From the Department of Immunology, Lerner Research
Institute, Cleveland Clinic, Cleveland, Ohio 44195 and the
¶ Department of Medical Biochemistry, Ohio State University,
Columbus, Ohio 43210
Received for publication, July 31, 2000, and in revised form, October 6, 2000
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ABSTRACT |
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Rat neuronal NO synthase (nNOS) contains an
Akt-dependent phosphorylation motif in its reductase
domain. We mutated a target residue in that site (Ser-1412 to Asp) to
mimic phosphorylation and then characterized the mutant using
conventional and stopped-flow spectroscopies. Compared with wild-type,
S1412D nNOS catalyzed faster cytochrome c and ferricyanide
reduction but displayed slower steady-state NO synthesis with greater
uncoupling of NADPH oxidation. Paradoxically, the mutant had faster
heme reduction, faster heme-NO complex formation, and greater heme-NO
complex accumulation at steady state. To understand how these behaviors
related to flavin and heme reduction rates, we utilized three soybean
calmodulins (CaMs) that supported a range of slower flavin and heme
reduction rates in mutant and wild-type nNOS. Reductase activity and
two catalytic parameters (speed and amount of heme-NO complex
formation) related directly to the speed of flavin and heme reduction.
In contrast, steady-state NO synthesis increased, reached a plateau, and then fell at the highest rate of heme reduction that was obtained with S1412D nNOS + CaM. Substituting with soybean CaM slowed heme reduction and increased steady-state NO synthesis by the mutant. We
conclude the following. 1) The S1412D mutation speeds electron transfer
out of the reductase domain. 2) Faster heme reduction speeds intrinsic
NO synthesis but diminishes NO release in the steady state. 3) Heme
reduction displays an optimum regarding NO release during steady state.
The unique behavior of S1412D nNOS reveals the importance of heme
reduction rate in controlling steady-state activity and suggests that
nNOS already has a near-optimal rate of heme reduction.
Nitric oxide (NO)1 is
synthesized by one inducible and two constitutively expressed NO
synthases (NOSs). The constitutive NOSs (endothelial (eNOS) and
neuronal synthases (nNOS)) are Ca2+-responsive and have
multiple functions in physiology and pathology (1, 2). All NOSs are
homodimers whose subunits contain an N-terminal oxygenase domain that
binds heme, (6R)-tetrahydrobiopterin (H4B), and
L-arginine (Arg), and a C-terminal reductase domain that
binds FMN, FAD, and NADPH (3, 4). A CaM binding motif is located
between the oxygenase and reductase domain, and its occupancy triggers
electron transfer between the FMN and heme (5). This enables NOSs
to catalyze the NADPH- and O2-dependent oxidation of Arg to generate NO and citrulline, with
N-hydroxyarginine (NOHA) being formed as an enzyme-bound
intermediate (3, 4).
The NOSs share many structural features but appear to differ in how
their catalysis is regulated. For example, in rat nNOS we have
established that heme-NO complex formation governs its rate of NO
synthesis during the steady state by setting up a condition where the
O2-dependent breakdown of the ferrous-NO
complex becomes a rate-limiting step (6, 7). This greatly shifts the
apparent Km,O2 value of the enzyme
and thus has physiologic impact (8). In nNOS, ferrous heme-NO complex
formation is rapid (6, 9) and results from reduction of a ferric
heme-NO species that is an immediate product of catalysis (10). In
contrast, eNOS does not build up detectable ferrous heme-NO complex
during steady-state catalysis (11), and its slow NO synthesis is
associated with slow heme reduction (9, 11).
To better understand the relationship between heme reduction rate, NO
complex formation, and NO release, we developed a kinetic model for
nNOS catalysis. The companion paper by Santolini et al. (12)
describes our kinetic model in detail and demonstrates that it can
accurately simulate initial and steady-state features of nNOS catalysis
including heme-NO complex formation, a concomitant deflection in NADPH
oxidation and NO synthesis, and an increase in enzyme apparent
Km,O2 values. Further simulations predicted that a complex relationship should exist between rates of
heme reduction and NO synthesis during the steady state (12). Specifically, there should be an optimal rate of heme reduction beyond
which NO synthesis is actually diminished, and nNOS appears to be set
near its optimum heme reduction rate. Although this concept is of
fundamental importance, only limited information is available on how
heme reduction rate affects NO synthesis by nNOS. These include studies
with CaM-troponin C chimeras (13), CaM point mutants that contain
dysfunctional Ca2+-binding sites (14), and NOS chimeras
whose reductase domains are swapped (15). In the only study where heme
reduction rates were measured (13), slower rates were obtained relative
to CaM, and NO synthesis was proportional to the heme reduction rate
(i.e. was slower). Unfortunately, no information was
provided in these studies regarding nNOS heme-NO complex formation or
how the enzymes might respond if heme reduction became faster than that
obtained with CaM.
