Buffer B minus L-arginine. Neuronal NOS was eluted with 10 ml of Buffer B minus L-arginine plus 10 mM NADPH. The NADPH was removed by passing the protein over a small calmodulin affinity column. The protein was concentrated (if necessary) to a concentration of greater than 1 mg/ml, aliquoted into small vials, and quickly frozen in liquid nitrogen. The enzyme was stored at -70 °C. Multiple freeze-thaw cycles were avoided in using the protein.

Enzyme Assays

The activities of the protein preparations were determined by measuring either the production of NO using the conversion of HbO to met-Hb or by measuring the conversion of L-[H]arginine to L-[H]citrulline(27) . The conditions of the NO assay were 50 mM HEPES (pH 7.5), 10 µM BH, 100 µM DTT, 100 µM EDTA, 2 mM CaCl, 10 µg/ml calmodulin, 5-10 µM HbO, 100 µM NADPH, 1-2 µg of nNOS, and 10 µML-arginine. The initial rates of NO production were determined at 37 °C from A ( = 38 mM) or, if catalase was present, from A ( = 12 mM)(27) . The conversion of L-arginine to L-citrulline was determined under the following conditions: 50 mM HEPES (pH 7.5) containing 100 µM BH, 100 µM DTT, 100 µM EDTA, 2 mM CaCl, 10 µg/ml calmodulin, 100 µM NADPH, 0.5 µg of nNOS, 16 nML-[2,3,4,5-H]arginine (20 µCi/ml), and 0.5-50 µML-arginine in a final volume of 50 µl. The assay was initiated by the addition of nNOS and carried out at 25 °C. The reaction was quenched after 5 min by the addition of 200 µl of ice-cold 0.1 M HEPES (pH 5.5) containing 5 mM EGTA, and the samples were boiled for 1 min. The samples were then passed over a 1-ml Dowex 50W-X8 column (Na form), and the eluents were collected in individual scintillation vials. The column was washed two times with double-distilled HO, and the eluent from the first wash was pooled with the eluent obtained during sample loading. BioScint scintillation mixture (10 ml) was added to each sample, and the amount of radioactivity present was determined by liquid scintillation counting. A control sample containing either no NADPH or no nNOS was included for background determination. The reduction of cytochrome c by nNOS was performed at 25 °C in a volume of 400 µl using an extinction coefficient of 21 mM cm at 550 nm. The reaction mixture contained 50 mM HEPES (pH 7.5), 100 µM EDTA, 100 µg/ml bovine serum albumin, 2 mM CaCl, 4 µM FAD, 4 µM FMN, 10 µM BH, 100 µM DTT, 500 µM NADPH, 50 µM cytochrome c, 50 units of catalase, and 1 µg of NOS and was initiated by the addition of cytochrome c. Some assays contained, as indicated, 10 µML-arginine and/or 10 µg/ml calmodulin.

Protein Determination

Protein concentrations were determined by the Bradford protein assay (28) or micro-assay (Bio-Rad) using bovine serum albumin as a standard. Concentrations of heme-containing NOS were determined from the absorption spectrum of the protein using the extinction coefficients = 72 mM cm for the ferric enzyme and = 76 mM cm for the ferrousCO complex(29) .

SDS-PAGE

Electrophoresis was carried out using a Pharmacia Phast Gel system according to the manufacturer's instructions. Homogenous 7.5% gels were used with SDS buffer strips and were stained with Coomassie Blue. Western blotting was carried out using the Phast Transfer system, and nNOS was detected using an antibody raised against the heme domain of nNOS and an alkaline phosphatase detection system.

