(Received for publication, October 21, 1996, and in revised form, December 17, 1996)
From the Department of Internal Medicine, Vascular Biology Research Center and Division of Hematology, University of Texas Health Science Center, Houston, Texas 77030
Nitric oxide (NO) and L-citrulline are formed from the oxidation of L-arginine by three different isoforms of NO synthase (NOS). Defining amino acid residues responsible for L-arginine binding and oxidation is a primary step toward a detailed understanding of the NOS reaction mechanisms and designing strategies for the selective inhibition of the individual isoform. We have altered Glu-361 in human endothelial NOS to Gln or Leu by site-directed mutagenesis and found that these mutations resulted in a complete loss of L-citrulline formation without disruption of the cytochrome c reductase and NADPH oxidase activities. Optical and EPR spectroscopic studies demonstrated that the Glu-361 mutants had similar spectra either in resting state or reduced CO-complex as the wild type. The heme ligand, imidazole, could induce a low spin state in both wild-type and Glu-361 mutants. However, unlike the wild-type enzyme, the low spin imidazole complex of Glu-361 mutants was not reversed to a high spin state by addition of either L-arginine, acetylguanidine, or 2-aminothiazole. Direct L-arginine binding could not be detected in the mutants either. These results strongly indicate that Glu-361 in human endothelial NOS is specifically involved in the interaction with L-arginine. Mutation of this residue abolished the L-arginine binding without disruption of other functional characteristics.
Nitric oxide (NO)1 has been identified as an important signal mediator that is involved in many physiological or pathophysiological processes. NO is produced together with L-citrulline through a two-step oxidation of L-arginine by three different NO synthase isoforms. The endothelial and neuronal isoforms are constitutively expressed, and their activities are regulated by calcium and calmodulin (Ca2+/CaM) (1, 2). The third NOS isoform is induced in response to cytokines or lipopolysaccharide, and its activity is independent of Ca2+/CaM (3). Despite the modest sequence homology and different regulation among the three NOS isoforms, they share a similar cofactor composition and possess a bidomain structure (4-8). The C-terminal reductase domain is homologous to NADPH-cytochrome P450 reductase and has binding regions for NADPH, FAD, and FMN. The N-terminal oxygenase domain containing heme, BH4, and L-arginine binding sites is a P450-type hemoprotein, but does not show sequence homology to other known P450s. A CaM binding module exists near the center of the NOS sequences (9), and binding of Ca2+/CaM facilitates electron transfer from the reductase to the oxygenase domain (10, 11).
Because of the obvious impact of NO on human health, a detailed understanding of the NOS reaction mechanism is essential for selective pharmacological intervention against individual isoforms. The oxygenase active site domain, including the proximal heme thiolate ligand, the distal heme pocket, and the substrate binding region is the center of NOS catalysis. Defining the amino acid residues making up the heme binding region and substrate oxidation site is a key step in unraveling the biochemistry of NO synthesis. The proximal heme thiolate ligand was first predicted by McMillan et al. (12) and subsequently confirmed by site-directed mutagenesis and spectral analysis for three NOS isoforms (13-16). However, due to the lack of a three-dimensional NOS structure and the lack of sequence homology of the NOS oxygenase domain to other P450s, it has been difficult to identify residues in the distal heme pocket responsible for L-arginine binding. Recently, a large fragment comprising residues 558-721 in nNOS was initially proposed to contain the BH4 binding site, but later shown to participate in binding of an Arg-analogue, NG-nitro-L-arginine (17). Amino acid residues 327-490 and 337-500 are the corresponding regions for human endothelial and murine macrophage isoforms of NOS, respectively. Analysis of these sequences revealed high sequence similarities in residues 340-380, 350-390, and 571-611 for human eNOS, iNOS, and nNOS, respectively. Active site studies using Arg-based analogs and heme ligands as probes strongly suggested that the guanidino head group is critical for L-arginine binding to NOS (18-20), presumably via polar or anionic residues. To identify such residues, we examined the effect of mutations of several anionic residues in the 340-380 region in human endothelial NOS; one residue, Glu-361, was shown to be specifically involved in L-arginine binding.
