(Received for publication, May 9, 1995; and in revised form, July 24, 1995)
From the
Neuronal NO synthase (NOS) is a flavin-containing hemeprotein
that generates NO from L-arginine, NADPH, and O.
NO has recently been proposed to autoinhibit NOS. We have investigated
whether a NOS heme-NO complex forms during aerobic steady-state
catalysis. Visible and resonance Raman spectra recorded during
steady-state NO synthesis by NOS showed that the majority of enzyme
(70-90%) was present as its ferrous-nitrosyl complex.
Ferrous-nitrosyl NOS formed only in the coincident presence of NADPH, L-arginine, and O
. Its level remained constant
during NO synthesis until the NADPH was exhausted, after which the
complex decayed to regenerate ferric resting NOS. Stopped-flow
measurements revealed that the buildup of the ferrous-NO complex was
rapid (<2 s) and caused a 10-fold decrease in the rate of NADPH
consumption by NOS. Complex formation and decay could occur several
times with no adverse affect on its subsequent formation or on NOS
catalytic activity. Neither enzyme dilution nor NO scavengers
(superoxide and oxyhemoglobin) diminished formation of ferrous-nitrosyl
NOS or prevented the catalytic inhibition attributed to its formation.
The ferrous-nitrosyl complex also formed in unfractionated cell cytosol
containing neuronal NOS upon initiating NO synthesis. We conclude that
a majority of neuronal NOS is converted quickly to a catalytically
inactive ferrous-nitrosyl complex during NO synthesis independent of
the external NO concentration. Thus, NO binding to the NOS heme may be
a fundamental feature of catalysis and functions to down-regulate NO
synthesis by neuronal NOS.
Nitric oxide (NO) ()is a widespread mediator of
physiological and pathophysiological
processes(1, 2, 3, 4, 5) .
NO is synthesized by a family of enzymes termed NO synthases (NOSs).
These enzymes are homodimers that catalyze the stepwise oxidation of L-arginine to NO plus L-citrulline(3, 4, 6, 7) .
The NOSs contain FAD, FMN, H
biopterin, and iron
protoporphyrin IX (heme) prosthetic groups. The flavins facilitate
transfer of electrons from NADPH to the heme iron(8) . In
neuronal NOS, the flavin-to-heme electron transfer is triggered by
calmodulin (CaM) binding(9, 10) , which occurs in
response to elevated Ca
concentration(3) .
The NOS heme iron plays a central role by binding and activating
O
at the catalytic site. This ultimately leads to oxygen
incorporation into both products of the enzyme
reaction(4, 7) . Indeed, NO synthesis from either L-arginine or N
-hydroxy-L-arginine is inhibited by
CO, and spectral evidence demonstrates that CO inhibits the reaction by
coordinating to the heme
iron(11, 12, 13, 14) . In addition
to binding O
or CO, the heme iron of neuronal NOS can bind
NO as a sixth ligand in both the ferrous and ferric states under an
anaerobic atmosphere to generate stable NOS heme iron-NO complexes (15) .
Characterization of the heme iron in neuronal NOS has recently been accomplished by employing techniques such as optical absorption (9, 10, 11, 12, 13) , electron paramagnetic resonance(12) , and resonance Raman spectroscopy(14, 15) . The heme iron is axially coordinated to the protein via a cysteine thiolate, as is the case for cytochrome P450s, and is predominantly five-coordinate and high spin in ferric NOS. Substrate appears to bind directly above the heme group and can interact with ligands bound to the heme iron, such as NO or CO(14, 15) . During catalysis of NO synthesis, such positioning may sterically specify substrate hydroxylation events that are catalyzed by the heme iron.
The binding of NO to neuronal NOS is particularly important in view of reports that NO can inhibit NOS activity(16, 17, 18, 19, 20, 21) . Studies with neuronal NOS purified from rat cerebellum indicated that NO inhibits by interacting directly with enzyme rather than with any of the soluble cofactors or substrates(19) . These studies also showed that NO generated by NOS caused an inhibition of catalysis(16, 19) , suggesting that NO may act as a negative feedback modulator of NOS, possibly through its interaction with enzyme-bound heme. This view was recently substantiated by optical absorption and resonance Raman spectroscopic evidence that showed neuronal NOS ends up as its ferrous heme-NO complex following NO synthesis under oxygen-limited conditions(15) . However, these studies did not address whether NO binds to neuronal NOS during normal aerobic catalysis and whether such a reaction explains the catalytic inhibition attributed to NO.