Given the above, we were interested in reports on serine/threonine
kinase Akt-dependent phosphorylation of Ser-1179 in bovine eNOS (16) and the analogous Ser in human eNOS (17). This residue is
located in the NADPH-FAD module near the C terminus of the reductase
domain. Cotransfection of the Akt-dependent kinase with eNOS led to phosphorylation at Ser-1179, increased NO production, and
allowed the enzyme to be activated at lower Ca2+
concentrations. Mutation of Ser-1179 to Asp, which mimics
phosphorylation by permanently introducing a negative charge, also
resulted in a 2-3-fold increase in NO synthesis and faster cytochrome
c reduction (16-18). These results suggest that the
mutation may increase electron transfer in eNOS and can explain how
eNOS is activated in endothelium in response to shear stress. An Akt
phosphorylation motif (RXRXX(S/T)) is also
present at the same site in rat nNOS and contains the corresponding
residue Ser-1412. However, cotransfection of the Akt-dependent kinase with nNOS did not result in increased
NO release (16), so it is puzzling why rat nNOS responded differently than eNOS.
To address this and to investigate further how electron transfer and
catalysis are related, we generated the S1412D mutant nNOS and
thoroughly characterized its electron transfer properties, heme-NO
complex formation, and initial and steady-state catalytic behaviors.
The experimental results and simulations using our kinetic model
precisely explain the mutant phenotype and greatly expand our
understanding of how heme reduction and NO complex formation combine to
regulate NOS catalysis.
Materials--
All regents and materials were obtained from
Sigma or sources previously reported (19). Soybean CaM proteins ScaM-1,
ScaM-4, and M144V ScaM-1 were cloned, expressed, and purified as
described previously (20, 21).
Molecular Biology--
Restriction digestions, cloning,
bacterial growth, and transformation and isolation of DNA fragments
were performed using standard procedures. Site-directed mutagenesis was
done using the Quick Change polymerase chain reaction in
vitro Mutagenesis Kit from Stratagene. Incorporated mutations were
confirmed by DNA sequencing at the Cleveland Clinic Core Facility. DNA
containing the desired mutation was transformed into Escherichia
coli for protein expression.
Oligonucleotides used to incorporate the S1412D mutation in nNOS were
synthesized by Integrated DNA technologies. A silent mutation coding
for a restriction site was incorporated into the oligonucleotides to
aid in screening. The mutation (bold), silent restriction site
(underlined), and the oligonucleotides used in the process were as
follows: S1412D-BsiWI sense,
GTCACCCTCCGTACGTATGAAGTGACCAACCGCCTTAGATCTGAGGACATCGCCTT; S1412D antisense, AAGGCGATGTCCTCAGATCTAAGGCGGTTGGTCACTTCATACGTACGGAGGGTGAC.
Expression and Purification of Wild-type and nNOS
Mutant--
Wild-type rat nNOS and the S1412D mutant both contained a
His6 tag attached to their N termini. They were
overexpressed in E. coli strain BL21 (DE3) using a modified
pCWori vector and purified by sequential chromatography on
Ni2+-nitrilotriacetic acid and 2',5'-ADP-Sepharose resins
as described (19). The ferrous-CO adduct absorbing at 444 nm was used
to quantitate heme protein content using an extinction coefficient of
74 mM NO Synthesis, NADPH Oxidation, Ferricyanide Reduction, and
Cytochrome c Reduction--
Steady-state activities of wild-type and
mutant nNOS were determined separately at 25 °C using
spectrophotometric assays that were described previously in detail
(19). To assay catalytic activities versus Ca2+
concentration, the standard assay mixtures were modified to contain 20 µM EDTA, and different amounts of CaCl2 were added.
H2O2-dependent NOHA
Oxidation--
H2O2-dependent nNOS
oxidation of NOHA to nitrite was assayed in 96-well microplates at
25 °C as described previously (19) with modification. The assay
volume was 100 µl and contained 40 mM EPPS, pH 7.6, 250 nM nNOS, or mutant, 1 mM NOHA, 1 mM
dithiothreitol, 25 units/ml superoxide dismutase, 0.5 mM
EDTA, and 4 µM H4B. Reactions were initiated
by adding 30 mM H2O2 and stopped
after 10 min by adding 1300 units of catalase. Nitrite was detected at
550 nm after adding the Griess reagent (100 µl) and quantitated based on nitrite standards.
Heme and Flavin Reduction--
The kinetics of flavin and heme
reduction were analyzed at 10 °C as described previously (13, 19)
using a diode array detector and stopped-flow apparatus from Hi-Tech
Ltd. (model SF-61) equipped for anaerobic work. Heme reduction was
followed by formation of the ferrous-CO complex, and the kinetics were
determined by absorbance change at 444 nm. Reactions were initiated by
rapid mixing an anaerobic, buffered, CO-saturated solution containing 50 µM NADPH with an anaerobic CO-saturated solution
containing wild-type or mutant nNOS (2 µM), 40 mM EPPS buffer, pH 7.6, 10 µM
H4B, 0.3 mM dithiothreitol, 5 mM
Arg, 4 µM CaM, or 20 µM soybean CaM
proteins, and 1 mM Ca2+. Flavin reduction was
monitored under the same conditions at 485 nm. Signal-to-noise ratios
were improved by averaging at least 10 individual mixing experiments.
