From the Institute for Chemical Reaction Science, Tohoku University, Sendai 980-8577, Japan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Nitric-oxide synthase (NOS) is a flavohemoprotein
that has a cytochrome P450 (P450)-type heme active site and catalyzes
the monooxygenation of L-Arg to
NG-hydroxy-L-Arg (NHA)
according to the normal P450-type reaction in the first step of NO
synthesis. However, there is some controversy as to how the second step
of the reaction, from NHA to NO and L-citrulline, occurs
within the P450 domain of NOS. By referring to the heme active site of
P450, it is conjectured that polar amino acid(s) such as Asp/Glu and
Thr must be responsible for the activation of molecular oxygen in NOS.
In this study, we have created Asp-314 Ala and Thr-315
Ala
mutants of neuronal NOS, both of which had absorption maxima at 450 nm
in the spectra of the CO-reduced complexes and studied NO formation
rates and other kinetic parameters as well as the substrate binding
affinity. The Asp-314
Ala mutant totally abolished NO formation
activity and markedly increased the rate of
H2O2 formation by 20-fold compared with the
wild type when L-Arg was used as the substrate. The NADPH oxidation and O2 consumption rates for the Asp-314
Ala
mutant were 60-65% smaller than for the wild type. The Thr-315
Ala mutant, on the other hand, retained NO formation activity that was
23% higher than the wild type, but like the Asp-314
Ala mutation,
markedly increased the H2O2 formation rate. The
NADPH oxidation and O2 consumption rates for the Thr-315
Ala mutant were, respectively, 56 and 27% higher than for the wild
type. When NHA was used as the substrate, similar values were obtained. Thus, we propose that Asp-314 is crucial for catalysis, perhaps through
involvement in the stabilization of an oxygen-bound intermediate. An
important role for Thr-315 in the catalysis is also suggested.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Nitric oxide synthase (NOS)1 is an important heme enzyme responsible for production of nitric oxide (NO) (Refs. 1-8 and references therein). NOS is composed of an oxidative domain and a reductase domain. The oxidative domain has an active site with a cytochrome P450 (P450)-like thiol-coordinated heme iron complex. The heme distal site of the oxidative domain should be one of the substrate binding site(s) and a site for molecular oxygen binding. The heme distal site must also play an important role in the activation of molecular oxygen before its reaction with the substrate. For P450s, it has been suggested that conserved Asp/Glu and neighboring Thr residues at the heme distal site are very important for the activation of molecular oxygen (9-12).
Fig. 1 shows a comparison between selected amino acid sequences of NOSs and P450s near the N-terminal site. Asp-314 and Thr-315 (numbered for neuronal NOS) are well conserved for all NOSs so far isolated. It is not unreasonable to match these amino acids of NOS with well conserved Glu/Asp and Thr residues (Glu-318 and Thr-319 of P450d or Asp-251 and Thr-252 of P450cam) of P450s. These polar amino acids of P450s play a very important role in the activation of molecular oxygen (9-12). Therefore, it was considered worthy to mutate Asp-314 and Thr-315 of neuronal NOS (nNOS) to study their effects on kinetic parameters associated with activation of O2.
|
In this study, we present data obtained for the Asp-314 Ala and
Thr-315
Ala mutants of nNOS including rates for NO formation, O2 consumption, H2O2 formation,
NADPH oxidation, and cytochrome c reduction. On the basis of
this work, we propose that Asp-314 and perhaps Thr-315 are crucially
involved in the activation of molecular oxygen during the catalytic
turnover of nNOS.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials--
The cDNA for rat nNOS was a kind gift of Dr.
S. H. Snyder. Site-directed mutagenesis and expression of the wild
type and mutant nNOS proteins in yeast (Saccharomyces
cerevisiae), and subsequent purification of expressed nNOS
proteins was carried out as described previously (13-16). The purified
enzymes were dialyzed against 50 mM Hepes-HCl (pH 7.4)
buffer containing 10 µM H4B, 10 µM dithiothreitol, 0.1 mM EDTA, and 10%
glycerol. The CO-reduced complexes of both mutants gave optical
absorption bands at 450 nm, suggesting that the denatured form, P420,
did not result from the mutations. The concentrations of nNOS were
determined optically from the CO-reduced reduced difference spectrum
using
444-467 nm = 55 mM
1
cm
1 as determined by the pyridine hemochromogen method
(17). Protein concentrations were determined by the Bradford protein
assay using bovine serum albumin as a standard. Purified wild type and
mutant proteins were more than 95% pure as judged by
SDS-polyacrylamide gel electrophoresis and Western blotting analysis.