Thus, our current objective was to determine whether L-arginine-derived NO would complex with the heme iron of neuronal NOS during enzyme catalysis under aerobic, steady-state conditions. Experiments were also conducted to determine if formation of a heme iron-NO complex was related to inhibition of NO synthesis, including stopped-flow analysis to examine the kinetic relationship between nitrosyl complex formation and inhibition of neuronal NOS by NO. Our results indicate that NO binding to the heme iron is rapid, quantitatively significant, and an integral part of the enzyme's normal catalytic processing and functions to regulate NO synthesis by neuronal NOS.
where k and k
are
first-order rate constants; t is time; A and B are amplitude factors; and e is 2.718. describes a process that takes place via single exponential
decay; represents the sum of two independent exponentials
for a parallel process.
Figure 1:
Formation of a ferrous-NO
complex during steady-state NO synthesis by neuronal NOS. The upper
panel contains spectral scans done on a 1-ml air-saturated
solution containing 40 mM Bis-Tris buffer, pH 7.4, 3
µM CaM, 0.9 mM EDTA, 4 µM Hbiopterin, 200 µM DTT, and 1 µM NOS at 15 °C. The solid line is resting enzyme. The dashed line was recorded after adding 24 nmol of NADPH. The dotted line was recorded after initiating NO synthesis by
adding 1 mM Ca
. The dashed and dotted
line was recorded after NO synthesis had stopped due to NADPH
depletion by NOS. The experiment shown is representative of five. The inset contains the spectrum of NOS prior to (solid
line) and during (dotted line) catalysis of NADPH
oxidation in the absence of L-arginine. The reaction was
started by the addition of 120 µM NADPH, and the scan was
completed when the NADPH concentration had reached 25 µM.
Conditions were otherwise identical to those described for the upper panel. The lower panel depicts spectral scans
of an anaerobic solution containing 40 mM Bis-Tris buffer, pH
7.4, 4 µM H
biopterin, 200 µM DTT,
and 1.3 µM NOS at 15 °C. The solid line is
resting NOS. The dashed line was recorded after adding excess
dithionite to reduce the NOS flavins and heme iron. The dotted line was recorded after the addition of NO gas to form the
ferrous-nitrosyl complex of NOS. The experiment shown is representative
of five.
The inset of the upper panel of Fig. 1contains a spectrum of ferric resting NOS and a spectrum of the same sample undergoing steady-state catalysis of NADPH oxidation in the absence of L-arginine. In this circumstance, the NOS heme iron-catalyzed oxygen reduction still occurs but results in superoxide formation instead of NO synthesis(25) . In this case, no ferrous-nitrosyl complex was observed. Instead, the spectrum is similar to that obtained previously for neuronal NOS in its reduced ferrous-deoxy state, which displays a lack of flavin absorbance and a Soret absorbance of lower extinction centered at 412 nm(8, 13, 14, 15) . This indicates that formation of the ferrous-nitrosyl complex was dependent on L-arginine conversion to NO.
To quantitate the amount of
ferrous-nitrosyl NOS that formed during steady-state NO synthesis, we
derived an estimated extinction coefficient for ferrous-nitrosyl NOS
standards that were generated as depicted in the lower panel of Fig. 1. The solid line is the spectrum of
resting neuronal NOS under an anaerobic atmosphere of nitrogen. The dashed line is the spectrum recorded after adding excess
dithionite, which completely reduced the NOS flavins and heme
iron(13, 14) . The dotted line is the
spectrum that was recorded after adding NO gas to form the
ferrous-nitrosyl complex of neuronal NOS. Given that the estimated
extinction coefficient for ferric neuronal NOS at 398 nm is 72,000 M cm
(12) , we
calculate an estimated extinction at 436 nm for ferrous-nitrosyl NOS of
48,200 ± 800 (n = 4). The ratio of the estimated
extinction coefficients for ferric NOS and ferrous-nitrosyl NOS (1.49)
is similar to that obtained for other thiolate-coordinated hemeproteins
such as cytochrome P450cam (26) and cytochrome
P450nor(27) . Using our estimated extinction coefficient for
ferrous-nitrosyl NOS, we calculate that
90% of neuronal NOS was in
its ferrous-nitrosyl form during steady-state NO synthesis in the
experiment depicted in the upper panel of Fig. 1.
