Reconstitution of Pterin-free Inducible Nitric-oxide Synthase*

Inducible nitric-oxide synthase (NOS) was expressed and purified in the absence of 6(R)-tetrahydro-l-biopterin (H4B). Pterin-free NOS exhibits a Soret band (416–420 nm) characteristic of predominantly low spin heme and does not catalyze the formation of nitric oxide (·NO) (Rusche, K. M., Spiering, M. M., and Marletta, M. A. (1998) Biochemistry 37, 15503–15512). Reconstitution of pterin-free NOS with H4B was monitored by a shift in the Soret band to 396–400 nm, the recovery of ·NO-forming activity, and the measurement of H4B bound to the enzyme. As assessed by these properties, H4B binding was not rapid and required the presence of a reduced thiol. Spectral changes and recovery of activity were incomplete in the absence of reduced thiol. Full reconstitution of holoenzyme activity and stoichiometric H4B binding was achieved in the presence of 5 mm glutathione (GSH). Preincubation with GSH before the addition of H4B decreased, whereas lower concentrations of GSH extended, the time required for reconstitution. Six protected cysteine residues in pterin-free NOS were identified by labeling of NOS with cysteine-directed reagents before and after reduction with GSH. Heme and metal content of pterin-free and H4B-reconstituted NOS were also measured and were found to be independent of H4B content. Additionally, pterin-free NOS was reconstituted with 6-methylpterin analogs, including redox-stable deazapterins. Reconstitution with the redox-stable pterin analogs was neither time- nor thiol-dependent. Apparent binding constants were determined for the 6-methyl- (50 μm) and 6-ethoxymethyl (200 μm) deazapterins. The redox-stable pterin analogs appear to bind to NOS in a different manner than H4B.

The respective roles of the heme and H 4 B cofactors in the NOS mechanism are not yet clear. The first step of the reaction, the hydroxylation of arginine, appears to require the involvement of the heme due to the inhibition of the reaction by CO (10). However, hydrogen peroxide and iodosobenzene fail to support this reaction (18), and pterin-free NOS does not catalyze arginine hydroxylation in the presence of either hydrogen peroxide or NADPH (19). Thus, H 4 B appears to play a role in the first step of the reaction. H 4 B has been proposed to participate in electron transfer in NOS. Bec et al. (20) infer a role for H 4 B in the reduction of the ferrous dioxygen complex of the heme by one electron to form the heme-derived oxidant, which would result in a pterin radical. Raman et al. (21) subsequently suggested, based on structural evidence, that the H 4 B-binding site could stabilize a pterin radical cation (21). Direct evidence of a pterin radical, which is proposed to be formed in the arginine reaction, has been obtained by rapid freeze-quench electron paramagnetic resonance studies of the iNOS heme domain (22). Evidence to support heme catalysis in the oxidation of NHA to citrulline and ⅐NO consists of inhibition of the reaction by CO (23) and studies of peroxide-dependent catalysis (18,24). Additionally, a direct role for the heme in the second step of the reaction is supported by the fact that oxidation of NHA is catalyzed by pterin-free NOS (19). However, the reaction of NHA catalyzed by pterin-free NOS yields different products than the reaction catalyzed by H 4 B-bound NOS. It is not clear what the role of H 4 B may be in the oxidation of NHA. It is apparent, though, that H 4 B performs one or more crucial functions in the mechanism of NOS.
Structural roles for H 4 B have also been proposed based on the observed effects of H 4 B on substrate affinity for NOS (25,26), enzyme oligomeric structure (27)(28)(29), heme spin state equilibrium (30 -35), and heme midpoint potential (36). The NOS crystal structures with H 4 B bound have provided some clues as to the cause of these effects (21,37,38). H 4 B binds near the heme, the nitrogens of the pyrimidine ring interacting with a heme propionate group. H 4 B also interacts with residues from the symmetry-related subunit, forming a link between the subunits of the dimer. In addition, the structures revealed a metalbinding site at the dimer interface in which a zinc atom is coordinated to four cysteine residues, two from each monomer (21,38).