All three enzymes showed a similar dimer formation pattern in low
temperature SDS-polyacrylamide gel electrophoresis conducted in the
presence of L-Arg and H4B as described
previously (18), suggesting that the mutations did not alter the
dimer-monomer equilibrium of the enzyme. The Soret spectral changes of
the mutants caused by adding dithiothreitol were the same as those of
the wild type, suggesting that the H4B binding site(s) was
retained in the mutants (19). The heme content of both purified mutants
estimated from reduced-CO
reduced difference spectra using the
calculated for the wild type agreed well with those calculated
using the pyridine hemochromogen assay, indicating that the mutations
did not result in heme loss and that a 1:1 heme:protein ratio is
retained by the mutants.
Assays of Enzyme Activity--
The rate of NO formation was
determined from the NO-mediated conversion of oxyhemoglobin to
methemoglobin, monitored at 401 nm using a methemoglobin minus
oxyhemoglobin extinction coefficient of 38 mM1 cm
1 (2). The NADPH
oxidation rate was determined spectrophotometrically as an absorbance
decrease at 340 nm, using an extinction coefficient of 6.22 mM
1 cm
1. Unless otherwise
indicated, assays were carried out at 25 °C in 50 mM
Hepes-HCl (pH 7.4) buffer containing 0.1 mM NADPH, 5 µM each of FAD and FMN, 10 µg/ml calmodulin, 1 mM CaCl2, 10 units/ml catalase, 10 units/ml
superoxide dismutase, 5 µM H4B, 5 µM dithiothreitol, and 0.05-0.2 nmol of NOS in the
presence or absence of 0.5 mM L-Arg or NHA.
Catalase and superoxide dismutase were added to avoid reactions between
NO and active oxygen species such as O
2 and
H2O2 that may also be constantly formed during
the catalysis (16, 20). Even in the absence of these enzymes, the same
activities were obtained, although the linearities of the assays were
shorter. Cytochrome c reductase activity was determined by
monitoring the absorbance at 550 nm using a
red-ox = 21 mM
1 cm
1. The
H2O2 generation rate was measured by the
formation of ferric thiocyanate under similar conditions as described
above without catalase and superoxide dismutase present. The
O2 consumption rate was determined with a Clark-type oxygen
electrode under the same conditions as the H2O2
generation assay.
UV-Visible Spectroscopy-- Absorption spectra were measured in 50 mM Tris-HCl (pH 7.4) containing 10 µM H4B, 10 µM dithiothreitol, 0.1 mM EDTA, and 10% glycerol with a Shimadzu UV-2500 spectrophotometer at 25 °C.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fig. 2 shows optical absorption
spectra of the wild type (A), the Asp-314 Ala mutant
(B), and the Thr-315
Ala mutant (C). The
ferric complex of the wild type had a Soret absorption peak at around
400 nm, which was shifted to 395 nm on addition of L-Arg as
reported previously (15). On the other hand, for the ferric Asp-314
Ala and Thr-315
Ala mutants, the Soret peaks were observed at 440 and 395 nm, respectively, and were not altered on addition of
L-Arg. The addition of L-Arg to
imidazole-induced low spin complexes of wild type NOS changes the spin
state to high spin, reflecting substrate binding to the active site
(21). The addition of excess L-Arg clearly changed the spin
state from imidazole-bound low spin (with a 427-nm absorption peak) to
high spin (with a 395-nm absorption peak) for the Thr-315
Ala
mutant (not shown). The absorption peak observed at 455 nm for the
Asp-314
Ala mutant CO-reduced complex in the absence of
L-Arg was shifted to 450 nm on the addition of
L-Arg (Fig. 2B), suggesting that this mutant
does have L-Arg binding affinity. Thus, it is evident that
neither mutation causes a loss of substrate binding affinity under
these conditions. The CO-reduced complexes of both mutants in the
presence of L-Arg showed typical P450-type absorption peaks at 450 nm, the same as the wild type, suggesting that the heme active
sites of the both mutants are well preserved in the ferrous form.