A
replica experiment was done to characterize independently the
NOSNO complex formed during steady-state NO synthesis in a
resonance Raman cell. As depicted in Fig. 2, the resonance Raman
spectrum of neuronal NOS during NO synthesis displayed a
ferrous-nitrosyl stretching mode at 549 cm
. The
frequencies and relative intensities of the porphyrin modes along with
this ferrous-nitrosyl stretching mode are identical to those in the
spectrum of ferrous-nitrosyl NOS generated with reagent NO in the
presence of L-arginine (15) but differ considerably
from those of ferric-nitrosyl NOS(15) . These spectral features
were not observed if NADPH and O
were omitted from the
reaction. Thus, the resonance Raman data confirm independently that
neuronal NOS generates its ferrous-nitrosyl complex during aerobic NO
synthesis.
Figure 2:
Resonance Raman spectrum of
ferrous-nitrosyl NOS generated during NO synthesis. Resonance Raman and
optical absorption spectra were recorded for a reaction carried out in
a sealed Raman cell containing air-saturated 40 mM Hepes, pH
7.4, 40 µM NOS, 330 µM Ca,
50 µM CaM, 40 µM H
biopterin, 120
µM DTT, and 5 mML-arginine. The sample
was excited at 441.6 nm to obtain the resonance Raman spectra before (trace b) or after (trace a) adding 80 µM NADPH to initiate NO synthesis by NOS. The inset contains
absorption spectra recorded before (dashed line) and after (solid line) the introduction of
NADPH.
Figure 3:
Relationships among ferrous-nitrosyl NOS
formation and catalysis of NADPH oxidation and citrulline formation
from L-arginine. The top panel shows the formation
and decay of the ferrous-nitrosyl complex at 436 nm over time and
concurrent NADPH oxidation at 340 nm in 0.4-ml reactions that contained
1 µM NOS in the presence (solid and dashed
lines) or absence (dotted line) of 20 nmol of L-arginine. In all cases the reactions were started by adding
24 nmol of NADPH at time zero. The bottom panel shows the
amounts of L-arginine remaining () and citrulline formed
(
) in replica reactions as determined by HPLC analysis on
10-µl aliquots removed and quenched with acid at the indicated
times. The data shown are representative of three similar
experiments.
The bottom panel of Fig. 3depicts the loss of L-arginine and formation of
citrulline which occurred in a replica experiment. Conversion of L-arginine to citrulline was linear, consistent with the
observed linear rate of NADPH oxidation, and ceased when NADPH was
oxidized completely. The amount of NADPH added to the cuvette (24 nmol)
enabled the conversion of 8 nmol of L-arginine to 8 nmol
of citrulline. Thus, approximately 3 NADPH were oxidized per mol of
citrulline formed in this experiment, which is twice the minimum amount
of NADPH required for neuronal NOS to generate 1 mol of citrulline from L-arginine(24) . Together the data show that a
constant degree of ferrous-nitrosyl complex formation occurs during
steady-state turnover and is associated with a constant rate of NADPH
oxidation and citrulline production by NOS.
Figure 4:
Stopped-flow analysis of ferrous-nitrosyl
complex formation, decay, and NADPH oxidation. The top panel depicts the absorbance change at 436 or 340 nm over time for a
reaction that was initiated by rapidly mixing a buffered solution
containing 2 µM NOS, 200 µM DTT, 4
µM Hbiopterin, 15 µg/ml CaM, 0.9 mM EDTA, 1.1 mM Ca
, and 0 (dotted
line) or 100 µM (solid lines) L-arginine with an equal volume of buffer solution containing
36 µM NADPH at 15 °C. The lower panel and its inset depict the absorbance change that occurred within the
first 5 and 0.4 s of the reaction, respectively. The results shown are
representative of three independent
experiments.
