From the § Howard Hughes Medical Institute,
¶ Division of Medicinal Chemistry and Department of
Biological Chemistry, University of Michigan,
Ann Arbor, Michigan 48109-0606
Received for publication, July 31, 2000, and in revised form, September 11, 2000
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ABSTRACT |
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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.
Nitric-oxide synthase
(NOS,1 EC 1.14.13.39) has
been characterized in three isoforms: a membrane-associated,
constitutive enzyme from the vascular endothelium; a soluble,
constitutive enzyme from neuronal cells; and an inducible enzyme best
characterized from murine macrophages (1). All of the isoforms catalyze
the formation of nitric oxide (·NO) and citrulline from
L-arginine (2-4). The reaction requires NADPH and
O2 and proceeds via the intermediate
NG-hydroxy-L-arginine (NHA) (5, 6).
NOS is homodimeric and binds an equivalent each of FAD, FMN (7-9), and
protoporphyrin IX heme (10-12) per subunit. Additionally, each NOS
subunit binds 1 equivalent of tetrahydrobiopterin (H4B),
which is required for full activity (9, 13, 14). Calmodulin binds to
the constitutive isoforms reversibly in response to the intracellular
calcium concentration (15, 16) and nearly irreversibly in an apparently
Ca2+-independent manner to the inducible isoform (4,
17).
The respective roles of the heme and H4B 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,
H4B appears to play a role in the first step of the
reaction. H4B has been proposed to participate in electron transfer in NOS. Bec et al. (20) infer a role for
H4B 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
H4B-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 ·NO 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 H4B-bound NOS. It is not clear what the role of
H4B may be in the oxidation of NHA. It is apparent, though,
that H4B performs one or more crucial functions in the
mechanism of NOS.
Structural roles for H4B have also been proposed based on
the observed effects of H4B on substrate affinity for NOS
(25, 26), enzyme oligomeric structure (27-29), heme spin state
equilibrium (30-35), and heme midpoint potential (36). The NOS crystal
structures with H4B bound have provided some clues as to
the cause of these effects (21, 37, 38). H4B binds near the
heme, the nitrogens of the pyrimidine ring interacting with a heme
propionate group. H4B also interacts with residues from the
symmetry-related subunit, forming a link between the subunits of the
dimer. In addition, the structures revealed a metal-binding 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 H4B 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 H4B 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 H4B as well as the 6-methyl analogs, including
redox-stable deazapterins (Fig. 1).
Reconstitution was assessed by spectral changes, quantitation of bound
H4B, and recovery of ·NO-forming activity.
Materials and General Methods--
Escherichia coli
JM109 competent cells were purchased from Promega. Ampicillin,
chloramphenicol, and isopropyl-
Stock solutions of H4B or MPH4 were prepared in
100 mM Hepes (pH 7.4) either in an anaerobic chamber (Coy
Laboratory Products, Inc.) or in buffer containing 100 mM
dithiothreitol (DTT). H4B concentrations were determined by
dilution into 50 mM Hepes (pH 8), and the absorbance was
recorded at 297 nm ( 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 2',5'-ADP-Sepharose 4B resin and
4 ml of DEAE Bio-Gel A resin. The eluate was concentrated
(Ultrafree-15) to <2 ml and frozen at Enzyme Assays--
The formation of ·NO was followed by the
oxyhemoglobin assay (
The amino acid products of the pterin-free NOS reaction
(i.e. oxidation of NHA to citrulline,
N Reconstitution of Pterin-free NOS with
H4B--
Pterin-free NOS (5 µM) was
reconstituted with H4B (50 µM) in the
presence of 690 µM 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 µl) were withdrawn from the cuvette at
various times and assayed at 37 °C for ·NO formation from arginine
by the oxyhemoglobin assay.
Reconstitutions of pterin-free NOS (1-3 µM) with 50 µM H4B 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 H4B 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
H4B 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 H4B with or
without GSH. Aliquots (4 µg of NOS in 5 µl) 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 H4B (or thiol) was not added.
Therefore, H4B in the assays (0.5 µM) 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 H4B with or without GSH (0.5 and 50 µM, 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
MPH4--
Pterin-free NOS was reconstituted with
MPH4 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 MPH4 was 500 µM.