|
Table I summarizes the various parameters obtained for the wild type and mutant enzymes. The NO formation rate, 72.1 µmol/min/µmol NOS heme (450 nmol/min/mg NOS protein), obtained for the wild type is comparable to the value previously reported (1-8, 16). In the presence of L-Arg, the NADPH oxidation and O2 consumption rates obtained for the wild type are essentially coupled to the rate of NO formation, considering that oxidation of 1.5 mol of NADPH and consumption of 2 mol of O2 generates 1 mol of NO.
|
More H2O2 was generated with the mutants both
in the absence and presence of the substrate compared with the wild
type (Table I). In the P450cam system, mutations at the
first layer of residues above the heme generate more
H2O2 than those at the second and third layers
with uncoupled substrates (22). Uncoupling occurs when mutations are
introduced so as to alter water distribution in the P450cam
active site and/or the stability of the oxygenated heme intermediate.
Altered polarity at the heme pocket caused by mutation of nNOS Asp-314
and Thr-315 residues would favor charge separation at the iron,
promoting the release of H2O2 or O2. Thus, it is suggested that Asp-314 and Thr-315 must be located close to
the heme.
The Asp-314 Ala mutant abolished the NO formation capability but
increased the rate of H2O2 formation by 20-fold
compared with the wild type in the presence of L-Arg. The
NADPH oxidation rate and O2 consumption rate observed with
the Asp-314
Ala mutant in the presence of L-Arg were
lower than observed with the wild type by 35 and 41%, respectively.
Note that this mutant retained its cytochrome c reductase
activity, indicating that the structure of the reductase domain is well
preserved. The kinetic values obtained for the Asp-314
Ala mutant
are reminiscent of Thr-252 mutants of P450cam. This residue
of P450cam is located at the heme distal site and is
suggested to be important in the activation of molecular oxygen
(9-11). The Thr-252
Ala mutation of P450cam abolished
monooxygenase activity and markedly increased the
H2O2 formation rate. The O2
consumption rate obtained for this mutant of P450cam was
48% that of the wild type value, whereas the NADH oxidation rate was
comparable to that of the P450cam wild type. Similarly,
kinetic values obtained for the Asp-314
Ala mutant of nNOS also
appear to correspond with kinetic parameters obtained for the Thr-268
Ala mutant of cytochrome P450BM3 (23). However, the
results obtained for the Asp-314
Ala mutant of nNOS differ from the
kinetic parameters obtained for the Asp-251
Ala and Asp-251
Asn
mutants of P450cam (or the Glu-318
Ala mutant of
P450d) in that the monooxidation activity, O2
consumption, and NADH oxidation rates with the P450cam
mutants were extremely low, less than 1% that of the wild type values;
therefore no marked increase in the rate of
H2O2 formation was detected (24-26). In the
P450 reaction cycle, protonation of dioxygen is required before the
O-O bond can be cleaved and the active oxygen species responsible for
the monooxidation reaction can be generated (9-13, 23-26). The
distal-site threonine is thought to stabilize the oxygen-bound intermediate through the formation of a hydrogen bond to the iron-bound oxygen. H2O2 formation by the Thr mutants of
P450cam and P450BM3 could be caused by
breakdown of a putative iron-peroxo complex before the formation of the
high-valent iron-oxo species, since the loss of the Thr hydroxyl group
reduces the stability of the oxygen-bound intermediate (9-11, 23). An
extra bound water molecule in the Thr mutants of P450cam
and P450BM3 may be responsible for uncoupling catalysis by
supplying a proton and aiding the release of water and
H2O2. Therefore, we conjecture that for nNOS,
the carboxylate proton of Asp-314 or a neighboring water proton
stabilizes the oxygen-bound intermediate in a similar fashion to
Thr-252 of P450cam and Thr-268 of P450BM3,
although this would require a low pKa for nNOS
Asp-314.