During the preequilibrium
phase of the reaction, one can see that the rate of NADPH oxidation
appeared to be deflected to a slower value as buildup of the
ferrous-nitrosyl complex neared its steady-state value. This phase of
the reaction is expanded in the lower panel of Fig. 4,
which shows the absorbance changes at 436 and 340 nm that occur over
the first 5 s of the reaction. The 340 nm absorbance decreased at a
rate of 0.01 s for the first 1.4 s of the reaction,
then slowed to a rate of 0.001 s
for the duration of
the reaction. This 10-fold decrease in rate took place when formation
of the ferrous-nitrosyl complex neared completion, as determined by the
gain in 436 nm absorbance over time. Together the kinetic data suggest
that a buildup of the ferrous-nitrosyl complex causes inhibition of
electron flux through neuronal NOS.
The lower panel inset of Fig. 4shows the change in absorbance at 436 nm that
takes place during the first 0.4 s of the reaction. The initial
decrease in 436 nm absorbance was best fit to a single exponential
equation, giving an apparent rate constant of 71 s.
This absorbance decrease can be attributed to NADPH reduction of the
NOS flavins(8) , which precedes heme reduction and NO
synthesis. The subsequent absorbance increase at 436 nm is the same as
that depicted in the lower panel and was best fit to a
two-exponential equation, giving apparent rate constants of 12
s
over the first 0.5-s time period and 1.2
s
between 0.5 and 5 s of the reaction. Together, the
data indicate that a buildup of the ferrous-nitrosyl complex is rapid,
biphasic, and occurs much faster than its decay under these
experimental conditions.
Figure 5: Ferrous-nitrosyl complex formation and decay during consecutive rounds of NO synthesis. A cuvette containing buffered reaction solution, 1 µM NOS, and no NADPH had equal amounts of NADPH added (24 nmol) six consecutive times to initiate six rounds of NO synthesis at 15 °C. Ferrous-nitrosyl complex formation and decay were monitored as an increase and decrease in absorbance at 436 nm over time. The experiment shown is representative of three.
Fig. 6shows the effect of a xanthine oxidase/hypoxanthine superoxide-generating system on ferrous-nitrosyl NOS formation during NO synthesis. In panel A both NADPH (24 nmol) and xanthine oxidase were added to the reaction at time zero to initiate concurrent NO synthesis by NOS and superoxide production by xanthine oxidase. The conditions were such that the measured rate of superoxide production by xanthine oxidase in the reaction (30 µM/min) was approximately three times the rate of NO synthesis (9 µM/min) by NOS. Panel B is the control reaction, which received only NADPH at time zero. Cuvette buffers contained 1 mM hypoxanthine in both cases. The results show that the presence of a superoxide generator did not affect the amount of ferrous-nitrosyl complex formed nor the time required to complete the reaction.
Figure 6: Effect of a superoxide-generating system on ferrous-nitrosyl NOS formation during NO synthesis. Generation of ferrous-nitrosyl NOS was monitored at 436 nm in reactions run at 15 °C which contained 1.25 µM NOS in 0.4 ml of reaction buffer supplemented with 1 mM hypoxanthine in all cases. In panel A, both NADPH (24 nmol) and a titered amount of xanthine oxidase were added to the reaction at the indicated time to initiate concurrent NO synthesis and superoxide production. The amount of xanthine oxidase added produced a measured rate of superoxide production of 30 µM/min in the cuvette. Panel B is a control reaction that received only NADPH at the indicated time. The results shown are representative of two independent experiments.
Fig. 7depicts the reaction time
courses for identical NOS reactions that were carried out either in the
presence or absence of excess oxyhemoglobin (25 µM; 10
nmol). The time course in the absence of oxyhemoglobin was followed by
monitoring ferrous-nitrosyl complex formation and decay at 436 nm and
is depicted as the solid line in the figure. Because it was
not possible to observe directly the formation of a ferrous-nitrosyl
complex at 436 nm in a reaction containing 25 µM
oxyhemoglobin, ()we instead monitored the absorbance
increase at 401 nm (dashed line in Fig. 7) which
reflects the NO-mediated conversion of oxyhemoglobin to methemoglobin
and can be used to quantitate both the duration and quantity of NO
synthesis(22) .
Figure 7: Effect of oxyhemoglobin on NOS catalytic activity. Reactions were run at 15 °C and contained 0.5 µM NOS in 0.4 ml. The solid line depicts the time course of ferrous-nitrosyl complex buildup and decay at 436 nm in a reaction that contained no oxyhemoglobin. The dashed line depicts NO generation over time in an identical reaction that also contained 10 nmol (25 µM) of oxyhemoglobin. NO synthesis was started in both cases by adding a limiting amount (10 nmol) of NADPH at time zero. The data shown are representative of four similar experiments.
