From the Department of Biochemistry, the University
of Texas Health Science Center at San Antonio, San Antonio, Texas
78284-7760 and the ¶ Department of Molecular Biology and
Biochemistry, the University of California,
Irvine, California 92697-3900
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ABSTRACT |
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Recently, we obtained x-ray crystallographic data
showing the presence of a ZnS4 center in the
structure of Escherichia coli-expressed bovine endothelial
nitric-oxide synthase (eNOS) and rat neuronal nitric-oxide synthase
(nNOS). The zinc atom is coordinated by two CXXXXC motifs,
one motif being contributed by each NOS monomer (cysteine 326 through
cysteine 331 in rat nNOS). Mutation of the nNOS cysteine 331 to alanine
(C331A) results in the loss of NO· synthetic activity and also
results in an inability to bind zinc efficiently. Although prolonged
incubation of the C331A mutant of nNOS with high concentrations of
L-arginine results in a catalytically active enzyme, zinc
binding is not restored. In this study, we investigate the zinc
stoichiometry in wild-type nNOS and eNOS, as well as in the
C331A-mutated nNOS, using a chelation assay and electrothermal
vaporization-inductively coupled plasma-mass spectrometry. The data
reveal an approximate 2:1 stoichiometry of heme to zinc in
(6R)-5,6,7,8-tetrahydro-L-biopterin-replete, wild-type nNOS and eNOS and show that the reactivated C331A mutant of
nNOS has a limited ability to bind zinc. The present study substantiates that the zinc in NOS is structural rather than catalytic and is important for maintaining optimally functional, enzymatically active, constitutive NOSs.
The constitutively expressed isoforms of nitric-oxide synthase
(NOS),1 endothelial
nitric-oxide synthase (eNOS; NOS III) and neuronal nitric-oxide
synthase (nNOS; NOS I), catalyze the NADPH-dependent conversion of L-arginine to L-citrulline with
the concomitant formation of nitric oxide (NO·) (1-5). NOS
isoforms are bidomain global structures in nature, being composed of a
flavin-containing C-terminal reductase domain and an N-terminal
oxygenase domain (6). The oxygenase domain contains iron protoporphyrin
IX (7-10) and binding sites for tetrahydrobiopterin (BH4)
and the substrate, L-arginine (11). All of the
aforementioned cofactors are required for full enzymatic activity; thus
cofactor binding would be expected to alter the enzyme function.
Site-directed mutagenesis studies have indicated that cysteine 99 of
human eNOS is involved in BH4 binding to that isoform (12)
and mutation of iNOS cysteine 109 leads to diminished BH4 binding (13). Additionally, deletion of the entire CXXXXC
motif (Fig. 1) in bovine eNOS causes a dramatic loss of enzyme
stability (14). To test the possibility of altered cofactor binding in nNOS, we mutated cysteine 331 (the homologous residue to cysteine 99 of
human eNOS) to alanine and have shown that this mutation affects
L-arginine binding and reductase-to-heme electron transfer (15, 16). Prolonged incubation of the nNOS C331A mutant with high
concentrations of L-arginine is required for substrate
binding to this mutant. However, once L-arginine binding is
restored, BH4 can then bind, resulting in efficient
flavoprotein-to-heme electron transfer. This reactivated mutant, with
BH4 and L-arginine bound, possesses the same
electron transfer properties and enzymatic activity as wild-type nNOS
(16).
In addition to cysteine 331, cysteine 326 of nNOS is also
phylogenetically conserved (Fig. 1).
Recently, we have obtained x-ray crystallographic data showing the
presence of a tetrahedral tetrathiolate zinc (ZnS4) center
in the trypsin-cleaved heme domain of eNOS (17) and also
nNOS,2 which is formed by the
CXXXXC motifs from two NOS subunits. Because full enzymatic
activity of the C331A mutant can be achieved by prolonged incubation
with L-arginine (16), we were interested in what role zinc
plays in NOS structure/function. Therefore, considering that mutations
within the CXXXXC motif should disrupt the ZnS4
center, we have determined the amount of zinc bound to wild-type forms
of nNOS and eNOS, as well as to various constructs of each and the
C331A-mutated nNOS. Furthermore, we have investigated the time course
required for L-arginine activation of the C331A-mutated nNOS.