Reconstitution of Pterin-free NOS with
Deazapterin--
Increasing concentrations of DPZH4 or
EtOMeDZPH4 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 DZPH4-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 DZPH4 (530 µM) 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 HiTrapTM 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.
H4B-reconstituted NOS samples were incubated with 50 µM H4B and 5 mM GSH for 3 h
at 15-20 °C before desalting. Pterin-free NOS samples were desalted
as isolated. Inductively coupled plasma/MS (Plasmaspec III, Leeman
labs, Hudson, NH) analysis of samples (3-4 µM, by the
Bradford protein assay) was carried out by Dr. Ted J. Huston (Dept. of
Geological Sciences, University of Michigan, Ann Arbor). In the buffer
controls, zinc was either not detected or was <2% 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
Pterin Content--
The pterin content of NOS samples was
determined by HPLC analysis with a Nova-Pak C18 column (3.9 × 150 mm, 4 µm; Waters) equipped with an Alltima C18-LL guard column (5 µm; Alltech) using a Hewlett Packard 1090 Series II HPLC with a diode
array detector. The mobile phase consisted of 50 mM sodium
acetate, 5 mM citric acid, and 50 µM EDTA (pH
6.0) (adapted from Refs. 46 and 47). Samples (25 µl) were eluted with
a 0-20% linear gradient of methanol over 9 min, followed by a
20-80% linear gradient of methanol over 3 min and a 1-min wash with
100% methanol (0.5 ml/min). Detection wavelengths were 297 ( Reconstitution of Pterin-free NOS with H4B--
As
previously reported (19), pterin-free NOS contained ferric, mostly low
spin (Soret at ~418 nm) heme and did not catalyze the formation of
·NO from arginine or NHA in the absence of H4B. 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
H4B. Reconstitution in the presence of DTT was evaluated by
spectral changes and changes in ·NO-forming activity (Fig. 2). H4B 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, H4B-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
H4B, 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 H4B binding correlated with the increase in specific activity (37 °C) from 0.50 (all time points, including the zero time
point, measured with 10 µM H4B, 0.14 mM DTT) to 1.25 µmol/min/mg (Fig. 2B),
equivalent to observed holoenzyme activity.
The complications arising from the use of DTT or
The thiol dependence of H4B 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 µM H4B resulted in
more rapid spectral shifts (15 °C, Fig. 5A). In addition, the
oxygen-substituted GSH analog, GOH, with 50 µM
H4B did not facilitate NOS spectral shifts over those with H4B alone. Third, the effect of an anaerobic preincubation
of pterin-free NOS with GSH before initiation with H4B,
versus simultaneous addition of H4B and GSH on
the H4B-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
MPH4--
MPH4 (Fig. 1) reconstitution of
pterin-free NOS required considerably higher concentrations of this
pterin. MPH4 at 30 µM did not effect any
change in the pterin-free NOS spectrum. Reconstitutions were carried
out at 25 °C with 500 µM MPH4 in the
presence or absence of 5 mM GSH. Similar to
H4B, MPH4 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
MPH4 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, DZPH4
(Fig. 1), were added to pterin-free NOS. DZPH4 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 ± 13 µM
(n = 4). In the presence of 0.2-0.8 mM
arginine, the spectral KD,app measured
for DZPH4 binding dropped to 34 ± 3 µM
(n = 6). This increase in pterin affinity in the
presence of substrate is similar to, although less than, that reported
for H4B affinity, where the KD for
H4B was 250 and 37 nM in the absence and
presence of arginine, respectively (25). Arginine binding to
DZPH4-bound NOS resulted in a shift of the Soret peak to
395 nm. The spectral binding constant measured for this conversion was
18.6 ± 5.9 µM (n = 2), as compared
with 8-13 µM for murine macrophage NOS purified in the
presence of H4B (26). EtOMeDZPH4 similarly
shifted the NOS Soret band from 419 to 400 nm (data not shown). The
KD,app determined for this conversion,
however, was 200 ± 10 µM, 4-fold higher than that
measured for the 6-methyl analog.
The effect of DZPH4 on the NADPH-dependent
reaction catalyzed by pterin-free NOS was also determined. Pterin-free
NOS oxidizes NHA to citrulline,
N Zinc, Heme, and Pterin Content--
Pterin-free and
H4B-reconstituted NOS were analyzed for zinc, heme, and
pterin content. Essentially no difference in the heme or zinc content
of pterin-free and H4B-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 H4B
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 (>90%), with only a small shift in the enzyme elution profile
upon H4B reconstitution (data not shown). As expected,
H4B was not detected in pterin-free NOS preparations.