The time course of absorbance change was fit to single or multiple
exponential equations using a nonlinear least square method provided by
the instrument manufacturer.
Kinetics of Ferrous Heme-NO Buildup and Concurrent NADPH
Oxidation--
Experiments were done at 10 °C in a Hi-Tech 51MX
stopped-flow instrument equipped with a variable wavelength detector.
To initiate NO synthesis, an air-saturated solution that contained 40 mM EPPS, pH 7.6, 2 µM nNOS or mutant, 0.4 mM dithiothreitol, 2 mM Arg, 20 µM H4B, 4 µM CaM, or 20 µM soybean CaM proteins, 40 µM NADPH, and
0.5 mM EDTA was rapid mixed with a buffered solution containing 2.4 mM Ca2+. Absorbance at 436 nm
was monitored to follow ferrous heme-NO formation and at 340 nm to
follow NADPH oxidation (7). The concentration of ferrous heme-NO
complex formed during NO synthesis was estimated from the absorbance
change at 436 nm using an extinction coefficient of 49,800 M Simulations--
These were run as described in detail in the
accompanying paper (12). Iterative computations were made using the 13 rate equations that describe the kinetic model. Simulations used the same rate constants listed in (12) unless specified otherwise. All rate
constants were determined at 10 °C.
To simulate the buildup of heme-NO complex using heme reduction rates
determined for CaM and the ScaM proteins, the time step used was 50 µs over a 3-s period, generating 60,000 points for each case. The
curves were plotted and analyzed to determine a rate of complex
formation in each case, which corresponds to the slow phase of ferrous
heme-NO buildup. We removed the initial lag phase (up to 300 ms) from
each simulated kinetic trace by using a floating origin:
y = A + B
exp( The S1412D nNOS used in our study was purified to homogeneity and
had the same molecular mass as wild-type nNOS (Fig.
1). This rules out potential artifacts
due to contaminating reductase domain. The mutant displayed the same
absorbance spectrum in the presence of Arg and H4B as wild
type, and its ferrous CO-bound spectrum contained a Soret peak at 445 nm (data not shown). These properties indicate that mutation of
Ser-1412 did not perturb enzyme structure, substrate, and prosthetic
group binding or heme electronic environment.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
cm
1
(A444-A500).
1 cm
1
(6), whereas the amount of NADPH oxidation was determined using an
extinction coefficient of 6,220 M
1 cm
1
at 340 nm. Signal-to-noise ratios were improved by averaging six
consecutive scans. Each experiment was performed three separate times.
k(x
xo)). The Origin© software used was based on the Marquardt algorithm. To simulate percentage of ferrous-NO complex achieved at steady state, simulations were run using a 1-ms time step for each heme reduction rate obtained with CaM and the ScaM proteins. Ratios of NADPH oxidized
per NO released were calculated under the same simulation conditions
(see Ref. 12 for details). Simulated rates of NO release at different
O2 concentrations were calculated using a time step of 1 ms
except for O2 concentrations above 2 mM where the time step was 0.1 ms. The heme reduction rate was set to values we
obtained with CaM in this report (3.9 s
1 for
wild-type and 5.4 s
1 for S1412D mutant). For
O2 reaction with the Fe(II) heme-NO complex we used a
pseudo-first order rate constant (0.19 s
1)
that was reported for nNOS by Adak et al. (7) at a single O2 concentration (half air-saturated) at 10 °C. We then
varied this rate constant in direct proportion to the simulated
O2 concentration.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Purification of S1412D nNOS followed by
SDS-polyacrylamide gel electrophoresis. Lane 1, Coomassie-stained molecular mass markers; lane 2, wild-type
nNOS standard; lanes 3-6, S1412D mutant after final
2',5'-ADP purification step, after elution from the
Ni2+-nitrilotriacetic acid resin; after ammonium sulfate
precipitation, and after the crude extract, respectively.
NOS Catalytic Activities--
We compared catalysis by wild-type
and S1412D nNOS by measuring their rates of
H2O2-dependent NOHA oxidation,
cytochrome c or ferricyanide reduction, and NO production.
The wild-type and mutant enzymes catalyzed
H2O2-dependent oxidation of NOHA at
similar rates (8.4 and 9.0 min1,
respectively), consistent with their oxygenase domains being identical
and this reaction not requiring electrons from the reductase domain.
Both cytochrome c and ferricyanide reduction was faster in
the mutant compared with wild-type in the presence or absence of
Ca2+/CaM (Table I). The
mutant did not synthesize NO without CaM and with CaM had a rate of NO
synthesis that was 25% diminished compared with wild-type enzyme
(Table I). We conclude that the mutant displays slower NO synthesis,
but this cannot be explained by defects in oxygenase domain catalysis
or electron transfer through the reductase domain.