Formation of Iron-Aryl Complexes

To form a phenyl-iron complex of nNOS, 1-2 µl of a stock solution of phenyldiazene was added to either 0.5 or 1 ml of a 1 mg/ml solution of nNOS (3-6 nmol) in 50 mM HEPES (pH 7.5) containing 10% glycerol and 10 µM BH. In some experiments the incubation also contained 0.5 mML-arginine. The stock solution was made immediately prior to use by mixing 1 µl of phenyldiazene carboxylate azo ester in 100 µl of an aqueous 1 N NaOH solution. The formation of a phenyl-iron complex was followed spectrophotometrically by the appearance of a maximum at 470 nm and a trough at 416 nm in a difference spectrum. When complex formation reached a maximum, five 1-µl aliquots of a 63 mM potassium ferricyanide solution were added to the cuvette over a period of 20 min to shift the phenyl group of the complex to the pyrrole nitrogens. The reaction was then added to 10 ml of an ice-cold solution of 5% sulfuric acid in acetonitrile and left overnight at 4 °C. The N-phenylprotoporphyrin IX regioisomers were isolated by removing the organic phase, adding 2 ml of an aqueous 5% sulfuric acid solution, and extracting the porphyrin adducts three times with CHCl. The combined organic extracts were concentrated to dryness on a rotary evaporator, and the residue was taken up in 100 µl of HPLC solvent A (6:4:1 methanol/water/acetic acid) for analysis. The products were analyzed by HPLC as described previously(21, 30) .

Inhibition Studies with Phenyldiazene

The phenyl-iron complex was formed as described above in a cuvette at 25 °C. At each time point a spectrum of the solution was taken, and an aliquot of the reaction was removed and added to the reaction mixture described above for the determination of citrulline formation. The reaction was allowed to proceed for 5 min before quenching with the EGTA stop mix. The samples were analyzed as described above. To determine the effect of phenyldiazene on the kinetics of cytochrome c reduction, an aliquot of nNOS (typically 10 µg) was placed on ice, and 1 µl of phenyldiazene solution was added. The stock solution of phenyldiazene was prepared as above except immediately before use it was diluted 1:50 with 50 mM HEPES (pH 7.5) to buffer the NaOH added in preparing the solution. Aliquots were taken at various time points and added to a reaction mixture as described above.


RESULTS

Expression of nNOS in E. coli

Catalytically active NOS has been heterologously expressed in mammalian cells and, more recently, in insect cell systems(9, 12, 13, 31, 32, 33, 34, 35, 36, 37) . Although expression of the separate heme and flavin domains in E. coli has been reported(38) , expression of an intact, catalytically active NOS in E. coli has not been described. The first task addressed in this study was therefore expression of nNOS in E. coli.

Cloning of an nNOS gene into pCWori, transformation of E. coli DH5 cells with the resulting vector, and growth of the cells at low temperature (22 °C) resulted in expression of functional nNOS as determined by Western blotting and activity assays. Purification of the enzyme in the presence of a mixture of proteolytic enzyme inhibitors was achieved by two-step affinity chromatography, first on a calmodulin-Sepharose 4B column and subsequently on a 2`,5`-ADP-Sepharose column (Table 1). The procedure typically yields 1 mg of protein/g of wet cells that is greater than 80% pure by SDS-PAGE and absorption spectroscopy. The principal contaminants of the protein are shown by SDS-PAGE and Western blotting to be proteolytic fragments of nNOS (Fig. 3). When cultures were grown at temperatures above 30 °C, a large amount of insoluble nNOS was produced, but no soluble protein was obtained. As the incubation temperature was decreased, the total amount of nNOS produced also decreased, but the amount of soluble nNOS increased. Increasing the cell density at the time the cells were harvested by increasing the rotation rate, aeration, or growth time after induction by IPTG enhanced the degree of proteolytic digestion. For example, when growth was allowed to continue longer than 20 h, a 70-kDa SDS-PAGE band that cross-reacts with the nNOS antibody increased in intensity with respect to the band for the full-length protein (see Fig. 3). Under controlled conditions, however, the E. coli system is capable of delivering large amounts of purified, catalytically active nNOS. Efforts to further optimize the expression and purification protocols are continuing.




Figure 3: SDS-PAGE analysis of the purified nNOS isolated from E. coli: lane A, molecular weight standards; lane B, crude cell lysate; lane C, sample after calmodulin-Sepharose column; lane D, final sample after the 2`,5`-ADP- Sepharose column. The gel has been stained with Coomassie Blue. The full-length nNOS and hydrolytic fragments are labeled as 1 and 2, respectively. Western blots indicate both bands react with polyclonal antibodies to nNOS.