Incubation of pterin-free NOS with H4B did result in the
binding of H4B to NOS, as evidenced by the detection of 0.8 H4B per NOS monomer. The H4B 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.
Pterin-free NOS exhibits a Soret band (416-420 nm) characteristic
of predominantly low spin heme. Upon incubation of NOS with H4B and arginine, the Soret band shifts to 398-400 nm
(30-35). We have studied the process and requirements of
H4B 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
H4B, and H4B 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 H4B 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
H4B 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
·NO-forming activity (Fig. 3B). We also carried out
extensive characterizations of these forms of the enzyme by
H4B, heme, and metal quantitation as well as
cysteine-labeling experiments. In the absence of reduced thiol, H4B 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 H4B (19). For ease of comparison in the activity
reconstitution experiments, however, zero time points were designed to
control for H4B 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
H4B with or without GSH at concentrations that corresponded
to those in assays of the timed incubations.
These results have indicated that facile and complete H4B
reconstitution requires the presence of reduced thiol. Because these experiments were carried out under strictly anaerobic conditions and
formation of oxidized pterin in H4B-only studies was not
observed in the spectral experiments (<2%), 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 H4B 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 H4B-reconstituted NOS (data not shown). Apparently, a thiol functions to reduce an NOS moiety, which in the
oxidized form, interferes with H4B reconstitution. This
proposal is supported by the dependence of H4B
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 H4B
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 H4B 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 H4B and reduced thiol. This disulfide bond would then
interfere with H4B binding and recovery of activity,
perhaps by constraining the structure of the enzyme. The previously
observed effects of GSH to stimulate H4B-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 pterin-binding 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 H4B binding, as supported by the results
presented here.
Another interesting result of these studies is the observation
that the redox-stable pterin analogs, DZPH4 and
EtOMeDZPH4, did not bind to pterin-free NOS in the same
fashion as H4B. The absorbance changes effected by the
redox-stable analogs were similar to those occurring upon
H4B 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 H4B is in the substituent at the C6 position.
To control for changes in the C6 substituents, the reconstitution
properties of the redox-active MPH4 compared with those of
DZPH4 were examined. These compounds both have a 6-methyl
group in contrast to the 6-dihydroxypropyl group of H4B.
MPH4 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 H4B. The
higher concentrations of MPH4 (as compared with
H4B) required for reconstitution are similar to those
needed to support NOS catalysis (13). DZPH4, however, is
unable to support NOS catalysis (13). Both compounds, MPH4
and DZPH4, are racemic, which may in part explain the
observed lowered affinity compared with 6(R)-H4B
(25). However, the 200-fold decrease in the affinity of
DZPH4 for NOS may also be due to differences in the pterin side chain. The 6-ethoxymethyl substituent of EtOMeDZPH4
results in an even greater decrease (400-fold as compared with
H4B) 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 H4B. The structures of MPH4 and DZPH4 also differ only in the absence
of the N5 atom in DZPH4. Therefore, the differences in
binding between MPH4 and DZPH4 cannot be
attributed to the C6 substituent. DZPH4 lacks a hydrogen
bond donor. The mode of DZPH4 binding may be different from
that of H4B, particularly in light of the different
hydrogen bonding pattern and the difference in reconstitution. Although crystal structures of 7,8-dihydrobiopterin and 4-amino-H4B
bound to NOS are indistinguishable from H4B-bound NOS (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 H4B (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-H4B 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 DZPH4 in addition to
the absence of the interactions of a 6-dihydroxypropyl side chain may
allow DZPH4 to bind in a different orientation compared
with biopterin molecules with a nitrogen at the 5 position. The
redox-stable pterin analogs most likely bind near the heme, however,
because the analogs affect the heme spin-state equilibrium, and
DZPH4 inhibits the pterin-free NOS reaction with NHA and NADPH.