The linear increase in absorbance at 401 nm indicates that oxyhemoglobin was scavenging NO as it was released into solution by neuronal NOS. A total of 3 nmol of NO was scavenged in the reaction. In spite of this, the times required to complete the reactions were approximately identical (1.8 min) in the presence or absence of oxyhemoglobin. This indicates that oxyhemoglobin scavenging of NO did not prevent the inhibition of catalysis attributed to ferrous-nitrosyl complex formation.
Figure 8:
Effect of dilution on ferrous-nitrosyl
complex formation and NOS catalytic activity. Ferrous-nitrosyl complex
formation and decay were monitored over time at 436 nm for reactions
run at 15 °C containing NOS at 1 µM (solid
line), 0.5 µM (dashed and dotted line), or
0.25 µM (dotted line). NADPH (24 nmol) was added
to initiate NO synthesis at time zero in each case. The inset shows citrulline production over time in replica reactions
containing 1 (), 0.5 (
), or 0.25 (
) µM NOS in which 10-µl aliquots were removed at the indicated
times and quenched with acid prior to HPLC analysis. The results shown
are representative of two identical
experiments.
To examine if NO-mediated catalytic inhibition would be maintained at enzyme concentrations below which direct observation of the ferrous-nitrosyl complex is possible, we compared the specific rates of NADPH oxidation during NO synthesis in reactions containing 10-200 nM neuronal NOS in the presence or absence of 20 µM oxyhemoglobin. The specific rates of NOS NADPH oxidation remained constant over the full range of dilution, and added oxyhemoglobin did not increase measurably the rate NOS NADPH oxidation in any case (data not shown). This suggests that catalytic inhibition attributed to ferrous-nitrosyl complex formation remains constant even at relatively low NOS concentrations and remains resistant to oxyhemoglobin scavenging.
Figure 9:
Formation of ferrous-nitrosyl NOS in
unfractionated cell supernatant. Ferrous-nitrosyl NOS formation and
decay were followed over time at 436 nm in unfractionated R293 cell
supernatant that contained 0.4 µM NOS. R293 cell
supernatant (200 µl, 1.85 mg) was mixed with 100 µl of 40
mM Bis-Tris buffer, pH 7.4, containing 12 µM H
biopterin, 3 mML-arginine, 600
µM DTT, 1 µM CaM, and 0.9 mM EDTA.
Reactions were run at 15 °C and initiated by adding 17 nmol of
NADPH at time zero, with (solid line) or without (dotted
line) Ca
(1.1 mM final concentration)
to initiate NO synthesis. NADPH oxidation in a replica reaction that
received NADPH plus Ca
was followed at 340 nm (dashed line).
We attempted to reduce the catalytic inhibition attributed to
ferrous-nitrosyl complex formation by adding the NO scavenger
oxyhemoglobin or superoxide. These scavengers react with NO at near
diffusion-controlled rates (28, 29) and are presumed
to have significantly lowered the solution concentration of NO in the
enzyme reactions. However, neither oxyhemoglobin nor a
superoxide-generating system increased NOS catalytic activity in our
system, as determined by comparing NO or citrulline formation, NADPH
oxidation, or the lifetime of the ferrous-nitrosyl complex. In
reactions that contained the superoxide generator we observed no
decrease in the degree of ferrous-nitrosyl complex formation during the
steady state. Thus, failure of the scavengers to increase neuronal NOS
activity appeared to be linked to their inability to decrease
ferrous-nitrosyl complex formation. This suggests that NO formed within
the active site of neuronal NOS may have an opportunity to bind to the
ferrous heme iron before it equilibrates with the solution as a whole.
An examination of the time required for complex buildup using
stopped-flow supports this contention (Fig. 4). Based on the the
final NOS concentration (1 µM) and the reported
stoichiometry of NO synthesis (1.5 NADPH oxidized/NO formed; (24) ) and the amount of NADPH utilized during the time
required for complex to build up to the steady-state level (3.5
µM), we estimate that only 2 NO had been generated per
heme by the time maximal complex formation and the 10-fold deflection
in rate of NADPH oxidation were observed. Because significant complex
formation can be seen even at earlier time points, it seems likely that
NO generated during catalysis can bind to the NOS heme iron before it
equilibrates in solution. This suggests that NO remains in or near the
distal heme pocket rather than diffusing out of the protein and may
bind to the heme in a geminate fashion, similar to when NO rebinds to
heme proteins following
photodissociation(34, 35, 36) . The NO that
does escape from the heme pocket can diffuse a considerable distance
from the point source in solution(37) . However, our data
indicate that this free NO is not involved in forming the
ferrous-nitrosyl complex during steady-state catalysis.