Chemicals--
4-(2-Pyridylazo)resorcinol disodium salt (PAR),
Trizma Base, nitrilotriacetic acid (NTA), EDTA, L-arginine,
and 2-mercaptoethanol were obtained from Sigma. BH4 was
obtained from RBI (Natick, MA). Sodium chloride and glycerol were
purchased from EM Science (Gibbstown, NJ). Chelex® 100 resin was
purchased from Bio-Rad. Yeast extract and tryptone were from Difco Labs
(Detroit, MI). Zinc chloride was from Aldrich. Guanidine HCl was from
ICN Biomedicals (Aurora, OH).
nNOS, eNOS, and Constructs--
nNOS and the C331A-mutated nNOS
were prepared as described by Roman et al. (18) and
Martásek et al. (16), respectively, and portions of
the preparations were incubated overnight either in the presence of 10 mM L-arginine or 10 mM
L-arginine and 0.5 mM BH4 after
affinity chromatography and before FPLC purification. Wild-type eNOS
was prepared as described by Martásek et al.
(19). Enzymes and constructs were purified by FPLC in Chelex®
100-treated buffer consisting of 50 mM Tris, 100 mM NaCl, 1 mM 2-mercaptoethanol, 0.5 mM L-arginine, and 10% glycerol, pH 7.8.
ETV-ICP-MS Analysis--
All labware used for handling samples
for metal determination was soaked overnight in 4 M nitric
acid and rinsed with Chelex®-treated water before use. Enzymes were
purified by FPLC in EDTA-free Chelex®-treated buffer, concentrated,
and subsequently passed over a 10-cm column of Chelex®-100 and
reconcentrated. ETV-ICP-MS analysis was performed by Delony Langer and
Dr. James Holcombe (Department of Chemistry and Biochemistry,
University of Texas at Austin). Standard deviations were calculated
from the uncertainties in the slopes of the calibration curves. All
results were derived from three replicates, plotted, and analyzed using
a least squares fit. Metal concentrations were corrected for the metal
contribution of the buffer, which, as a percentage of signal, was 29%
for zinc and 18% for copper.
Chelation Assay--
Zinc content of the constitutive NOS
isoforms and constructs was also measured by the PAR assay essentially
as described by Crow et al. (20) with modifications. PAR has
a low absorbance at 500 nm in the absence of Zn2+ (Fig.
2). However, in the presence of
Zn2+, the absorbance at 500 nm increases dramatically as
the PAR2Zn2+ complex is formed. Enzyme (1 nmol
in a volume of 20 µl) was added to a rapidly stirred 3-ml cuvette
containing 100 µM PAR in Chelex® 100-treated 50 mM Tris, 100 mM NaCl, pH 7.8, and, for some
experiments, including Chelex® 100-treated 7 M guanidine
HCl. In some assays, oxidants were added to facilitate Zn2+
release. Assays were run at 23 °C in rapidly stirred cuvettes, and
the total assay volume was 1.5 ml. Near the end of the assay, 1 mM NTA was added. Under these assay conditions, NTA
selectively chelates Zn2+ from the
PAR2Zn2+ complex and causes a decrease in
absorbance at 500 nm, which is used to quantitate the amount of zinc
that has been released from the protein. Following the NTA addition, 1 mM EDTA was added to confirm that no other metal was being
chelated in addition to zinc. The extinction coefficient for
PAR2Zn2+ was calculated, using a standard
solution of ZnCl2, to be 87.7 mM Activity Assay--
NOS activity was measured using the
hemoglobin capture assay in 50 mM Tris, 100 mM
NaCl, pH 7.8. The rate of methemoglobin formation from oxyhemoglobin
was measured as the difference in absorbance change between 401 and 411 nm/min as described by Sheta et al. (6) except that an
extinction coefficient of 60 mM Protein Determination--
The protein concentration for the
enzymes and constructs was determined on the basis of heme content by
reduced CO difference spectra using an extinction coefficient of 100 mM With nNOS-wt holoenzyme, 90% of the zinc (based on 1 zinc atom/2
hemes) was released in the presence of 7 M guanidine HCl and 1 mM H2O2 (Table
I). Results of the chelation assay were corroborated by ETV-ICP-MS analysis (Table
II) and confirmed that zinc and not
copper was bound to nNOS. Likewise, the trypsin-cleaved, dimeric heme
domain of nNOS-wt contained a similar zinc content (89-92%). The zinc
content (89-92%) of the trypsin-cleaved heme domain of nNOS, derived
from the full-length nNOS holoenzyme, contrasts sharply to the zinc
content of the heme domain construct (residues 1-714) of nNOS (
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Fig. 1.