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Electron flux between the reductase and oxygenase domains can be a critical determinant of NO synthesis (9, 11, 13-15). We therefore compared steady-state rates of NADPH oxidation by CaM-bound S1412D and wild-type nNOS under a number of different conditions (Table II). S1412D nNOS had faster rates of NADPH oxidation than wild-type in all cases. This is consistent with the mutant catalyzing faster electron transfer from its reductase domain to external acceptors such as ferricyanide or cytochrome c. However, it cannot account for the observed decrease in NO synthesis. In fact, the concomitant decrease in NO synthesis and increase in NADPH oxidation led to a more uncoupled activity during the steady state, with the calculated NADPH/NO ratio rising to 3.5 for the mutant instead of 2 for wild-type nNOS (Tables I and II).
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Ca2+-dependent Activation of Wild-type and
Mutant nNOS--
Two different groups (16, 17) reported that
phosphorylation at the analogous residue in eNOS (Ser-1179) or
substitution with Asp increased enzyme affinity toward Ca2+
during CaM activation of NO synthesis. As shown in Fig.
2, at lower Ca2+
concentrations the nNOS mutant showed higher steady-state NO synthesis
rates compared with wild-type, but this disappeared sharply as
Ca2+ concentrations increased. The greatest difference in
activity was seen at 13 µM Ca2+ (Fig. 2,
upper inset), which was a suboptimal concentration
for both enzymes. In contrast, when the experiment was repeated using cytochrome c reduction to measure activity, mutant activity
remained consistently higher than wild-type across the same range of
Ca2+ concentrations. We conclude that the mutation causes
enhanced response to Ca2+ in nNOS, but this only occurs at
suboptimal Ca2+ concentrations and only when activity is
measured by steady-state NO synthesis.
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Heme and Flavin Reduction-- We next examined the kinetics of flavin and heme reduction at 10 °C by stopped-flow spectroscopy. Observed rate constants for heme and flavin reduction are listed along with other data in Tables III and IV. Traces in Fig. 3 panel A illustrate heme reduction in mutant and wild-type nNOS in response to CaM. Heme reduction was monophasic and achieved the same extent of reduction in both enzymes but was 1.4 times faster in the mutant. Thus, a slower steady-state NO synthesis activity in the mutant is actually associated with a faster rate of heme reduction. Panels B and C compare rates of flavin reduction in the presence or absence of CaM. Flavin reduction was biphasic in all cases and was faster in the mutant only in the absence of CaM. This indicates that faster cytochrome c reduction in the mutant is only associated with faster flavin reduction in the CaM-free state.
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Relationships between Flavin and Heme Reduction and Enzyme Behavior-- To understand better the mutant phenotype, we examined how different rates of flavin and heme reduction would impact mutant behaviors relative to wild type. For this purpose we utilized two native soybean calmodulins (ScaM1 and ScaM4) and a point mutant of ScaM1 (M144V), which were shown by Johnson and colleagues (20, 21) to bind to nNOS with good affinity but support different rates of cytochrome c reduction and NO synthesis in the steady state. Our hope was that these "CaM analogs" would behave like CaM-troponin C chimeras in supporting different rates of flavin and heme reduction in mutant and wild-type nNOS (13). As shown in Tables III and IV, this was the case because the ScaM proteins and CaM each supported different rates of flavin and heme reduction in the two enzymes.
Table III shows how different rates of flavin reduction affect
cytochrome c reductase activity in mutant and wild-type
nNOS. Flavin reduction was biphasic in all cases, with each phase
representing one-third to two-thirds of the total absorbance change
depending on the CaM used in the experiment. Although the two nNOS
started with different basal rates of flavin reduction, they achieved identical rates in response to each CaM and thus showed the same rank
order of response (CaM > ScaM4 > M144V ScaM1 > ScaM1)
and range of enhancement (100% with CaM to 50% with ScaM1). In both mutant and wild-type nNOS the cytochrome c reductase
activities increased according to the same rank order observed for
flavin reduction. As shown in Fig. 4, a
linear relationship exists between rates of flavin reduction and
cytochrome c reduction for both proteins. Moreover, Fig. 4
clearly shows that cytochrome c reduction was consistently
greater in S1412D nNOS, although it displayed the same rate of flavin
reduction as wild-type nNOS in response to any given CaM protein.
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Table IV shows that heme reduction by CaM and the ScaM proteins was stimulated according to the same rank order as for flavin reduction (CaM > ScaM4 > M144V ScaM1 > ScaM1), with heme reduction being consistently faster in S1412D nNOS in response to each protein. Because this exactly matches what we observed for cytochrome c reduction, it suggests that electron transfer to cytochrome c or the nNOS heme occurs through mechanisms that are affected in the same way by the S1412D mutation.