Expression and purification of pterin-free NOS has enabled studies examining the role of the reduced pterin cofactor. The ability to reconstitute this enzyme with H 4 B and recover all physical and catalytic properties complements previous studies of pterin-free NOS reactivity (19). These studies support a role for reduced thiol in the reconstitution of H 4 B as also observed by Sono et al. (39). We have studied the process and requirements of pterin reconstitution of the full-length iNOS in the absence of substrate utilizing H 4 B as well as the 6-methyl analogs, including redox-stable deazapterins (Fig. 1). Reconstitution was assessed by spectral changes, quantitation of bound H 4 B, and recovery of ⅐NO-forming activity.
Purification of iNOS-Expression and purification of pterin-free iNOS were as described previously (19) with the following modifications. Supernatant derived from the cell pellets of 6 liters of culture was purified using 3 g of 2Ј,5Ј-ADP-Sepharose 4B resin and 4 ml of DEAE Bio-Gel A resin. The eluate was concentrated (Ultrafree-15) to Ͻ2 ml and frozen at Ϫ80°C with 50% glycerol. Subsequently, the sample was thawed, diluted to Ͻ3 ml, and loaded onto a gel filtration column (S200 16/60) equilibrated with 100 mM Hepes (pH 7.4), 200 mM NaCl, and 10% glycerol. NOS was eluted with the same buffer, concentrated to ϳ10 M, and frozen in aliquots with 50% glycerol. The gel filtration step removed a small amount of contaminating reductase domain and aggregated NOS (eluting in the void volume). Protein concentration was determined by the Bradford protein assay using bovine serum albumin as a standard. NOS purified by this method (ϳ13 mg in a single preparation) was greater than 95% pure, as judged by SDS-polyacrylamide gel electrophoresis stained with Coomassie Blue R-250. The oligomeric state of pterin-free and H 4 B-reconstituted samples of NOS (30 l) was analyzed as described previously (19) by analytical gel filtration on a Tosohaas GFC 300 GL (15 cm, 5 m) column.
Enzyme Assays-The formation of ⅐NO was followed by the oxyhemoglobin assay (⌬⑀ 401 ϭ 60,000 M Ϫ1 cm Ϫ1 for the formation of methemoglobin) on a Beckman DU 640 spectrophotometer with a Peltier temperature controller. Assays contained 1 mM L-arginine, 100 M NADPH, 6 M oxyhemoglobin, up to 10 M H 4 B, 100 mM Hepes (pH 7.4) and were initiated by the addition of 3-5 g of pterin-free NOS (500 l total assay volume). Assays at 15, 25, or 37°C were controlled by setting the Peltier unit to the specified temperature and incubating the buffer containing appropriate concentrations of arginine and NADPH in a Neslab RTE-111 circulating water bath at the specified temperature.
The amino acid products of the pterin-free NOS reaction (i.e. oxidation of NHA to citrulline, N ␦ -cyanoornithine, and NO Ϫ ) were analyzed by reverse phase HPLC of o-phthalaldehyde derivatives with absorbance detection, as described previously (19,24). The reactions (25 l total volume) consisted of 100 mM Hepes (pH 7.4), 1 mM NHA, 50 M phenylalanine (HPLC internal standard), 300 M NADPH, 7.4 g of pterin-free NOS with and without 530 M DZPH 4 (final concentrations). DZPH 4 or an equivalent volume of Hepes was added to pterin-free NOS at 25°C 1-2 min before initiation of the reaction with NHA and NADPH. The reactions were quenched after 4 min at 25°C by the addition of 15 l of 6 mM o-phthalaldehyde, 86 mM ␤-mercaptoethanol, and 1.0 M potassium borate (pH 10.4) to a vial containing 25 l of sample or standard. After a 4-min derivatization time, 25 l of the reaction solution was injected. NADPH was omitted from control assays for determination of background citrulline (Ͻ2%) in the NHA stock.