Sequence alignment of nitric-oxide synthases
from different species in the region where rat nNOS cysteine 331 was
mutated to alanine detailing the phylogenetically conserved
CXXXXC motif. Also, note the conserved cysteine
326 in rat nNOS.
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1
cm
1 at pH 7.8. The results reported in Table I represent
the means ± S.D. of three or four determinations except for those
obtained with nNOS heme domain (nNOSwt-HD with BH4).
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Fig. 2.
Absorbance spectra of PAR in the absence
(solid line) and presence (dotted
line) of 5 µM zinc
chloride. Note the increase in absorbance at 500 nm following zinc
addition to PAR, indicating formation of the
PAR2Zn2+ complex.
1 was used.
All spectrophotometric assays were conducted on a Shimadzu 2101-PC
dual-beam spectrophotometer.
1 cm
1 for a
of
444-475 (21). This method of protein determination probably
underestimates the actual protein concentration because apoprotein
(minus heme) exists in all preparations to some extent. However, the
average heme content of the NOS preparations used in this laboratory is
~80%.3
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20%, data not shown) expressed in Escherichia coli. These
observations are consistent with the results of Crane et al.
(22), who crystallized the E. coli-expressed heme domain of
iNOS (residues 66-498) and did not observe enzyme-bound zinc but
rather identified a disulfide bond located in the analogous position
where Raman et al. (17) found the ZnS4 center in
the eNOS heme domain. When the zinc content of the BH4-free
nNOS-wt holoenzyme was examined, a zinc content of only 66% was
observed, suggesting that in the absence of bound BH4,
disruption or destabilization of the ZnS4 center occurs. In
contrast to the zinc contents observed with nNOS-wt holoenzyme and the
trypsin-cleaved nNOS-wt heme domain, only 5 and 19% of the theoretical
zinc content was obtained with the C331A nNOS holoenzyme mutant
preincubated with L-arginine or preincubated with
L-arginine and BH4, respectively. These data strongly suggest that mutation of cysteine 331 of nNOS results in
disruption of the ZnS4 center and prevents stoichiometric
zinc binding. However, because prolonged incubation of the nNOS C331A mutant as isolated with high concentrations of L-arginine
is sufficient to restore enzymatic activity (16), these findings
strongly implicate zinc in a structural rather than a catalytic role.
eNOS-wt holoenzyme behaved in a similar manner to nNOS with respect to zinc content. In the BH4-repleted state, eNOS was 94%
replete with zinc versus only 76% repletion in the absence
of bound BH4. Again, data from the ETV-ICP-MS analysis of
eNOS-wt holoenzyme were used to confirm that zinc and not copper was
present in eNOS (Table II). Additionally, x-ray crystallographic data
of both the BH4-free and BH4-bound eNOS heme
domain show the presence of zinc (17).