We next examined how different heme reduction rates affect the kinetics
of heme-NO complex buildup, the percentage of heme-NO complex attained
at steady state, and steady-state NO synthesis in mutant and wild-type
nNOS. These three parameters are good determinants of NOS catalytic
behavior (see Ref. 12). Results are summarized in Table IV. Heme-NO
complex buildup was followed at 436 nm in the stopped-flow instrument.
It occurred within the first seconds after initiating NO synthesis and
was best described by a two-exponential function in all cases, with the
slow phase representing the majority of absorbance change. As may be
seen in Fig. 5, panel A, the
fast and slow phases were both linearly proportional to the rate of
heme reduction, with data from wild-type and mutant enzymes fitting
well along the same two lines. This indicates that heme-NO complex
formation is directly linked to the heme reduction rate. Fig. 5,
panel B, shows that there was also a consistent increase in
percentage heme-NO complex attained at steady state. Thus, it is clear
that both rate and percentage of heme-NO complex buildup increases with
heme reduction rate in wild-type and S1412D nNOS.
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In contrast, the relationship between the heme reduction rate and
steady-state NO synthesis was more complex. For wild-type nNOS the
steady-state activity increased with heme reduction. In contrast, for
S1412D nNOS the activity increased, reached a plateau, and then fell at
the highest rate of heme reduction (5.4 s1,
Table IV). Thus, SCaM4 supported a greater steady-state rate of NO
synthesis in S1412D nNOS than did CaM itself, despite SCaM4 supporting
a slower rate of heme reduction. When all steady-state NO synthesis
rates were plotted versus speed of heme reduction (Fig. 5,
panel C), the distribution suggested that an optimal rate of
heme reduction exists for steady-state NO synthesis, and this optimum
is exceeded in CaM-bound S1412D nNOS.
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DISCUSSION |
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The S1412D mutant is quite valuable because it helps demonstrate how rates of flavin and heme reduction relate to the initial and steady-state catalytic behavior of nNOS. In the steady state, the mutant displayed increased NADPH oxidase and cytochrome c reductase activities but somewhat slower NO synthesis. Remarkably, this differed from the analogous S1179D eNOS mutant, which displayed an increase in all three activities (16-18). We first discuss how the S1412D mutation alters electron transfer in and out of the nNOS reductase domain, and then we discuss the effect of the mutation on heme reduction in light of our kinetic simulation model (see accompanying paper by Santolini et al. (12)) that can explain the behavior of the S1412D nNOS and why it differs from the analogous eNOS mutant.
Reductase Domain Electron Transfer-- Before discussing the effects of the mutation, it is important to review some general properties of reductase domain catalysis. All NOS reductase domains are composed of an NADPH-FAD module and an FMN module (22). As established for nNOS by Mayer and co-workers (23) and Gachhui et al. (24), electron transfer to cytochrome c and ferricyanide occurs via direct electron transfer from the reductase domain and does not require heme reduction or superoxide generation. Cytochrome c binds with high affinity to the FMN module and accepts electrons exclusively from this site, whereas ferricyanide accepts electrons from either FAD or FMN (9, 23-25).
Our results are the first to show that modification of Ser-1412 alters both flavin reduction and reductase activities in any NOS. However, it is important to note that S1412D nNOS displayed faster flavin reduction relative to wild type only in the CaM-free state, and this was associated with faster reduction of cytochrome c and ferricyanide. Upon CaM binding, the mutant lost its advantage regarding speed of flavin reduction but still displayed greater cytochrome c reductase activity than wild type. This relationship held when any of three ScaM proteins substituted for CaM. We conclude that electron transfer to cytochrome c is directly related to the speed of flavin reduction (see Table IV). However, in CaM-bound nNOS Ser-1412 primarily influences this activity through a mechanism that does not involve changing the speed of flavin reduction. Although the details are not understood, we suspect that phosphorylation at Ser-1412 alters a physical interaction in the reductase domain that enhances electron transfer out of the FMN module. One mechanism would have the mutation make the FMN group more accessible to external electron acceptors, and we are currently testing this possibility. Because Ser-1412 is in the NADPH-FAD module and therefore "upstream" from the FMN module, the Asp mutation must alter an interaction between these two modules even in the presence of CaM. Recent data suggest the C terminus of the NADP-FAD module could be a candidate for interacting with the FMN module (26). In any case, the S1412D mutation seems to speed electron transfer to the nNOS heme and to cytochrome c by the same mechanism, because changes in these two parameters correlate precisely in the mutant and wild-type enzymes (see Tables III and IV).