Reconstitution of Pterin-free NOS with H 4 B-Pterin-free NOS (5 M) was reconstituted with H 4 B (50 M) in the presence of 690 M DTT (or 2.5 mM ascorbate) at 25°C. Spectra were recorded at various times on a Cary 3E UV-visible spectrophotometer (Varian) with a Neslab RTE-111 circulating water bath. Aliquots (5 l) were withdrawn from the cuvette at various times and assayed at 37°C for ⅐NO formation from arginine by the oxyhemoglobin assay.
Reconstitutions of pterin-free NOS (1-3 M) with 50 M H 4 B and 1 or 5 mM GSH or 5 mM GOH at 15°C were carried out anaerobically. Concentrated stock solutions of NOS were diluted in the anaerobic chamber with anaerobic 100 mM Hepes (pH 7.4) and placed in an anaerobic cuvette. Anaerobically prepared H 4 B and GSH were pipetted onto the side of the cuvette (above, and not mixed with the sample), and the cuvette was sealed with a silicone/Teflon septum. The cuvette was equilibrated to 15°C by immersion in the circulating water bath attached to the spectrophotometer. An initial spectrum of the enzyme was recorded before initiation with H 4 B with or without GSH by mixing. Mixing was accomplished by tilting the cuvette to mix the sample with the drops suspended on the side. Spectra were subsequently recorded at various times. A similar procedure was followed to monitor changes in NOS activity, with the exception that an anaerobic reaction vial was used instead of a cuvette. The reaction vial was incubated in a circulating water bath at 15°C before and after initiation with H 4 B with or without GSH. Aliquots (4 g of NOS in 5 l) were removed from the reaction vial with a gas-tight syringe and used to initiate aerobic oxyhemoglobin assays. Assays were carried out with arginine at 15°C, and additional H 4 B (or thiol) was not added. Therefore, H 4 B in the assays (0.5 M) was derived from the enzyme incubation. Similarly, the GSH concentration in the assays reflected the 100-fold dilution of the enzyme aliquot. Zero time points were obtained by assaying pterin-free NOS with these same concentrations of H 4 B with or without GSH (0.5 and 50 M, respectively). For longer incubations in the absence of reduced thiol, samples were prepared in multiple reaction vials and assayed at various time points, as repeated piercing of the silicone/ Teflon septum resulted in compromised anaerobiosis.
Reconstitution of Pterin-free NOS with MPH 4 -Pterin-free NOS was reconstituted with MPH 4 in the presence or absence of 5 mM GSH (25°C). These anaerobic spectral experiments were carried out similarly to those described above with the exception that the concentration of MPH 4 was 500 M.
Reconstitution of Pterin-free NOS with Deazapterin-Increasing concentrations of DPZH 4 or EtOMeDZPH 4 were added to pterin-free NOS at 25°C. Spectra were recorded initially and after each addition. An apparent binding constant was calculated from non-linear analysis of the spectral difference data. In addition, an apparent binding constant for arginine binding to DZPH 4 -bound NOS was determined by the spectral changes occurring with increasing substrate concentration (an absorbance increase at 395 and a decrease around 416 nm). NOS in the presence of DZPH 4 (530 M) was assayed (25 and 37°C) with NHA as the substrate by an HPLC assay of amino acid products as described above.
Metal Analysis and Heme Content-Samples for analysis of zinc content were prepared by desalting on a HiTrap column (Sephadex G-25 superfine) equilibrated with metal-free buffer. The buffer, 20 mM Hepes (pH 7.4), was prepared by passage over a Chelex-100 column and was stored in acid-washed glass bottles. detected or was Ͻ2% of the concentration of metal in the protein samples. The heme content of the same samples was determined by HPLC analysis with myoglobin standards using a Beckman System Gold HPLC and a previously described method (43). The mobile phase consisted of 0.1% aqueous trifluoroacetic acid, and samples were eluted with a 0 -75% linear gradient over 20 min of 0.1% trifluoroacetic acid in acetonitrile.