Zinc release by constitutive NOS isoforms and constructs in response to
denaturant and H2O2
80% heme-replete and considering the
possibility that heme-free enzyme might possess the ability to bind
zinc, the stoichiometry of zinc:heme could be slightly lower. Values
are expressed as the means ± S.D. of three or four
determinations. The range of two determinations is shown for nNOS-wt HD
(BH4+).
ETV-ICP-MS analysis of copper and zinc content of eNOS and nNOS
The nNOS C331A mutant is inactive as isolated, but activity can be
restored by prolonged incubation with L-arginine. Fig. 3 shows the time course for activation of
the nNOS C331A mutant upon exposure to 10 mM
L-arginine. The mutant enzyme progressively became more
active with time. However, maximal activity (255 nmol/min/mg) was not
reached for 6-8 h following exposure to L-arginine.
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In contrast to the recent findings of Perry and Marletta (23), which showed that copper is bound to E. coli-expressed nNOS-wt holoenzyme as isolated, we found no significant amount of copper in our preparations (copper to heme ratios: nNOS 0.01 and eNOS 0.03; Table II) and feel that zinc is the naturally occurring metal in the E. coli-expressed constitutive isoforms of NOS. This finding was confirmed by both the chelation assay performed in this laboratory, by x-ray crystallographic structure data (17),2 and by ETV-ICP-MS analysis performed by an independent laboratory. Perry and Marletta (23) concluded that copper and zinc are the relevant metals in nNOS and iNOS, respectively. Our crystallographic work on eNOS (17) and nNOS2 unambiguously establishes that a ZnS4 center is present at the bottom of the dimer interface in both proteins. It has also been shown that zinc can be successfully incorporated into human iNOS.4 Our biochemical characterization lends support to the x-ray crystallographic evidence for a structural zinc in all NOS isoforms. Our earlier work (24) has already provided evidence for a zinc-mediated inhibition of nNOS (Ki = 30 µM). It is conceivable that a metal binding site in intact NOS, rather than the ZnS4 site, may be responsible for this effect. The high affinity of zinc for the tetrathiolate ligands (Kd = nanomolar range) further supports a view that the zinc-mediated inhibition previously observed (24) arose from a site different from that of the ZnS4 center. It is conceivable that the work by Perry and Marletta (23) has also implicated a metal site different from the zinc site (ZnS4) discussed in the present study. Culture conditions are important and can play a critical role in which metal is incorporated into the protein. However, the quantity of copper present in the culture medium is not germane to this issue because copper cannot substitute for zinc and utilize a tetrathiolate liganding geometry. A survey of all the mononuclear copper sites in the Brookhaven Protein Data Bank reveals that the preferred geometry for copper sites in proteins is tetragonal or trigonal (25). Tetrahedral geometry at the copper site is very rare, and there is no report in the literature, to the best of our knowledge, involving CuS4, either in model compounds or in proteins. It should be noted that the only other metal that can substitute for zinc in the metal tetrathiolate is iron. Both physiological relevance and coordination geometry criteria would be satisfied if iron were to substitute for zinc. Having narrowed the number of metals to two, the issue of culture conditions that would favor one metal over the other can now be addressed. There are numerous examples in the literature addressing the problem of metal homeostasis and heterologous expression in E. coli. There are three examples worth noting. First is rubredoxin, containing a FeS4 center, which when expressed in E. coli leads to a mixture of 70% ZnS4 and 30% FeS4 containing protein (26). No zinc-containing rubredoxin is isolated from the original host, Clostridium pasteurianum. Crystal structures of both the natural FeS4 rubredoxin and the ZnS4 rubredoxin show the ability of the thiolates to coordinate either metal with similar geometry (27). Second, expression of azurin, a copper protein in E. coli, leads to a mixture of copper-azurin and zinc-azurin (28). In this protein, however, the native copper is coordinated via two histidines and a methionine. Zinc is able to substitute for copper in a trigonal geometry. Once again, crystal structures of both copper- and zinc-azurin reveal the possible need for a copper chaperone in vivo for maintaining metal homeostasis. Third, ribonucleotide reductase R2 subunit, when expressed in E. coli, is produced as an apoprotein and exogenous Fe2+ needs to be added to obtain a catalytically active protein (29). Thus, it is conceivable that NOS, when expressed in E. coli, incorporates zinc, whereas the endogenous source may contain iron. We can, however, narrow down the possibility to two metals, zinc or iron (see above). Also, as has been established for metallothionein, there remains the possibility that the ZnS4 site in NOS may be thermodynamically stable but kinetically labile, thus facilitating exchange with iron. However, in our crystallographic experiments, we did not find any evidence for the ability of iron to displace zinc (17). Therefore, we do not feel that iron is the endogenous metal in E. coli-expressed NOS. We are currently investigating the identity of the metal bound to NOS expressed in mammalian cells.