The S1412D mutation may increase electron transfer from NADPH to FAD in CaM-free nNOS by a different mechanism. We suspect the S1412D mutation partly relieves a repression that is exerted by the FMN module on the NADPH-FAD module. This repression of "upstream" electron transfer was first described by Adak et al. (19), who showed that several FMN module mutants had faster electron transfer between NADPH and FAD. The repression in wild-type nNOS is fully relieved by CaM binding or by FMN removal (19), and our current results show that the ScaM proteins can also relieve the repression but to lesser degrees than CaM. It is also clear that phosphorylation of Ser-1412 should only provide partial relief of repression compared with CaM binding or FMN removal. It is tempting to speculate that Ser-1412 is located at a site in the NADPH-FAD module that interacts with a repressor element from the FMN module. Although the repressor element has not been identified, a good candidate is a 40-amino acid insert present in the FMN module of nNOS that inhibits ferricyanide and cytochrome c reduction in the CaM-free state (27-29). Perhaps this element inhibits electron transfer between NADPH and FAD through interaction with Ser-1412.
Catalytic Control in S1412D nNOS--
From the perspective of NO
synthesis there is only one relevant change in the S1412D mutant, its
faster heme reduction. But our analysis of catalytic parameters shows
apparent contradictions. Indeed, how can a faster heme reduction give
rise to greater heme-NO complex formation yet slower NO synthesis in
the steady state? These paradoxical effects are in fact related to
heme-NO complex formation during NO synthesis and can be understood
within the context of our kinetic model for nNOS (12). An abbreviated
kinetic model is illustrated in Fig. 6
and involves a forward pathway that generates the ferric nNOS heme-NO
complex and citrulline as products (10). This heme-NO species then
partitions between two paths to regenerate ferric enzyme. One involves
NO dissociation from the ferric heme (step B), and the other involves
reduction of the heme-NO species to form the ferrous heme-NO complex
(step A'). Importantly, the reductive path is futile because little or
no NO is released from the ferrous heme-NO complex before it reacts
with oxygen (step C). Thus, partitioning between productive and futile
pathways reflects relative rates of NO dissociation from the ferric
heme versus its reduction to the ferrous heme-NO species.
Because NO dissociation and heme reduction occur at similar speeds in
nNOS (~5 and 3.6 s1 at 10 °C,
respectively), and oxidation of the ferrous heme-NO complex is
comparatively slow (~0.19 s
1), a majority
of nNOS is present as the ferrous heme-NO complex during steady-state
NO synthesis (6-8).
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Computer simulations of our kinetic model use rate constants derived for nNOS at 10 °C that are described in the accompanying article (12). As shown there, simulations accurately reproduce the initial and steady-state behaviors of wild-type nNOS and the behaviors of the hyperactive mutant W409F (7, 30). We therefore utilized the kinetic model to understand the pre-steady-state and steady-state behaviors of S1412D nNOS. Because this mutation is in the reductase domain, it is reasonable to assume that it only affects the speed of heme reduction. In fact, several data obtained with mutant and wild-type nNOS distributed along a single line when plotted as a function of heme reduction rate (see Fig. 5) strongly supported this assumption. The nNOS heme can receive an electron from the reductase domain at two different steps in the kinetic model as follows: during NO biosynthesis (step A in Fig. 6) and after the ferric heme-NO complex forms (step A' in Fig. 6). We assumed that the rate of heme reduction for both steps was identical in S1412D nNOS, as appears to be the case for wild-type nNOS.2 We then ran simulations of the kinetic model using heme reduction rates that were measured in response to CaM and the three ScaM proteins (see Tables III and IV).
Our experimental results showed that a linear relationship exists
between the rates of heme reduction and heme-NO complex buildup in the
first seconds of NO synthesis (Fig. 5). The fast phase of this reaction
likely involves NO binding to ferric heme as soon as it is made (10,
31). Thus, as the heme reduction rate increases, NO is made faster, and
the ferric heme-NO product should form more quickly. In addition, since
the heme-NO complex we followed here is ferrous (Soret peak at 436 nm),
we suspect that reduction of the ferric heme-NO product also increases
in speed exactly as does heme reduction in the biosynthetic pathway. To
test these assertions, we utilized our kinetic model to simulate the
kinetics of heme-NO complex buildup at different heme reduction rates,
using a 50-µs time step over a 3-s period (see "Experimental Procedures"). Simulated curves for four heme reduction rates are depicted in the inset of Fig.
7A. The lag phase we observe
is due to our model having difficulty resolving the very rapid initial phase (the first 300 ms; discussed in Ref. 12). Despite this, we could
fit the subsequent mono-exponential portion of each curve (which
reflects the slow phase of heme-NO complex formation) to obtain
simulated rates for heme-NO complex buildup at each heme reduction
rate. As shown in panel A of Fig. 7, the simulated rates actually distribute along the same line as the experimental values for
slow phase heme-NO complex buildup in S1412D and wild-type nNOS. Thus,
results from the simulation are consistent with (a) a faster
intrinsic NO synthesis for the S1412D mutant, (b)
ferric heme-NO complex buildup prior to NO release, and (c)
equivalent reduction rates for ferric and ferric-NO species within a
given enzyme.