Labeling of Cysteine Residues in NOS-Pterin-free NOS in 0.5 M Tris and 5 M guanidine HCl (pH 7) was reacted with a 10-fold molar excess (23 mol of cysteine/mol of NOS) of N-ethylmaleimide for 75 min at 37°C. The reaction was quenched with a 10-fold molar excess (over protein thiol plus N-ethylmaleimide) of DTT and incubated at 37°C for 1 h, then diluted into 0.5 M Tris and 5 M guanidine HCl (pH 8) with a 10-fold molar excess (over total thiol concentration) of iodoacetamide. After 1 h at 37°C, the reaction was quenched with a 10-fold molar excess of ␤-mercaptoethanol. The concentration of guanidine HCl was decreased by dilution and concentration with an Ultrafree-15 (Biomax-30 kDa NMWL; Millipore), and the resulting sample was gelpurified (Novex 3-8% Tris acetate gels). Bands corresponding to NOS from multiple lanes were excised, washed with 50% acetonitrile, and submitted to the Harvard Microchemistry Facility (Harvard University) for tryptic digestion and peptide sequencing. Sequence analysis was performed by microcapillary reverse-phase HPLC nanoelectrospray tandem mass spectrometry (LC/MS/MS) on a Finnigan LCQ quadrupole ion trap mass spectrometer. The MS/MS were then correlated with the known iNOS sequence using the algorithm Sequest, developed at the University of Washington (44) and programs developed at the Harvard facility (45).
Pterin Content-The pterin content of NOS samples was determined by HPLC analysis with a Nova-Pak C18 column (3.

Reconstitution of Pterin-free NOS with H 4 B-As previously
reported (19), pterin-free NOS contained ferric, mostly low spin (Soret at ϳ418 nm) heme and did not catalyze the formation of ⅐NO from arginine or NHA in the absence of H 4 B. When purified in the higher protein concentrations achieved in this report, the enzyme was mostly dimeric (determined by analytical gel filtration; data not shown). Pterin-free NOS could be reconstituted with H 4 B. Reconstitution in the presence of DTT was evaluated by spectral changes and changes in ⅐NO-forming activity (Fig. 2). H 4 B and DTT were added simultaneously to pterin-free NOS. The first spectrum after initiation was consistent with partially DTT-bound NOS (48 -50), with absorbance maxima at 380 and 457 nm ( Fig. 2A). This spectrum converted to that of high spin, H 4 B-bound NOS (395 nm) over time with three distinct isosbestic points (430, 488, and 530 nm). These changes reflect the dissociation of DTT from the active site, binding of H 4 B, and formation of high spin heme. Difference spectra were calculated, and the absorbance changes were plotted versus time. The changes in the spectrum of pterin-free NOS with H 4 B binding correlated with the increase in specific activity (37°C) from 0.50 (all time points, including the zero time point, measured with 10 M H 4 B, 0.14 mM DTT) to 1.25 mol/min/mg (Fig. 2B), equivalent to observed holoenzyme activity.
The complications arising from the use of DTT or ␤-mercaptoethanol (namely, binding to the pterin-free NOS heme) were circumvented by the use of GSH. The spectrum (or activity) of pterin-free NOS was not affected by the addition of up to 5 mM GSH. However, the addition of H 4 B with 5 mM GSH effected a shift of the Soret from 418 to 398 nm (Fig. 3A). The conversion from low to high spin was direct (three isosbestic points; 410, 465, 528 nm) and increased over time (t1 ⁄2 Ϸ 20 min). The spectral changes again correlated with recovery of ⅐NO-forming activity (Fig. 3B). NOS assays at 15°C resulted in a maximal specific activity of approximately 0.25 mol/min/mg. The specific activity of these samples assayed at 37°C was approximately 1.1 mol/ min/mg, corresponding to full reconstitution of ⅐NO-forming activity. These reconstitution studies were carried out at 15°C to maintain enzyme stability over long incubation times. In the absence of GSH, anaerobic H 4 B reconstitution of pterin-free NOS resulted in only a small shift in the spectrum and a corresponding partial recovery of activity over a similar time frame (Fig.  4A). When the changes in absorbance became minimal, the addition of 5 mM GSH (final concentration) resulted in an increase in the absorbance changes similar to that observed when H 4 B and GSH are added simultaneously (Fig. 4B).