Figure SI:
To assess if the model presented in Fig. SIis reasonable we have performed computer simulations of the reaction. The simulations were carried out by solving the simultaneous rate equations for the population of each intermediate iteratively. The rate constants used in the simulations were either inferred or derived from the measurements reported here and are listed below.
The initial concentrations of NADPH and NOS were 18 and 1 µm,
respectively. The simulations, shown in Fig. 10, of the
reactions in Fig. SIqualitatively account for all of our
experimental observations. Specifically, the ferrous-NO complex builds
up rapidly during the initial phase of the reaction and then remains at
a near constant level (0.95 µM) until the NADPH is
depleted (top panel of Fig. 10). There is an initial
rapid consumption of NADPH during complex buildup which is followed by
a slower rate of consumption (bottom panel of Fig. 10).
After an initial burst, citrulline production continues linearly until
the NADPH is depleted. These qualitative calculations based on the
model in Fig. SIare consistent with all of the essential
features of the experimental observations. Simulations carried out in
the absence of formation of the inhibitory complex (not shown) reveal
that catalytic inhibition does not occur, and NADPH continues to be
depleted at the initial rapid rate.
Figure 10: Computer simulation of the reactions depicted in Fig. SI. The top panel displays the progress of the reaction for a 100-s duration, and the bottom panel follows the progress of the reaction for the first 5 s. The right y axis scale applies to the formation of the ferrous-NO complex (A) and the left y axis scale applies to NADPH consumption (B) and citrulline formation (C).
Although our model has
ferrous-nitrosyl NOS acting as a catalytically inactive species whose
formation is not directly involved in the generation of NO and
citrulline, we cannot rigorously exclude the alternative possibility
that ferrous-nitrosyl NOS is an intermediate in the pathway of NO
synthesis. However, this seems unlikely, given the stability of most
ferrous nitrosyl complexes (32) and that the rate of
ferrous-nitrosyl complex decay in our experiments (0.06
s) is too slow to account for the rate of NO
synthesis during the steady state.
As depicted in Fig. SI,
the degree of ferrous-nitrosyl NOS formation during the steady state
likely depends on several factors, including the rate of NOS heme iron
reduction, the concentration of O, the affinity of the
ferrous heme iron toward NO versus O
, and the rate
of ferrous-nitrosyl complex breakdown. Regarding heme iron reduction,
our spectral data suggest that very little neuronal NOS exists in
ferric form during steady-state NO synthesis. A considerable buildup of
ferrous-deoxy NOS (
70%) also occurs during its catalysis of NADPH
oxidation in the absence of substrate (see Fig. 1, inset), which involves O
binding to ferrous heme
and electron transfer to form superoxide(8, 25) . This
suggests that the slow step may involve O
access and
binding. Given that ferrous hemeproteins generally display a greater
affinity toward binding NO than their ferric
forms(32, 33) , the propensity of neuronal NOS to
generate ferrous heme iron during catalysis may increase its
susceptibility to NO.
Although the maximal solution NO
concentrations which could be achieved in our studies (0-15
µM) were less than the dissolved oxygen concentration
(300 µM initially), we observed significant
ferrous-nitrosyl NOS formation during NO synthesis in all cases. This
is consistent with ferrous iron having a greater affinity toward NO
than O
(32, 33) . Also, O
must
diffuse into the active site from solution, whereas NO is not subject
to this constraint. Ligand binding to the NOS heme iron may also be
influenced by substrate. L-Arginine decreases the rate of CO
binding to ferrous NOS by a factor of 12(10) . This is
consistent with resonance Raman studies showing that substrate binds
directly above the heme iron and can affect ligands coordinated to the
heme iron such as CO and NO (14, 15) . L-Arginine was also found to stabilize ferrous-nitrosyl
NOS(15) . How these and other factors interact to control NO
and O
binding to NOS during catalysis will require
continued investigation.