Characterization of nNOS Expressed in E. coli

The spectra of the purified, recombinant ferric protein in the presence and absence of L-arginine, and of the ferrous carbon monoxide complex of the protein, are shown in Fig. 4. In the absence of substrate, the enzyme has an absorbance maximum at 400 nm with a shoulder at 418 nm. Upon addition of either L-arginine or N-L-hydroxyarginine, the absorbance maximum shifts to 394 nm with a shoulder at 370 nm, in agreement with a shift in the equilibrium from low to high spin. Both forms have a charge-transfer band at 634 nm. The dithionite reduced, CO-bound form has an absorbance maximum at 444 nm. These spectral changes are the same as those seen with native nNOS(29, 39) .


Figure 4: Absorbance spectra of recombinant nNOS in the presence (-) and absence(- - - -) of L-arginine. The spectrum of the reduced, CO-bound protein minus the reduced protein is shown in the inset. The samples are in 50 mM HEPES (pH 7.5) containing 10 µM BH.



The recombinant protein is active with a V at 25 °C of 150-400 nmol NO minmg (25-65 nmol minnmol) nNOS and a Kof 0.8 µM (Fig. 5). The variability in the V correlates with the degree of proteolysis in the sample. An apparent loss of activity is seen in incubations with high concentrations (>10 µM) of L-arginine if the concentration of BH is less than 10 µM, as it is in the oxyhemoglobin activity assay. This affect has been observed previously and is due to product inhibition by NO(12, 40) . In agreement with results on the native enzyme, recombinant nNOS is completely inhibited by >5 µMN-nitro-L-arginine and is not inhibited by [D]-arginine. Furthermore, the enzyme is completely dependent for activity on exogenously provided calmodulin and CaCl, and the rate of the reaction is enhanced by exogenous BH. The addition of FAD and/or FMN does not greatly enhance the activity. The enzyme catalyses the reduction of cytochrome c at a rate similar to that reported for the native enzyme (6) and the rate is calmodulin-dependent: 44 µmolminmg with calmodulin versus 3.9 µmolminmg without.


Figure 5: Typical Michaelis-Menton plot of the activity of nNOS with increasing concentrations of L-arginine. The inset is a Hanes plot of the data with the axes [L-arginine]/ versus [L-arginine]. Conditions are as described under ``Experimental Procedures,'' using 0.2 µg of nNOS. The values calculated from the plot are V = 425 nmol citrulline/min/mg nNOS and K = 0.8 µM.



Inactivation of nNOS by Phenyldiazene

The formation of a phenyl-iron complex of nNOS can be followed spectrophotometrically in a difference spectrum by the absorbance increase at 470 nm and the corresponding decrease at 418 nm. Complex formation is much faster in the absence of L-arginine: the rate of complex formation decreases 3-fold, and the maximum degree of complex formation is diminished, in the presence of 50-500 µML-arginine. These results are consistent with a sterically restricted active site that is blocked by the presence of L-arginine. Monitoring the rate of formation of L-citrulline at different time points during the reaction of nNOS with phenyldiazene shows that enzymatic activity decreases in proportion to the extent of formation of the phenyl-iron complex (Fig. 6). As already noted, the rate of NOS-catalyzed cytochrome c reduction is greatly increased by the binding of calmodulin. Calmodulin binding promotes reduction of the heme iron, and this reduction is presumably blocked by the presence of inhibitors that bind to the iron. Formation of the phenyl-iron complex does not affect cytochrome c reduction, however (Fig. 7). This lack of inhibition is seen in the presence or absence of calmodulin, indicating that the cytochrome c reduction rate is unaffected by the state of the heme iron. An inability of inhibitors of the heme site to affect cytochrome c reduction has been reported with L-thiocitrulline, an inhibitor that coordinates to the heme iron(41) .


Figure 6: Time dependence of the formation of the iron-phenyl complex and the inhibition of NOS by phenyldiazene. The formation of the phenyl-iron complex () is determined by the absorbance at 470 nm and the citrulline-forming activity () with respect to the activity of the protein incubated similarly but without phenyldiazene.




Figure 7: Effect of phenyldiazene and calmodulin on the rates of L-citrulline formation and cytochrome c reduction by nNOS. The first three bars are for citrulline formation at 25 °C (in nmol/min/mg protein): in the absence of CaM (A), in the presence of 10 µg/ml CaM (B), and in the presence of 10 µg/ml CaM after preincubation with phenyldiazene (C). The absolute value for the bar in lane B is 125 nmolminmg. The second set of four bars are for cytochrome c reduction at 25 °C (in µmol minng): in the absence of CaM (D), in the absence of CaM after preincubation with phenyldiazene (E), in the presence of 10 µg/ml CaM (F), and in the presence of 10 µg/ml CaM after preincubation with phenyldiazene (G). The absolute value of the bar in lane F is 44 µmol minng. Preincubations with phenyldiazene were for 20 min.