The thiol dependence of H 4 B reconstitution was examined by several methods. First, the presence of 2.5 mM ascorbate did not facilitate reconstitution as observed with GSH (data not shown). Second, 5 mM compared with 1 mM GSH in the reconstitution with 50 M H 4 B resulted in more rapid spectral shifts (15°C, Fig. 5A). In addition, the oxygen-substituted GSH analog, GOH, with 50 M H 4 B did not facilitate NOS spectral shifts over those with H 4 B alone. Third, the effect of an anaerobic preincubation of pterin-free NOS with GSH before initiation with H 4 B, versus simultaneous addition of H 4 B and GSH on the H 4 B-dependent NOS spectral shift (15°C) was examined (Fig.  5B). Preincubation of pterin-free NOS with GSH resulted in a more rapid shift as compared with simultaneous addition of the same GSH concentration.
Reconstitution of Pterin-free NOS with MPH 4 -MPH 4 ( Fig.  1) reconstitution of pterin-free NOS required considerably higher concentrations of this pterin. MPH 4 at 30 M did not effect any change in the pterin-free NOS spectrum. Reconstitutions were carried out at 25°C with 500 M MPH 4 in the presence or absence of 5 mM GSH. Similar to H 4 B, MPH 4 in the absence of GSH effected only a small shift of the NOS spectrum. In the presence of GSH, however, reconstitution proceeded rapidly (data not shown). In the anaerobic MPH 4 experiment, extended incubation times resulted in the very slow reduction of the flavin cofactors of NOS. This was evidenced by decreases in absorbance between 450 and 500 nm.
Reconstitution of Pterin-free NOS with Deazapterin-Increasing concentrations of the redox-stable pterin analog, DZPH 4 (Fig. 1), were added to pterin-free NOS. DZPH 4 effected a concentration-dependent decrease in absorbance at 418 -422 nm and increase in absorbance at 396 -399 nm (Fig. 6). The absorbance changes at each concentration occurred rapidly (within the time required for mixing) and did not increase further. These changes did not require the presence of reduced thiol. The apparent spectral binding constant measured for this conversion was 50 Ϯ 13 M (n ϭ 4). In the presence of 0.2-0.8 mM arginine, the spectral K D,app measured for DZPH 4 binding dropped to 34 Ϯ 3 M (n ϭ 6). This increase in pterin affinity in the presence of substrate is similar to, although less than, that reported for H 4 B affinity, where the K D for H 4 B was 250 and 37 nM in the absence and presence of arginine, respectively (25). Arginine binding to DZPH 4 -bound NOS resulted in a shift of the Soret peak to 395 nm. The spectral binding constant measured for this conversion was 18.6 Ϯ 5.9 M (n ϭ 2), as compared with 8 -13 M for murine macrophage NOS purified in the presence of H 4 B (26). EtOMeDZPH 4 similarly shifted the NOS Soret band from 419 to 400 nm (data not shown). The K D,app determined for this conversion, however, was 200 Ϯ 10 M, 4-fold higher than that measured for the 6-methyl analog.
The effect of DZPH 4 on the NADPH-dependent reaction catalyzed by pterin-free NOS was also determined. Pterin-free NOS oxidizes NHA to citrulline, N ␦ -cyanoornithine, and NO Ϫ (19). Pterin-free NOS was incubated with 530 M DZPH 4 , and amino acid product formation (citrulline plus N ␦ -cyanoornithine) at 25°C was measured in the presence and absence of DZPH 4 . The specific activity of pterin-free NOS in the absence of DZPH 4 was 210 Ϯ 40 nmol/min/mg (n ϭ 4). In the presence of DZPH 4 , the specific activity dropped 48%, to 110 Ϯ 7 nmol/ min/mg (n ϭ 2).