Analysis of the zinc, heme, and H4B content of pterin-free
and H4B-reconstituted NOS was carried out to further
examine the differences between these two forms of the enzyme. However,
the presence or absence of H4B 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 H4B content and not
correlated to the heme stoichiometry. H4B content, when
reconstituted, and heme content were equivalent within error in these
samples. Heme binds to NOS in the absence of H4B, as
evidenced by results with the pterin-free samples. From the
stoichiometries observed, it appears that H4B may bind to
NOS only when heme is bound.
In the absence of reduced thiol, H4B reconstitution as
assessed by activity measurements corresponds to ~50% of the
activity recovered in the presence of thiol. As assessed by spectral
changes, however, H4B reconstitution in the absence of
thiol corresponds to ~25% 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 H4B bound (leading to
an increase in the base line observed for these experiments). The
former results in an underestimation of the extent of H4B
binding, and the latter may result in an overestimation of
H4B binding. In support of these interpretations, the
stoichiometry of H4B 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
H4B as evidenced by the electronic absorption spectrum, the
recovery of ·NO-forming activity, and the measurement of
H4B bound to the enzyme. In addition, heme and metal
content of NOS are not dependent on the presence of H4B.
H4B 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 H4B, perhaps indicating that these
deaza molecules bind to NOS differently than does H4B.
H4B 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
H4B to NOS is a complex process. Further studies on the
binding of H4B and pterin analogs will aid in the
elucidation of the role of H4B in NOS.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (9K):
[in a new window]
Fig. 1.
Pterin structures.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside were purchased from Roche Molecular Biochemicals. NHA was obtained from
Alexis Corp. 2',5'-ADP Sepharose 4B, the S200 16/60 gel filtration column, and HiTrapTM desalting columns were purchased from Amersham Pharmacia Biotech. DEAE Bio-Gel A, Coomassie Blue R-250, and Bradford protein dye reagent were purchased from Bio-Rad. Reaction vials and
silicone/Teflon septa were purchased from Pierce. Centrifugal filtration units (Ultrafree-15, Biomax-50K NMWL membrane) were purchased from Millipore. H4B and
6(R,S)-methyltetrahydropterin (MPH4)
were purchased from Dr. B. Schircks Laboratory (Jona, Switzerland). The
tri-peptide
-L-Glu-L-Ser-Gly (GOH) was a
generous gift of Dr. Richard Armstrong, Vanderbilt University. All
other reagents were purchased from Sigma.
= 8700 M
1 cm
1)
(40). MPH4 concentrations were determined by dilution into 0.1 N HCl, and the absorbance was recorded at 265 nm
(
= 14, 380 M
1
cm
1) (41).
6(R,S)-Methyl-5-deazatetrahydropterin
(DZPH4) was synthesized as described previously (13, 42)
and used as the monotrifluoroacetate salt. DZPH4 (4-6 mg)
was dissolved in 0.5 ml of 0.1 N NaOH and diluted to 4 ml
with 200 mM Hepes (pH 7.4). The pH was adjusted to 7.4 with
HCl, the concentration was determined by dilution into 100 mM Hepes (pH 8), and the absorbance was recorded at 279 nm
(
= 15, 500 M
1
cm
1) (42).
6(R,S)-Ethoxymethyl-5-deazatetrahydropterin
(EtOMeDZPH4) was a generous gift from Dr. Edward C. Taylor
(Princeton University) and was prepared similarly to
DZPH4.
80 °C with 50% glycerol.
Subsequently, the sample was thawed, diluted to <3 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 ~10
µM, 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 (~13 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
H4B-reconstituted samples of NOS (30 µl) was analyzed as
described previously (19) by analytical gel filtration on a Tosohaas
GFC 300 GL (15 cm, 5 µm) column.
401 = 60,000 M
1 cm
1
for the formation of methemoglobin) on a Beckman DU 640 spectrophotometer with a Peltier temperature controller. Assays
contained 1 mM L-arginine, 100 µM
NADPH, 6 µM oxyhemoglobin, up to 10 µM
H4B, 100 mM Hepes (pH 7.4) and were initiated
by the addition of 3-5 µg of pterin-free NOS (500 µl 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.
-cyanoornithine, and NO
) were
analyzed by reverse phase HPLC of o-phthalaldehyde
derivatives with absorbance detection, as described previously (19,
24). The reactions (25 µl total volume) consisted of 100 mM Hepes (pH 7.4), 1 mM NHA, 50 µM phenylalanine (HPLC internal standard), 300 µM NADPH, 7.4 µg of pterin-free NOS with and without
530 µM DZPH4 (final concentrations).