L-[2,3,4,5-3H]Arginine
(58 Ci/mmol) was purchased from Amersham Corp.
(6R)-5,6,7,8-Tetrahydro-L-biopterin
(BH4) was obtained from Research Biochemical International.
AG 50W-X8, cation-exchange resin, Bradford protein dye reagent, and
electrophoretic chemicals were products of Bio-Rad. Spodoptera
frugiperda (Sf9) cells, baculovirus transfer vector (pVL1392),
and BaculoGold viral DNA were obtained from Pharmingen. Grace's insect
cell culture medium was purchased from Life Technologies, Inc. NADPH,
CaM, and other chemicals were purchased from Sigma.
CaM-Sepharose and 2,5
-ADP-Sepharose were products of Pharmacia
Biotech Inc.
Soluble eNOS prepared by mutation of the second
amino acid, glycine, to alanine was described
elsewhere.2 The oligonucleotide primer for
mutation of Glu-361 Leu was: 5
-CATGAGCAC
CGGCAC-3
. This primer
introduces an AseI site (underlined) and mutates the GAG
codon to TTA (boldface). The primer for the mutation of Glu-361 to
glutamine was: 5
-AGCACTCAGATC
AGGAAC-3
. A
KpnI site (underlined) was inserted, and GAG codon was
changed to CAG (boldface). The basic strategy for polymerase chain
reaction-based mutagenesis was described previously (13). The mutations
were confirmed by sequencing using the dideoxy chain termination method and Sequenase 2.0 (U. S. Biochemical Corp.) (21).
Wild-type and mutated eNOS cDNAs were inserted into the EcoRI site of the pVL1392 transfer vector and used to generate recombinant virus in Sf9 cells. Procedures for preparation of crude cell homogenates and purification of recombinant proteins were essentially the same as described previously (22). Because the myristoylation site was removed, the proteins were purified directly from the soluble fraction without detergent treatment.
Assays of Enzyme ActivityNOS activity was assayed by
measuring conversion of L-[3H]arginine to
L-[3H]citrulline (22). NADPH oxidase activity
was measured as the decrease in absorbance at 340 nm before and after
addition of 0.5 µM calmodulin, using an extinction
coefficient of 6.22 mM1 cm
1.
Assays were performed at 37 °C in a cuvette containing 1 ml of 50 mM Tris-HCl buffer, pH 7.5, 1 mM
dithiothreitol, 0.1 mM EDTA, 0.1 mM EGTA, 10%
glycerol (Buffer A), with 2.5 mM CaCl2, 10 µM BH4, 4 µg of enzyme, and 100 µM NADPH in the presence or absence of 1 mM
L-arginine. Cytochrome c reductase activity was determined as the absorbance increase at 550 nm using a
red-ox of 21 mM
1
cm
1 as described previously (8).
Assay of L-arginine binding was performed by a previously reported procedure with slight modifications (23). A 100-µl reaction mixture containing Buffer A and 3 µg of purified enzyme was incubated on ice for 15 min with a serial concentration of unlabeled L-arginine (0-20 µM) and a fixed concentration of 3H-labeled L-arginine (2 µCi). The incubation was stopped by adding 0.15 ml of cold solution of bovine serum albumin prepared in Buffer A (20 mg/ml) and 0.75 ml of a 20% aqueous solution of polyethylene glycol. The mixtures were vortexed, incubated on ice for another 15 min, and then centrifuged at 12,000 × g at 4 °C for 20 min. The supernatant was removed by aspiration, the pellet was dissolved in 100 µl of H2O, and the radioactivity was determined by scintillation counting. Parallel experiments in the presence of 50 µM BH4 were carried out to assess the effects of BH4. Nonspecific binding (less than 3%) was measured in the absence of NOS and subtracted from the total binding.