Zinc, Heme, and Pterin Content-Pterin-free and H 4 B-reconstituted NOS were analyzed for zinc, heme, and pterin content. Essentially no difference in the heme or zinc content of pterinfree and H 4 B-reconstituted NOS was observed ( Table I). The heme stoichiometry was determined to be approximately 0.76 per monomer. Zinc was present at 0.5 per NOS monomer, consistent with the crystallographically observed zinc tetrathiolate center at the dimer interface of the endothelial and inducible NOS isoforms (21,38). It is interesting to note that the zinc stoichiometry was independent of H 4 B content. In these experiments 0.5 zinc was reproducibly bound to 1 NOS monomer, implying that pterin-free NOS retains the ability to bind zinc. This conclusion is consistent with the observation by analytical gel filtration that these preparations of pterin-free NOS were mostly dimeric (Ͼ90%), with only a small shift in the enzyme elution profile upon H 4 B reconstitution (data not shown). As expected, H 4 B was not detected in pterin-free NOS preparations. Incubation of pterin-free NOS with H 4 B did result in the binding of H 4 B to NOS, as evidenced by the detection of 0.8 H 4 B per NOS monomer. The H 4 B and heme stoichiometries in the same enzyme preparation are equivalent within error.
Cysteine Labeling-Pterin-free NOS cysteine residues were labeled by N-ethylmaleimide before reduction with DTT and by iodoacetamide after reduction. After tryptic digestion of the sample, MS/MS peptide sequencing identified which of the 23 cysteine residues of iNOS were labeled with each reagent. Six cysteine residues did not react with the first reagent (N-ethylmaleimide) and were only labeled by iodoacetamide after reduction. Two of these cysteine residues (Cys-104 and Cys-109) correspond to those reported to be the ligands in the zinc tetrathiolate center (21,38). Presumably, bound zinc would protect these cysteine residues until exposure to excess reduced thiol results in removal of the zinc ion. Two other cysteine residues in the heme domain, Cys-284 and Cys-378, as well as two in the reductase domain, Cys-565 and Cys-675, also did not react with the first reagent. DISCUSSION Pterin-free NOS exhibits a Soret band (416 -420 nm) characteristic of predominantly low spin heme. Upon incubation of NOS with H 4 B and arginine, the Soret band shifts to 398 -400 nm (30 -35). We have studied the process and requirements of H 4 B binding to pterin-free NOS in the absence of substrate. This is important in that L-arginine (or NHA) binding alone   effects a low to high spin shift of the NOS Soret band. In addition, L-arginine increases the apparent affinity of NOS for H 4 B, and H 4 B increases the apparent affinity for L-arginine. Thus in experiments containing both substrate and pterin cofactor, particularly those relying solely on spectral data, the influence of one compound cannot be isolated from the influence of the other. The shift of the pterin-free NOS Soret band occurred rapidly in the presence of H 4 B and reduced thiol (Fig.  3A) and did not require the presence of substrate as previously observed with the nNOS heme domain (39). Sono et al. (39) observed low to high spin shifts of the heme domain Soret upon extended (24 h to 10 days) incubations with H 4 B and DTT (with or without arginine). Since our experiments were carried out with the full-length enzyme, we were able to correlate the observed spectral changes with full recovery of ⅐NO-forming activity (Fig. 3B). We also carried out extensive characterizations of these forms of the enzyme by H 4 B, heme, and metal quantitation as well as cysteine-labeling experiments. In the absence of reduced thiol, H 4 B alone effects little change in the pterin-free NOS spectrum and achieves reconstitution of only approximately 50% of full activity (Fig. 4). It is important to remember that pterin-free NOS does not catalyze turnover of arginine in the absence of added H 4 B (19). For ease of comparison in the activity reconstitution experiments, however, zero time points were designed to control for H 4 B reconstitution that may occur upon L-arginine binding or turnover in the assay. This was accomplished by assaying pterin-free NOS in the presence of H 4 B with or without GSH at concentrations that corresponded to those in assays of the timed incubations.