DZPH4 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 µl of 6 mM
o-phthalaldehyde, 86 mM
-mercaptoethanol, and
1.0 M potassium borate (pH 10.4) to a vial containing 25 µl of sample or standard. After a 4-min derivatization time, 25 µl
of the reaction solution was injected. NADPH was omitted from control
assays for determination of background citrulline (<2%) in the NHA stock.
-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 gel-purified (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
(µLC/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).
max for H4B) and 280 nm (
max
for 7, 8-H2B). Under these conditions, H4B
elutes at 4 min, and 7,8-dihydro-L-biopterin (7, 8-H2B) elutes at 4.8 min. NOS samples were concentrated to ~40 µM with centrifugal filtration units
(Ultrafree-0.5, Biomax-10K NMWL membrane, from Millipore).
H4B-reconstituted NOS was prepared by incubation with 50 µM H4B and 0.7 mM DTT for 3 h at 4 °C. Samples (50-75 µl) were prepared for analysis by
desalting with Micro Bio-Spin P-30 columns (Bio-Rad) equilibrated with
10 mM DTT and 100 mM Hepes (pH 7.4). Control
experiments were carried out to ensure the efficiency of the desalting
procedure. H4B and DTT (50 µM and 0.7 mM, respectively) in 50-75-µl aliquots were desalted with the Micro Bio-Spin columns and analyzed for H4B.
H4B or 7,8-dihydro-L-biopterin was not detected
in these experiments, evidence that the method of desalting was
adequate. Guanidine HCl was added to desalted protein samples for a
final concentration of 0.67 M. Samples and standards
contained 10 mM DTT and were prepared immediately before analysis. 7,8-Dihydro-L-biopterin was not detected in the
standards, and only a small amount (<5% of H4B measured)
was detected in the NOS samples.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (21K):
[in a new window]
Fig. 2.
Reconstitution of pterin-free NOS with
H4B and DTT. Final concentrations of 50 µM
H4B and 690 µM DTT were simultaneously added
to ~5 µM pterin-free NOS. Spectra were recorded during
incubation at 25 °C, and aliquots were removed and assayed with
L-arginine (at 37 °C) by oxyhemoglobin oxidation at the
indicated times. A, in a representative experiment
(n = 3), the spectrum of pterin-free NOS (solid
line) converted initially to a spectrum consistent with partially
DTT-bound NOS (dashed line). The absorbance at 457 nm
decreased, and the absorbance at 395 increased to produce the spectrum
of H4B-bound, high spin NOS (dotted line) after
2 h. B, difference spectra were calculated, and the
absorbance changes (closed circles, left
axis) and the changes in specific activity (open
circles, right axis) were plotted versus
time.
-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 H4B 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
20 min). The
spectral changes again correlated with recovery of ·NO-forming
activity (Fig. 3B). NOS assays at 15 °C resulted in a
maximal specific activity of approximately 0.25 µmol/min/mg. The
specific activity of these samples assayed at 37 °C was
approximately 1.1 µmol/min/mg, corresponding to full reconstitution
of ·NO-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 H4B 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 H4B and GSH are added
simultaneously (Fig. 4B).
View larger version (21K):
[in a new window]
Fig. 3.
Reconstitution of pterin-free NOS with
H4B and GSH. Final concentrations of 50 µM H4B and 5 mM GSH were
simultaneously added to pterin-free NOS. Spectra were recorded during
incubation at 15 °C, and aliquots were removed and assayed with
L-arginine (at 15 °C) at the indicated times.
A, in a representative experiment (n = 5),
pterin-free NOS (thin, solid line
max417) converted to H4B-bound, high spin
NOS (thick, solid
line
max399) after 1.5 h with selected
intermediate spectra (dashed line, 5 min;
dotted line, 25 min) shown. The absorbance decreased at 417 nm and increased at 399 nm. B, absorbance changes
(closed circles, left axis) and the changes in
specific activity (open circles, right axis) were
plotted versus time.
View larger version (19K):
[in a new window]
Fig. 4.