Active Site Topology of nNOS

The nNOS phenyl-iron complex was directly transferred into an acidic acetonitrile solution or was oxidized with potassium ferricyanide prior to being transferred to the acetonitrile solution. When the protein complex was directly added to acidic acetonitrile and the porphyrin products were isolated and quantitated, the four possible regioisomers of N-phenylprotoporphyrin IX were obtained in equal amounts. Previous work has shown that there is no inherent regiospecificity of phenyl group migration when the phenyl-iron heme complex is free in solution(22) . Isolation of the N-phenylprotoporphyrin IX products provides clear evidence for phenyl-iron complex formation in the reaction with phenyldiazene. In contrast to these results, the N-phenylprotoporphyrin IX isomer ratio is not equal when the phenyl-iron protein complex is treated with ferricyanide prior to transfer to acidic acetonitrile. Although the precise values obtained are sensitive to the concentrations of phenyldiazene and ferricyanide that are employed, the ratio of the N-phenylprotoporphyrin IX regioisomers (N/N/N/N in order of elution from the HPLC column) is approximately 14:20:21:45 (Fig. 8). The N-isomer is consistently the most abundant and the N-isomer the least. These results indicate that migration of the phenyl group within the intact active site favors the nitrogen of pyrrole ring D, although there do not appear to be gross differences in the accessibilities of the four pyrrole ring nitrogens.


Figure 8: A typical HPLC trace of the separation of the four N-phenylprotoporphyrin IX regioisomers obtained from phenyldiazene-treated nNOS. The ratio of the N/N/N/N isomers (listed in order of elution from the HPLC column) is typically 14:20:21:45. Isocratic analyses were carried out on a Partisil ODS-3 5 micron column with a solvent mixture consisting of 20% solvent B (10:1 methanol/acetic acid) in solvent A (6:4:1 methanol/water/acetic acid) at a flow rate of 1 ml/min. The detector of the HPLC system was set to monitor the 416 versus 600 nm difference.




DISCUSSION

Expression in E. coli of catalytically competent nNOS makes the enzyme more readily available in large amounts and more accessible to site-specific mutagenesis than it currently is. This should facilitate elucidation of the detailed structure and mechanism of NOS. The recombinant nNOS obtained from E. coli has the same ferric and ferrous CO-complexed spectra as the enzyme from rat brain (Fig. 4). Furthermore, although the enzyme expressed in E. coli is free of calmodulin, reconstitution of the enzyme with exogenous calmodulin yields a protein with the same dependence on Ca and BH, a similar calmodulin-dependent ability to reduce cytochrome c (Fig. 7), and essentially the same Kand V for the oxidation of arginine as the native enzyme (Fig. 5). Furthermore, the recombinant enzyme is inhibited by high concentrations of arginine (presumably via the production of excess NO) and N-nitro-L-arginine in the same manner as the native enzyme. The recombinant and native enzymes thus appear to be identical.

Several classes of NOS inhibitors, in addition to NO itself (12, 33) , are currently known. Arginine analogues such as N-nitro-L-arginine and N-methyl-L-arginine (42) , and imidazole derivatives that coordinate to the heme iron(43, 44) , are the two classes of agents that have received the most attention. Limited information is available on other classes of inhibitors, including other heterocycles(45) , tetrahydrobiopterin analogues(46) , and electron acceptors that uncouple electron transfer between the flavin and heme domains(47) . Some of the arginine analogues (e.g.N-methyl-L-arginine) appear to be mechanism-based irreversible inhibitors(48, 49) . As shown here, phenyldiazene is an irreversible inhibitor of nNOS due to reaction with the heme to form a -bonded phenyl-iron complex (Fig. 2). Phenylhydrazine autooxidizes to phenyldiazene and should therefore also be an irreversible inhibitor of nNOS(50) , but the present studies were carried out with phenyldiazene to avoid ambiguities caused by the possible sensitivity of nNOS to HO formed by autooxidation of phenylhydrazine. Irreversible inhibition of nNOS by phenyldiazene, as suggested by the correspondence of phenyl-iron complex formation with loss of activity (Fig. 6), is due to interference with the catalytic action of the iron atom rather than to interference with the electron transfer capacity of the flavin groups. This is clearly shown by the fact that phenyl-iron complex formation does not result in loss of the ability of the flavin groups to accept electrons from NADPH of to transfer them to cytochrome c (Fig. 7).