These results have indicated that facile and complete H 4 B reconstitution requires the presence of reduced thiol. Because these experiments were carried out under strictly anaerobic conditions and formation of oxidized pterin in H 4 B-only studies was not observed in the spectral experiments (Ͻ2%), the presence of reduced thiol was not required to maintain a pool of reduced pterin. We considered the possibility that reduced thiol could function to chelate the NOS-bound zinc molecule, which might aid H 4 B binding by relaxing structural constraints. The zinc stoichiometry of pterin-free NOS incubated with GSH alone, however, was unchanged relative to either pterin-free or H 4 B-reconstituted NOS (data not shown). Apparently, a thiol functions to reduce an NOS moiety, which in the oxidized form, interferes with H 4 B reconstitution. This proposal is supported by the dependence of H 4 B reconstitution on thiol concentration (Fig. 5A), the increased rate of absorbance change observed upon preincubation of pterin-free NOS with GSH (Fig. 5B), and the inability of the oxygen-substituted GSH analog, GOH, to facilitate H 4 B reconstitution (Fig. 5A). These data as well as the inability of ascorbate to substitute for GSH imply that a protein disulfide must be reduced for facile H 4 B binding and recovery of activity. To examine this hypothesis, labeling of NOS cysteine residues before and after reduction with DTT was carried out. Of the six cysteine residues labeled only after reduction, two ligate a zinc atom in combination with the equivalent two cysteine residues from the opposing subunit (21,38) and may be expected to be protected from reaction until dissociation of the zinc atom, induced by high thiol concentrations and/or enzyme denaturation after prolonged incubations at 37°C. Examination of the NOS heme domain structures (21,37,38) or the cytochrome P450 reductase structure (51) does not explain the protection of the remaining two cysteine residues in the heme domain and the two in the reductase domain. It is unlikely but not impossible that some cysteine residues might be inaccessible to the labeling reagent due to structural constraints even though the reactions were carried out in 5 M guanidine HCl. However, based on the results presented in this study it is tempting to speculate that one or more disulfide bonds (intra-or intersubunit) involving these residues are formed during expression and/or purification of NOS in the absence of H 4 B and reduced thiol. This disulfide bond would then interfere with H 4 B binding and recovery of activity, perhaps by constraining the structure of the enzyme. The previously observed effects of GSH to stimulate H 4 B-bound neuronal NOS activity and stabilize activity over long (15-60 min) assays (52) was proposed to be due to reduction of protein thiols, which interact with the pterin-binding site of NOS. Although direct interaction of protein cysteine residues with the pterinbinding site is not observed in the NOS structures (21,37,38), the function of GSH in those studies may indeed have been in the reduction of a protein disulfide, which interfered with H 4 B binding, as supported by the results presented here.
Another interesting result of these studies is the observation that the redox-stable pterin analogs, DZPH 4 and EtOMeDZPH 4 , did not bind to pterin-free NOS in the same fashion as H 4 B. The absorbance changes effected by the redoxstable analogs were similar to those occurring upon H 4 B binding. However, the redox-stable analogs bound pterin-free NOS in a concentration-dependent (not time-dependent) manner, without a dependence on the presence of reduced thiol. One difference between the redox-stable analogs and H 4 B is in the substituent at the C6 position. To control for changes in the C6 substituents, the reconstitution properties of the redox-active MPH 4 compared with those of DZPH 4 were examined. These compounds both have a 6-methyl group in contrast to the 6-dihydroxypropyl group of H 4 B. MPH 4 has already been shown to reconstitute NOS activity (13) and, as reported here, reconstituted pterin-free NOS in a time-and thiol-dependent manner, similar to H 4 B. The higher concentrations of MPH 4 (as compared with H 4 B) required for reconstitution are similar to those needed to support NOS catalysis (13). DZPH 4 , however, is unable to support NOS catalysis (13). Both compounds, MPH 4 and DZPH 4 , are racemic, which may in part explain the observed lowered affinity compared with 6(R)-H 4 B (25). However, the 200-fold decrease in the affinity of DZPH 4 for NOS may also be due to differences in the pterin side chain. The 6-ethoxymethyl substituent of EtOMeDZPH 4 results in an even greater decrease (400-fold as compared with H 4 B) in affinity. The C6 substituent of the pterin thus appears to be an important determinant of binding affinity; this is supported by the multiple interactions of the biopterin side chain with protein residues observed in the NOS structures (21,37,38).