Thiol dependence of NOS spectral changes and
recovery of activity. Anaerobic incubations of pterin-free NOS at
15 °C were initiated by the addition of 50 µM
H4B with or without 5 mM GSH, final
concentrations. Spectral experiments (~3 µM NOS) and
enzyme assays with L-arginine were carried out as described
under "Experimental Procedures" A, from a
representative experiment (n = 5), absorbance changes
(open symbols) are plotted on the left axis, and
specific activity (closed symbols) is plotted on the
right axis. Pterin-free NOS was reconstituted either in the
absence (circles) or in the presence (squares) of
GSH. In the absence of H4B, NOS does not catalyze ·NO
formation (19). B, H4B was added to pterin-free
NOS in the absence (closed circles) or presence of 5 mM GSH (open circles), and the spectrum was
recorded over time. At 90 min after initiation with H4B, 5 mM GSH (final concentration) was added to the
H4B-only experiment (marked by an arrow). After
the addition of GSH, the absorbance changes accelerated and reached the
same maximum as observed when H4B and GSH were added
simultaneously.
View larger version (20K):
[in a new window]
Fig. 5.
Effects of increasing GSH concentration and
preincubation. A, H4B (50 µM)
was added either simultaneously with either 1 mM
(triangles) or 5 mM (squares) GSH or
with 5 mM GOH (circles). Spectra were recorded
over time, and difference spectra were calculated. Changes in
absorbance versus time from H4B and GSH (or GOH)
addition are plotted. In each experiment the NOS concentration was 1.8 µM. B, the rate of formation of the high spin
heme depended not only on the concentration of GSH but also on
preincubation with GSH. H4B and GSH (50 µM
and 5 mM, respectively) were added either simultaneously to
pterin-free NOS (open squares) or after pterin-free NOS was
incubated with 5 mM GSH for 1 h on ice (closed
circles).
View larger version (24K):
[in a new window]
Fig. 6.
Titration of pterin-free NOS with
DZPH4. The addition of DZPH4 to
pterin-free NOS (thin, solid line) caused a
concentration-dependent decrease in absorbance at 422 nm
and an increase in absorbance at 397 nm. Selected intermediate spectra
(dashed and dotted lines; 15 and 72 µM DZPH4, respectively) are shown as well as
the final spectrum (thick, solid line). In this
representative experiment (2.4 µM NOS), the
KD,app calculated from non-linear
analysis (inset) of the spectral difference data was 65 ± 2 µM.
-cyanoornithine, and NO
(19).
Pterin-free NOS was incubated with 530 µM
DZPH4, and amino acid product formation (citrulline plus
N
-cyanoornithine) at 25 °C was measured in
the presence and absence of DZPH4. The specific activity of
pterin-free NOS in the absence of DZPH4 was 210 ± 40 nmol/min/mg (n = 4). In the presence of DZPH4, the specific activity dropped 48%, to 110 ± 7 nmol/min/mg (n = 2).
Zinc, heme and H4B stoichiometry per NOS
monomer
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Melissa J. Clague for her part in the adaptation of the method for pterin quantitation, Dr. Joan M. Hevel for synthesizing DZPH4, and for their generous gifts, Dr. Richard Armstrong (GOH) and Dr. Edward C. Taylor (EtOMeDZPH4). In addition, we thank the Marletta lab members for their helpful critique of this manuscript.
![]() |
FOOTNOTES |
---|
* This research was supported by Howard Hughes Medical Institute and National Institutes of Health Grant 50414.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
734-764-2442; Fax: 734-647-5687; E-mail: marle@umich.edu.
Published, JBC Papers in Press, October 5, 2000, DOI 10.1074/jbc.M006860200
2 K. M. Rusche and M. A. Marletta, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
NOS, nitric-oxide
synthase;
iNOS, inducible NOS;
·NO, nitric oxide;
H4B, 6(R)-5,6,7,8-tetrahydro-L-biopterin;
MPH4, 6(R,S)-methyltetrahydropterin;
DZPH4, 6(R,S)-methyl- 5-deazatetrahydropterin;
EtOMeDZPH4, 6(R,S)-ethoxymethyl-5-deazatetrahydropterin;
DTT, dithiothreitol;
GSH, reduced glutathione;
GOH, -L-Glu-L-Ser-Gly;
NHA, NG-hydroxy-L-arginine;
HPLC, high
performance liquid chromatography;
MS, mass spectrometry;
NMWL, nominal
molecular weight limit.
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