To examine whether or not Asp-314 is involved in the second reaction
from NHA to NO, the same catalytic parameters were obtained for NHA as
the substrate (Table I). For wild type nNOS, the NO formation rate with
NHA was similar to the NADPH oxidation rate, implying that most of the
reaction from NHA to NO is of the P450-type. For the Asp-314 Ala
mutant, the tendency of the catalytic values with NHA was similar to
that with L-Arg (Table I). Thus, it is suggested the
Asp-314 is also involved in the second reaction from NHA to NO.
The rates of NO formation, NADPH oxidation, and O2
consumption obtained for the Thr-315 Ala mutant with
L-Arg were 23-56% higher than the wild type. The Thr-315
Ala mutation increased the formation of
H2O2 by 11-fold compared with wild type in the presence of L-Arg. These results differ from the
observations made for the Thr-252
Ala mutants of
P450cam and P450BM3, which exhibited low
monooxidation activities, less than 15% those of the wild type values,
and NADH oxidation and O2 consumption rates 25-48% those
of the wild type values (10, 11, 23). The NO formation rate with NHA
for the Thr-315
Ala mutant of nNOS was 17% higher than with
L-Arg (Table I) but was similar to the NADPH oxidation
rate. Thus, the role of Thr-315 of nNOS may be different from that of
Thr-252 of P450cam (10, 11). H2O2
produced by the Thr-315
Ala mutant of nNOS may in part be used to
facilitate the second reaction with NHA and generate extra NO, since NO
is known to be generated from NHA and H2O2 with
nNOS even in the absence of NADPH (27). A hydrogen atom of a water
molecule near Ala-315 in the Thr-315
Ala mutant of nNOS may also
directly serve as a hydrogen bond donor in the acid-base catalysis
required for O-O bond scission (12, 24-26, 28). Otherwise, the
Thr-315
Ala mutation may alter the conformation and/or orientation
of the substrate to allow easier access to active oxygen for more efficient catalysis to occur in the active site.
During the review process, an x-ray crystallographic study of the truncated oxidative domain (residues 115-498, equivalent to residues 337-720 of nNOS) of inducible NOS (iNOS) was published (34). It was claimed that L-Arg itself works as a proton donor to cleave the O-O bond of molecular oxygen for the reaction L-Arg to NHA because only one polar amino acid, Glu-371, exists in the active-site cavity, and this is in contact with L-Arg. However, the structure of half (residues 1-336) the oxidative domain at the N-terminal site of the nNOS was not yet determined. These missing residues are expected to contain sites very important in catalysis, such as for H4B binding, L-Arg binding, dimer formation, etc. (35-37). Deletion of the N-terminal residues of iNOS (residues 1-114) or endothelial NOS (residues 1-105, residues 1-336 of nNOS) resulted in a change in the heme-iron spin state from high spin to low spin (35, 36). This suggests that the N-terminal part of NOS is located close to the heme distal site in the native enzyme such that the N-terminal deletion induced an open space above the heme for a ligand such as a water molecule to coordinate to the heme iron, switching the spin state. In fact, the x-ray crystal structure shows that the heme of the truncated iNOS oxidative domain is orientated to face the vacant space and is exposed in places (34). In P450s, amino acids in contact with the substrate differ from those contributing to O-O scission; for NOS, the same situation also appears likely (9-14, 23-26).
In summary, the results of this study suggest that Asp-314 is crucially involved in the stabilization of an oxygen-bound intermediate in nNOS catalysis and that Thr-315 is located close to the heme. Since the nNOS reaction is composed of two stepwise monooxidations, further studies to clarify how the Asp-314/Thr-315 residues contribute to these two steps remains to be carried out.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a Grant from Takeda Science Foundation and by a Grant-in-aid from Ministry of Education, Science, Sports, and Culture of Japan for Priority Area (biometallics) 9235201 (to T. S.).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.: +81-22-217-5604
(or 5605); Fax: +81-22-217-5604; E-mail:
ikuko{at}icrs.tohoku.ac.jp.
1 The abbreviations used are: NOS, nitric oxide synthase; nNOS and iNOS, neuronal and induced NOS, respectively; P450, cytochrome P450; H4B, (6R)-5,6,7,8-tetrahydro-L-biopterin; NHA, NG-hydroxy-L-Arg.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|