The redox-stable pterin analogs also lack the nitrogen atom at the 5 position, which is present in H 4 B. The structures of MPH 4 and DZPH 4 also differ only in the absence of the N5 atom in DZPH 4 . Therefore, the differences in binding between MPH 4 and DZPH 4 (53), no structure with a 5-deazapterin bound has been determined. In the structure of the iNOS heme domain dimer (37), a hydrogen bond bridges the N5 of H 4 B (through a water molecule) to Arg-375 of the substrate binding helix, which participates in additional hydrogen bonds. Interestingly, Sono et al. (39) report that the 4-amino-H 4 B effects a thiol-dependent shift in the nNOS heme domain Soret. Apparently, the thiol-independent effects of the deazapterins are unique to changes at the N5 position. The absence of this hydrogen bond donor in DZPH 4 in addition to the absence of the interactions of a 6-dihydroxypropyl side chain may allow DZPH 4 to bind in a different orientation compared with biopterin molecules with a nitrogen at the 5 position. The redoxstable pterin analogs most likely bind near the heme, however, because the analogs affect the heme spin-state equilibrium, and DZPH 4 inhibits the pterin-free NOS reaction with NHA and NADPH.
Analysis of the zinc, heme, and H 4 B content of pterin-free and H 4 B-reconstituted NOS was carried out to further examine the differences between these two forms of the enzyme. However, the presence or absence of H 4 B made no difference in the binding of heme and zinc by NOS. NOS bound zinc with a highly reproducible stoichiometry of 0.5 per monomer, entirely consistent with the zinc atom bound at the dimer interface of the NOS heme domain (21,38). Zinc content was independent of H 4 B content and not correlated to the heme stoichiometry. H 4 B content, when reconstituted, and heme content were equivalent within error in these samples. Heme binds to NOS in the absence of H 4 B, as evidenced by results with the pterinfree samples. From the stoichiometries observed, it appears that H 4 B may bind to NOS only when heme is bound.
In the absence of reduced thiol, H 4 B reconstitution as assessed by activity measurements corresponds to ϳ50% of the activity recovered in the presence of thiol. As assessed by spectral changes, however, H 4 B reconstitution in the absence of thiol corresponds to ϳ25% of the spectral changes observed in the absence of thiol. The disparity in these two measurements may be explained by 1) the fact that pterin-free NOS is not completely low spin even in the absence of substrate and pterins and 2) that activity measurements must be carried out in the presence of substrate, which may in itself effect an increase in H 4 B bound (leading to an increase in the base line observed for these experiments). The former results in an underestimation of the extent of H 4 B binding, and the latter may result in an overestimation of H 4 B binding. In support of these interpretations, the stoichiometry of H 4 B bound to pterin-free NOS in the absence of reduced thiol was measured by HPLC assay at 0.32/NOS monomer. 2 In summary, pterin-free NOS can be reconstituted with H 4 B as evidenced by the electronic absorption spectrum, the recovery of ⅐NO-forming activity, and the measurement of H 4 B bound to the enzyme. In addition, heme and metal content of NOS are not dependent on the presence of H 4 B. H 4 B binding exhibits a dependence on the presence of a reduced thiol. The most likely explanation for this dependence is the requirement for reduction of a protein disulfide bond perhaps involving one or more of the cysteines identified in this study. The reconstitution characteristics of the redox-stable pterin analogs differ from those of H 4 B, perhaps indicating that these deaza molecules bind to NOS differently than does H 4 B. H 4 B does not seem to be required for the structural integrity of NOS, which has also been demonstrated crystallographically (21). The results presented here indicate that binding of H 4 B to NOS is a complex process. Further studies on the binding of H 4 B and pterin analogs will aid in the elucidation of the role of H 4 B in NOS.