Optical and EPR SpectroscopyOptical spectra were recorded by using a Shimadzu-2101 PC spectrophotometer. The ferrous heme-CO spectrum was obtained by flushing the sample with CO gas, followed by reducing the sample with dithionite solution. Spectral perturbation by L-arginine or imidazole was conducted as described by McMillan and Masters (24). Titration of the enzyme with imidazole was carried out by stepwise additions of a stock solution of imidazole. Binding isotherms were constructed by plotting the difference in absorbance at 432 and 394 nm as a function of imidazole concentration. Dissociation constant (Kd) values were estimated by fitting the data to a hyperbolic one-site binding model. EPR spectra were recorded on a Varian E-6 spectrometer with an Air Products liquid helium transfer line (25). A Hewlett-Packard HP5342 frequency counter was used to monitor the microwave frequency. Progressive power saturation of the flavin radical was detailed elsewhere (26). Half-saturation power, P1/2 was obtained by nonlinear regression to an equation of the form,
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(Eq. 1) |
Gel filtration chromatography
was carried out using a Superdex 200 column (Pharmacia). The column was
pre-equilibrated with phosphate-buffered saline. Thyroglobulin,
-globulin, bovine serum albumin, ovalbumin, and myoglobin were used
as molecular weight standards.
Sequence homology comparisons revealed that the putative L-arginine binding regions are highly conserved among the three human NOS isoforms (Table I). Several anionic residues in the region from residue 338 to 379 of human eNOS were mutated, and these mutants were expressed in Sf9 cells. The crude cell homogenates were prepared for L-citrulline formation assay and Western blotting. When the same amount of expressed proteins was used in assays, E342I and E377I retained most of L-citrulline forming activity as the wild-type enzyme. In contrast, E347L, E361L, and D369I mutants were almost inactive (Table II). These three eNOS mutants were overexpressed and purified. The optical spectra of purified protein were determined, and only the Glu-361 mutant preserved the native heme spectra. We thus further characterized the Glu-361 mutant. Besides E361L, E361Q was also constructed, and the mutant protein was purified. L-Citrulline formation, cytochrome c reduction, and NADPH oxidase activities of both purified Glu-361 mutants are summarized in Table III. Mutation of Glu-361 to Leu or Gln resulted in a complete loss of L-citrulline formation, but retained essentially all of the NADPH-cytochrome c reductase and NADPH oxidase activities. Similar to that of the wild type, the cytochrome c reduction rate of the Glu-361 mutants was enhanced 10-fold by the presence of Ca2+/CaM. NADPH oxidation was observed only in the presence of CaM for all these recombinant enzymes. When L-arginine was added to the reaction mixture, the NADPH oxidation rate was doubled for the wild type but was not increased for the two mutants.
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The rate of
NADPH oxidation by NOS directly reflects the rate of electron flux to
the heme center (10, 11). The two Glu-361 mutants exhibited similar
cytochrome c reductase and NADPH oxidase activities as the
wild type, indicating that electron transfer from flavin to cytochrome
c or to the heme center was not affected by mutation of
Glu-361 to Leu or Gln. Results from
L-[3H]arginine binding studies indicated that
L-arginine binding to wild-type eNOS was
concentration-dependent with a Kd of
~1.2 µM. The binding was not affected by the presence
of 50 µM BH4. In contrast, no
L-arginine binding was detected with either of the Glu-361
mutants (Fig. 1).
Optical and EPR Spectroscopic Characterization
Optical
spectroscopy was utilized to examine the heme environment of the E361L
mutant. The resting E361L mutant exhibited a Soret peak at 397 nm, a
charge-transfer band at 647 nm, and a flavin absorbance shoulder
between 450 and 475 nm (Fig. 2A). The
dithionite-reduced, CO-bound E361L displayed spectral peaks at 445 nm
and 550 nm (Fig. 2A). These characteristics are similar to
those observed for the wild-type enzyme.2 As shown in Fig.