As found previously for cytochrome P450, the phenyl group of the nNOS phenyl-iron complex shifts to the porphyrin nitrogens when the complex is oxidized in situ with ferricyanide. This provides independent support for the contention that a cysteine thiolate is ligated to the iron because thiolate ligation is required for phenyl group migration within the intact active site(15, 18, 19, 20, 21, 23) .

The ratio of the four N-phenylprotoporphyrin IX isomers derived from the N-phenyl heme group extracted from nNOS is N/N/N/N = 14:20:21:45 (Fig. 8). These results suggest an active site in which the region directly above the heme group is more open above pyrrole ring D than above pyrrole rings A, B, or C. The active site may be sterically congested because arginine slows down the rate of formation of the phenyl-iron complex, a finding that suggests that arginine and phenyldiazene compete for common space above the heme iron atom or within an access channel leading to the iron atom.

The formation of all four N-phenylprotoporphyrin IX isomers with nNOS differs from the pattern observed with most P450 enzymes, although there is limited similarity to the pattern (N/N/N/N = 13:67:13:07) observed with P450 (CYP102) in that all four isomers are formed in significant amounts, including the N isomer that is rarely observed(30) . Among P450 enzymes examined to date, only in the case of P450 does the phenyl group migrate significantly toward the nitrogen of pyrrole ring B(30) . Comparison of the crystal structure of P450 with those of P450 and P450 shows that in all three the heme group is held in the active site by the pincer action of two helices, one located above and the other below the heme group(51, 52) . The distal helix in all three enzymes sits directly above pyrrole ring B of the heme, although in P450 the helix is displaced away from the iron relative to its position in the other two enzymes. This makes the nitrogen of pyrrole ring B more accessible to the phenyl group than it is in other two P450 enzymes. The observation that N is formed as the minor isomer in the reaction of phenyldiazene with nNOS suggests that the heme in nNOS may be held in the active site crevice by a similar two-helix motif. This possibility is supported by a small extent of sequence identity between the residues surrounding the cysteine iron ligand in NOS and P450 3A(14) .

In sum, the structural and mechanistic links between nitric oxide synthases and cytochromes P450 are strengthened by the present demonstration that nNOS, like cytochrome P450, is (a) inactivated by phenyldiazene via formation of a phenyl-iron complex, (b) undergoes ferricyanide-mediated migration of the phenyl from the phenyl-iron complex to the porphyrin nitrogens within the intact active site, and (c) has a P450-like active site topology.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM25515. 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.

§
To whom correspondence should be addressed: School of Pharmacy, University of California, San Francisco, CA 94143-0446.

The abbreviations used are: NOS, nitric oxide synthase; nNOS, neuronal NOS from rat brain; BH, (6R)-5,6,7,8-tetrahydrobiopterin; heme, iron protoporphyrin IX regardless of the oxidation or ligation state; CaM, Ca-dependent calmodulin; Hb, methemoglobin; HbO, oxyhemoglobin; PMSF, phenylmethylsulfonyl fluoride; IPTG, isopropyl -D-thiagalactopyranoside; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; NaPi, a mixture of mono- and di-basic sodium phosphate buffer; HPLC, high performance liquid chromatography; N, N, N, and N are the isomers of N-phenylprotoporphyrin IX with the phenyl group on the nitrogens of, respectively, pyrrole rings A, B, C, and D.

S. Black and P. R. Ortiz de Montellano, manuscript submitted for publication.


ACKNOWLEDGEMENTS

We thank Solomon Snyder (Johns Hopkins University) for kindly providing the nNOS clone, Robert Fletterick (University of California, San Francisco) for the pCWori vector, and Dr. Stephen Black for initial work on subcloning of nNOS into Bluescript.


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