In earlier studies describing NO inhibition of neuronal NOS, the inhibition was found to occur gradually, was enhanced by superoxide removal, diminished by the NO scavenger oxyhemoglobin, and became irreversible under some circumstances(16, 17, 18, 19) . In our current study the ferrous-nitrosyl complex formed immediately after NO synthesis was initiated, reaching a steady state within 2 s. The complex was maintained at a constant level while NOS continued to catalyze NADPH oxidation and NO synthesis. Neither the degree of complex formation nor the enzyme specific activity was affected by enzyme dilution or by NO scavengers such as oxyhemoglobin and superoxide. Multiple rounds of complex formation and decay were not accompanied by a gradual loss of enzyme activity. Thus, the characteristics of inhibition which we attribute to ferrous-nitrosyl complex formation in our current system do not match those observed previously for NO-mediated inhibition of NOS.
The discrepancy between our experiments showing no effect of superoxide or oxyhemoglobin on the rate of NO synthesis during the steady state in comparison with prior reports showing strong effects is particularly intriguing. At present we can only postulate possible origins for these differences. The NOS concentrations used in most of our measurements were high (see Fig. 6and Fig. 7) in comparison with the earlier studies. Thus, in our studies of the influence of NO scavengers on the reaction, the NADPH was depleted within 2-3 min. In contrast, in the earlier studies the NOS concentration was much lower, and the NADPH concentration was higher so the reaction progressed for many tens of minutes. In addition, the temperature in our measurements was controlled at 15 °C, whereas in the earlier experiments it was 37 °C. These time and temperature differences could have a significant effect, considering that two phases of NO inhibition have been reported for cytochrome P450(31) . At early times NO reversibly inhibited P450 by coordinating to the ferrous iron of the heme just as occurs in neuronal NOS. However, a second phase was characterized at longer incubation times in which there was an irreversible loss of catalytic activity compared with enzyme never exposed to NO. It was proposed that the irreversible inhibition resulted from oxidation of certain amino acids in P450 by reactive oxides of nitrogen. Thus, neuronal NOS inhibition by NO could also display two phases: one phase in which NO coordinates to the ferrous heme iron prior to leaving the active site and is thereby not affected by the solution conditions; and a slower phase in which the NO escapes from the heme pocket to eventually react with the protein component of the enzyme. Additional experiments are needed to test these ideas.
To help determine if ferrous-nitrosyl complex formation is a general
property of neuronal NOS catalysis, we examined whether the inhibition
of NADPH oxidation which is associated with buildup of the
ferrous-nitrosyl complex would be maintained at lower enzyme
concentrations and if complex formation would still occur in an
unfractionated cell supernatant that contained neuronal NOS. We found
that the specific rate of NADPH oxidation remained unchanged even at
the lowest enzyme concentration tested (10 nM), and this rate
was not increased in the presence of oxyhemoglobin. This suggests that
the degree of complex formation is constant and independent of solution
NO over a wide range of enzyme concentrations. Ferrous-nitrosyl NOS
also formed during NO synthesis in an unfractionated cell supernatant
that was estimated to contain 0.4 µM neuronal NOS. Thus,
cytosolic constituents potentially capable of scavenging NO (thiols,
ascorbate, metalloproteins, superoxide) were unable to prevent
ferrous-nitrosyl complex formation in the supernatant, implying that
its formation is not restricted to purified systems and may also occur
in intact cells that express neuronal NOS. We have carried out similar
studies with inducible macrophage NOS and found that this isoform also
generates a nitrosyl complex during aerobic NO synthesis. However, in
contrast to neuronal NOS, catalytic inhibition related to nitrosyl
complex formation is prevented by added oxyhemoglobin. Thus, fundamental differences appear to exist between neuronal
and macrophage NOS regarding their autoinhibition due to nitrosyl
complex formation.
To conclude, we report that a majority of neuronal NOS quickly converts to its inactive ferrous-nitrosyl complex after initiating catalysis, causing the enzyme to operate at only a fraction of its maximum possible activity. Complex formation and associated catalytic inhibition are unaffected by enzyme dilution, NO scavengers, and cellular constituents and seem to arise from a reaction between NO and the ferrous heme iron within the enzyme's active site. That an enzyme would evolve to function largely autoinhibited is remarkable and suggests that there are biological roles for neuronal NOS which remain to be considered. These are under current investigation in our laboratories.