2, B and C, addition of imidazole shifted the Soret peak to 428 nm for both the wild-type and the E361L enzymes. The
Kd values for imidazole were calculated from the data of optical titration to be 102 and 50 µM for the
wild-type enzyme and the E361L mutant, respectively (Fig. 2,
B and C, insets). The low spin
spectrum of the imidazole complex of wild-type eNOS could be converted
back to high spin (394 nm) upon addition of L-arginine
(Fig. 2B). However, no spectral change was induced upon
addition of L-arginine to the E361L-imidazole complex (Fig. 2C). Two other ligands, 2-aminothiazole and acetylguanidine,
have also been found to cause type I spectral changes for wild-type eNOS, with Kd values of 125 and 0.45 µM, respectively (20). Addition of these two ligands
resulted in shifting of the wild type-imidazole complex to high spin,
but neither ligand altered the low spin state of the E361L-imidazole
complex (data not shown).
We further characterized the heme environment and the flavin component
of the E361L mutant by EPR spectroscopy. EPR spectrum of the resting
wild-type eNOS appears to contain both high and low spin hemes
(spectrum a in Fig. 3A). The high
spin heme exhibited g values of 7.67, 4.34, and 1.84 (vertical arrows). Components of the low spin signal were
observed at g values of 2.45, 2.30, and 1.87 (vertical
lines). Addition of 300 µM L-arginine to
the wild-type eNOS resulted in a disappearance of the low spin signals and an increased amplitude of the high spin signals (spectrum b in Fig. 3A). The g values of high spin
heme complex with L-Arg were 7.65, 4.33, and 1.84. The
E361L mutant (spectrum c in Fig. 3A) exhibited
essentially identical EPR spectra for both high and low spin heme as
the wild-type eNOS, except that the low spin heme signals of the
Glu-361 mutant are more pronounced than those of the wild type.
Addition of L-arginine to E361L enzyme did not alter the
proportion of low spin and high spin heme, nor did it change the
g values (spectrum d in Fig. 3A).
Addition of imidazole to the E361L mutant resulted in a complete shift
to the low spin state (spectrum e in Fig. 3A).
The presence of two discrete sets of g values (at 2.70, 2.30, 1.75 and 2.57, 2.30, 1.83) indicated that at least two low spin
heme species were present in the E361L-imidazole complex. A similar
heterogeneity was also seen in the imidazole complex of wild-type eNOS
(26). The sharp free radical signals observed in Fig. 3A are
enlarged in Fig. 3B, inset. This free radical
displayed an overall line width of 20 G and was centered at a
g value of 2.004, typical for a neutral flavin semiquinone radical (26, 31). A power saturation study of the flavin radical gave
the same P1/2 value (50 microwatts) for both
wild-type eNOS and the E361L mutant. This result indicated that
mutation of Glu-361 did not change the dipolar interaction between the
heme center and the flavin radical (Fig. 3B).
Dimeric Structure of the Wild-type eNOS and the E361L Mutant
L-Arginine binding has been reported to
influence the dimeric assembly of macrophage iNOS (32). The quaternary
structures of wild-type eNOS and the Glu-361 mutant were analyzed by
gel filtration chromatography. The two recombinant enzymes had
indistinguishable elution volumes (Fig. 4) with
molecular mass estimated to be 280 kDa. Given the subunit molecular
mass of 135 kDa deduced from SDS-polyacrylamide gel
electrophoresis,2 the chromatographic results indicate that
both recombinant enzymes were homodimeric.