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The amount of ferrous heme-NO complex attained at steady state also correlated with the rate of heme reduction (see Fig. 5). This parameter likely reflects enzyme partitioning between a productive regenerating cycle (release of NO from the ferric heme) and a futile one (reduction of the ferric-NO complex) (see Fig. 6). Thus, as heme reduction speeds up, the balance between these two paths should shift to favor more ferrous-NO complex formation and less NO release. This is well depicted by our model simulations. In Fig. 7, panel B, the measured and simulated percentages of ferrous heme-NO species present at steady state are plotted as a function of heme reduction rate. Although the simulated percentages are all slightly higher than the experimental values, both groups follow an identical distribution pattern that increases as heme reduction rate increases. Hence, an increase in the heme reduction rate will increase the percentage of enzyme that partitions into the futile cycle.
These two facets of enzyme catalysis (intrinsic rate of NO synthesis
and enzyme partitioning toward the ferrous heme-NO complex) are both
controlled by heme reduction, but they counterbalance one another to
different degrees depending on the rate of heme reduction. Thus, their
combined effect on steady-state NO synthesis is complex. This was noted
in the accompanying paper by Santolini et al. (12) who
simulated how steady-state NO synthesis varies with heme reduction
rate. The analysis predicted that NO synthesis should increase, reach a
maximum, and then fall as the rate of heme reduction increases. This
pattern in fact matches what we observed here for wild-type and S1412D
nNOS at different rates of heme reduction (see Fig. 5, panel
C). Thus, our kinetic model can explain why we observed an optimum
range of heme reduction for NO synthesis, and why the S1412D mutant has
slower steady-state activity. Specifically, faster heme reduction in
the mutant speeds its intrinsic NO formation (resulting in faster
ferric heme-NO complex formation) but also causes a greater proportion
of enzyme to partition into the futile cycle. Our data suggest that
heme reduction rates between 3.5 and 4.5 s1
strike an optimal balance between these opposing parameters to enable
peak NO synthesis by nNOS. Heme reduction in CaM-bound S1412D nNOS is
5.4 s
1 and therefore is too fast to strike an
optimal balance, resulting in slower NO synthesis during the steady
state. However, when its heme reduction is slowed to 4.6 s
1 by using ScaM4 in place of CaM, a near
optimal balance is regained, and the rate of NO synthesis is increased.
Simulation of our kinetic model using currently available rate
constants actually predicted an optimal rate for heme reduction between
2 and 3 s1 (12), which is slightly slower
than the experimental optimum we observed here (about 3.8 s
1, see Fig. 5, panel C). However,
it is important to realize that the optimum rate of heme reduction
depends on the following two facets of the kinetic mechanism, which
themselves are functions of multiple reactions: 1) partitioning between
productive and futile cycles, which depends on rates of ferric heme-NO
formation, NO dissociation, and reduction of the ferric heme-NO
complex, and 2) the relative rates of the productive and futile cycles, which depend on NO dissociation from the ferric heme and the reaction of O2 with the ferrous heme-NO complex. Thus, the
difference between simulated and experimental values for optimal rate
of heme reduction is probably reasonable given such a complex system.
Small inaccuracies in the kinetic constants if present can shift the
optimum rate for heme reduction (see Fig. 5C in Ref. 12).
Additional measurements and refinement of the simulation should
reconcile the relatively small difference between the theoretical and
experimental optimum and should also reveal mechanisms for
"tuning" the optimum.
Kinetic simulations also predict that the rate of NADPH oxidation will
reach a maximum as heme reduction rate increases in nNOS (12), but this
parameter does not fall off as fast as NO synthesis. Thus, with the
faster heme reduction there is a gradual decrease in catalytic
efficiency. This is because faster heme reduction favors cycling
through the futile cycle, which consumes NADPH without NO release and
therefore leads to increased uncoupling between NADPH consumption and
NO production. The simulation predicts S1412D nNOS should oxidize 3.65 NADPH per NO formed, which is quite similar to our experimental value
of 3.5. These values are greater than the ratio for wild-type nNOS (the
simulation predicts 2.8, the experimental value was 2.0) and signifies
that a greater proportion of mutant enzyme cycles through the futile
pathway during the steady state. In fact, the simulation predicts that 52% of S1412D nNOS cycles through the futile pathway in unit time, which is understandable given that its rate of heme reduction (5.4 s1) is about the same as its NO release (5 s
1) and gives a partition coefficient near 1.
Another parameter that is affected by this complex regulation is enzyme
apparent Km,O2. The apparent
Km,O2 measured during steady state
is actually a combined function of ferrous heme affinity for
O2 and the oxidation rate of the ferrous heme-NO complex,
which depends differently on O2 concentration (8). This
gives wild-type nNOS a surprisingly high apparent Km,O2 that enables NO release in a
graded manner across the entire physiologic concentration range for
O2 (0-200 µM) (8). We ran simulations to
examine how the S1412D mutant and wild-type nNOS might differ in their
steady-state NO synthesis as a function of O2 concentration
(Fig. 7, panel C). The rate of ferrous heme-NO oxidation was
assumed to vary in direct proportion to the O2
concentration, based on earlier data obtained with wild-type nNOS (8).