The C331A-mutated nNOS is inactive (as isolated), and its lack of
activity is reflected in its inability to bind cofactors efficiently
and stabilize an active conformation. L-Arginine activation of C331A nNOS is an interesting observation, but it is just that. The
work of Chen et al. (12), Ghosh et al. (13),
Rodríguez-Crespo et al. (14), and Martásek
et al. (16) all reveal altered cofactor binding, loss of
activity, and dramatic loss in protein stability resulting from the
loss of the zinc ligand. Therefore, it is clear that zinc-mediated
stabilization of the bottom of the dimer interface is key for catalytic
activity, albeit an indirect effect. The comparison of other
ZnS4 proteins with the NOS isoforms suggests that the
ZnS4 center facilitates protein-protein interaction(s). In
NOS, the ZnS4 center stabilizes the dimer interface and/or the flavoprotein-heme domain interface (17). Zinc is not needed for
folding because L-arginine is sufficient to restore
enzymatically active protein. If zinc were only essential for folding,
then the enzyme should function perfectly in its absence. Without
preincubation in the presence of high concentrations of
L-arginine, this is definitely not the case. Preincubation
with L-arginine must stabilize an active conformation.
However, it does not restore zinc binding. Therefore, zinc facilitates
but is not absolutely required for activity. The present study
substantiates that zinc content is influenced profoundly by pterin and
confirms a structural, rather than catalytic, role for zinc in
maintaining enzymatically active constitutive nitric-oxide synthases.
Furthermore, we demonstrate the one zinc/two heme stoichiometry in the
dimers of the constitutive NOS isoforms isolated from E. coli expression systems.
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Note Added in Proof |
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In support of our finding of zinc in the E. coli-expressed nitric-oxide synthases, Fischmann and colleagues (30) have reported the crystal structures of human inducible and endothelial nitric-oxide synthase domains in which they find a zinc tetrathiolate center at the dimer interface.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM52419 and NHLBI 30050 and Grant AQ1192 from the Robert A. Welch Foundation (to B. S. S. M.).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.
§ Supported by National Heart and Lung Cardiovascular Training Grant HL07350-19.
To whom correspondence should be addressed. Fax: 210-567-6984;
E-mail: masters{at}uthscsa.edu.
2 C. S. Raman, H. Li, T. L. Poulos, P. Martásek, and B. S. S. Masters, unpublished observations.
3 A.-L. Tsai, P. Martásek, L. J. Roman, and B. S. S. Masters, unpublished observations.
4 H. Li, manuscript submitted.
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ABBREVIATIONS |
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The abbreviations used are: NOS, nitric-oxide synthase; nNOS (NOS I), rat neuronal NOS; eNOS (NOS III), bovine endothelial NOS; ETV-ICP-MS, electrothermal vaporization-inductively coupled plasma-mass spectrometry; BH4, (6R)-5,6,7,8-tetrahydro-L-biopterin; NTA, nitrilotriacetic acid; PAR, 4-(2-pyridylazo)resorcinol; FPLC, fast protein liquid chromatography; wt, wild type.
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