Understanding of the NOS interaction with its substrate,
L-arginine, is important not only for developing highly
selective inhibitors, but also for elucidating the catalytic mechanism. L-Arginine analogs in conjunction with spectroscopic
studies to determine the factors contributing to the Arg-NOS
interaction yielded conflicting results. Some reports suggested that
positive charge is required for the tight binding of L-Arg
to NOS (33, 34). Others showed that positive charge may not be critical for the Arg-NOS interaction (35, 36). Studies with the Glu-361 mutant
in this report has provided a clearer insight into the nature of the
interaction of eNOS with arginine. Results described above indicate
that the Glu-361 residue is specifically involved in binding of
L-arginine to eNOS. 1) Neither E361L nor E361Q mutant binds
to L-[3H]arginine (Fig. 1). 2) In contrast to
the wild type, imidazole-derived low spin complexes of Glu-361 mutants
were insensitive to L-arginine and other type I ligands,
such as 2-aminothiazole and acetylguanidine (Figs. 2 and 3), consistent
with a defective L-arginine binding. 3) The NADPH oxidase
activity for wild type was increased 2-fold upon addition of
L-arginine, but no such change was observed for either
mutant. Arginine binding changed the heme spin states and likely
increased the redox potential. This change will facilitate the electron
transfer to heme center and increase NADPH oxidation rate (11). The
lack of change of NADPH oxidase activity for the Glu-361 mutant
reflects a binding defect of the substrate. However, mutation of
Glu-361 did not perturb other functional characteristics. The Glu-361
mutant preserved essentially all of the NADPH-cytochrome c
reductase activity and NADPH oxidase activity compared with the
wild-type eNOS (Table III). The Glu-361 mutants also exhibited
identical optical and EPR spectra for their resting, reduced CO complex
and imidazole complex as the wild type. The flavin radical and its
relaxation behavior on EPR are also preserved. Although the Glu-361
mutant contained more low spin heme than the wild type, this was
probably attributed to a slight difference in the solvent accessibility
of the heme in that the Glu-361 mutant had more molecules of
H2O/OH at the sixth position. Furthermore,
the Glu-361 mutant retained a homodimeric structure just like the wild
type (Fig. 4). On the other hand, mutations of other anionic residues
in the vicinity of the Glu-361 residue either had no effect or showed
multiple effects on eNOS. Mutations of residues Glu-347 and Asp-369
also led to a complete loss of L-citrulline formation;
however, the heme environment of these two mutants was disturbed. In
contrast, mutation of Glu-361 preserved the native heme spectra and
other functional characteristics. Loss of enzymic activity mainly
resulted from the defective L-arginine binding. These
results strongly indicate that Glu-361 plays an important role in
L-arginine binding.
Glu-361 residue interacts with L-arginine probably by ionic
interaction. 2-Aminothiazole and acetylguanidine, which share with
L-arginine a common guanidine structure but lack the amino acid function, could convert the low spin imidazole complex to a high
spin state in the wild type but not in the Glu-361 mutant, indicating a
direct interaction of the guanidine moiety of
L-arginine with Glu-361 residue. We have also shown that
mutation of Glu-361 to Gln destroyed the L-citrulline
formation and L-[3H]arginine binding,
indicating that the negative charge on -carboxylate of Glu-361
residue is required for the interaction with L-arginine. In
addition, Kd values of L-arginine
binding to wild-type eNOS determined in the buffers containing 0, 1, and 2 M NaCl are 1.1, 9.0, and 10.5 µM,
respectively, implicating that L-arginine binding affinity
decreased by increasing ionic strength. The result substantiates the
importance of charge interaction in L-arginine binding and
confirms our EPR studies (26) which imply that the distal heme pocket
of eNOS contains a much more polar surrounding than other P450s. In the
structure of L-arginine, the only cationic group which
could possibly form an ionic bond with the
-carboxylate of the
Glu-361 residue is the guanidinium nitrogen. A diagrammatic illustration of this specific interaction between Glu-361 and L-arginine is shown in Scheme I. Binding of
L-arginine to eNOS would orient the substrate so that its
side chain guanidine group is brought near the heme iron to facilitate
a monooxygenation reaction. A charge interaction between
-carboxylate of Glu-361 and the guanidinium of arginine provides the
major stabilization energy for the enzyme-substrate complex, as
replacement of Glu-361 by a glutamine also removed
L-arginine binding activity.
In summary, this work has demonstrated that Glu-361 residue in human eNOS is critically involved in the interaction with L-arginine. Mutation of this residue specifically impaired the L-arginine binding without disruption of other characteristics. Our study has provided important information about the nature of Arg-NOS interaction and will shed light on the substrate binding mechanism of eNOS and the other two isoforms.
We thank Dr. Graham Palmer for his kind support of the EPR facility. We also thank Dr. Richard J. Kulmacz for helpful discussions and reading this manuscript.