The simulation predicts that the apparent
Km,O2 value of the mutant will shift
to a somewhat higher value (738 µM) compared with wild
type (482 µM) (using heme reduction rates of 3.9 and 5.4 s1, respectively). This makes sense, because
the increased rate of heme reduction of the mutant causes a greater
buildup of ferrous heme-NO complex during steady state. In
air-saturated buffer (~250 µM O2), the
mutant rate of NO synthesis is predicted to be 84% that of wild type,
which is close to the 78% value calculated from our experimentally
determined rates. As shown in the inset of Fig. 7,
panel C, the simulation predicts the mutant will always display slower NO synthesis throughout the physiological concentration range of O2. This is because oxidation of the ferrous
heme-NO species remains rate-limiting for the futile cycle within this range of O2 concentrations. However, as the O2
concentration further increases to very high levels, the oxidation rate
of the ferrous heme-NO complex is predicted to eventually become faster
than the rate of heme reduction. At this point, the rate-limiting step in the futile cycle would switch to heme reduction, and under this
circumstance the simulation predicts NO synthesis by the mutant would
become faster than wild-type nNOS (Fig. 7, panel C).
Catalytic Control in eNOS and in Other nNOS Mutants--
Our
kinetic model may also explain why the analogous mutation in eNOS
(S1179D) increased steady-state NO synthesis along with NADPH oxidation
and cytochrome c reduction (16-18). Basically, heme
reduction in eNOS is much slower than in nNOS (<0.1
s1 at 10 °C; Ref. 11) and thus is in the
range where increasing it should speed NO synthesis according to the
kinetic model (12).
Two other reductase domain mutants have a similar catalytic phenotype as we observed here for S1412D nNOS. Both are nNOS deletion mutants, one missing the final 33 C-terminal residues (32) and the other missing a 42-residue insertion that is present in the FMN module (28). NADPH oxidation and cytochrome c reductase activities increased in both mutants relative to wild type, whereas their steady-state NO synthesis was either slower (28) or about the same (32). Rates of flavin and heme reduction were measured in the N-terminal deletion mutant under aerobic conditions and were found to be faster than wild type (32). However, neither group was able to explain the catalytic behavior of these mutants. We suspect that their behaviors reflect an increase in heme reduction rate above the optimum value for nNOS and will ultimately be understood based on our kinetic model.
Biologic Implications--
There is no obvious advantage to
speeding heme reduction in rat nNOS, because it already functions near
its optimum rate and is predicted to have faster steady-state NO
synthesis than the S1412D mutant throughout the physiologic
O2 concentration range. Indeed, our results suggest that
phosphorylation will increase NO synthesis relative to native enzyme
only within a narrow Ca2+ concentration range that is
suboptimal for full CaM binding. Thus, the Ser-1412 phosphorylation
site in rat nNOS is of uncertain physiologic consequence. On the other
hand, eNOS is set to respond differently to phosphorylation by
increasing NO synthesis in response to a faster heme reduction rate. It
will be interesting to see if auxiliary proteins that increase eNOS
activity such as HSP-90 also act by speeding heme reduction. For nNOS,
other mechanisms are available to increase its steady-state NO
synthesis. For example, in nNOS mutants W409F and W409Y the ferrous
heme-NO complex reacts 7 times faster with O2 (7), and
hyperactivity results despite their heme reduction being slower than
wild type (7).3 Although
factors that influence ferrous heme-NO complex oxidation are not
identified, our analysis suggests that modulation of this parameter
would be a good strategy to up-regulate NO release by nNOS.
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ACKNOWLEDGEMENTS |
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We thank Abby Meade, Ritsu Kondo, and D. J. Black for excellent assistance.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM51491 (to D. J. S.) and DK33727 (to D. J).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.
§ Fellow of the American Heart Association. To whom correspondence may be addressed: Immunology NB-3, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-6950; Fax: 216-444-9329; E-mail: adaks@ccf.org.
To whom correspondence may be addressed: Immunology NB-3,
Lerner Research Institute, Cleveland Clinic, 9500 Euclid Ave.,
Cleveland, OH 44195. Tel.: 216-445-6950; Fax: 216-444-9329; E-mail:
stuehrd@ccf.org.
Published, JBC Papers in Press, October 18, 2000, DOI 10.1074/jbc.M006857200
2 J. Santolini, S. Adak, and D. J. Stuehr, unpublished results.
3 S. Adak and D. J. Stuehr, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: NO, nitric oxide; NOS, nitric-oxide synthase; CaM, calmodulin; eNOS, endothelial NO synthase; EPPS, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid; H4B, (6R)-5,6,7,8-tetrahydro-L-biopterin; nNOS, neuronal NO synthase; ScaM, soybean calmodulin; NOHA, N-hydroxyarginine; Arg, L-arginine.
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