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INTRODUCTION |
Nitric-oxide synthases
(NOS)1 constitute a family of
heme proteins that catalyze conversion of L-arginine to
citrulline and nitric oxide (1). The production of nitric oxide in
specific cell types fulfills certain physiological roles for which each isoform is suited according to its structure and regulation (2). Neuronal NOS+ (nNOS) (1) isoforms are localized in brain
and in skeletal myotendinous junctions for the production of NO as a
neurotransmitter. Inducible NOS (iNOS), first found in macrophages and
induced by cytokines, produces NO for cytotoxic action. Endothelial NOS
(eNOS) produces NO as a vasodilator. It was reported some years ago
that NOS activity is inhibited by diatomic ligands such as carbon
monoxide (3-5), cyanide (3), and nitric oxide (3). Although CO
inhibition of NOS would be due to ligand binding with the ferrous heme
intermediate, the situation with NO is more complex. Griscavage
et al. demonstrated that NO inhibits and probably provides
negative feedback for all three isoforms: iNOS (6), eNOS (7), and nNOS
(8). The last of those studies made a particularly convincing case that NO is a powerful inhibitor and acts by interacting directly with the
ferric heme intermediate (8).
We previously reported the kinetics of CO association with nNOS and its
expressed heme domain as well as dissociation from the nNOS-CO complex
(9). From the rates, we estimated an equilibrium constant suggesting
that in vivo inhibition of NOS by CO is unlikely for
plausible physiological concentrations, unless there is great local
enhancement of CO concentration, as could possibly be created by heme
oxygenase activity. Nevertheless, in vitro studies of inhibition of NOS activity by CO are important for elucidating the
reaction mechanism of NOS activity. Reported here are similar studies
of the reaction of NO with both ferric and ferrous nNOS. These
provide additional insights into the mechanism of reactivity in
general, bear on the suggestions of Griscavage et al. (8) that NO autoregulates NOS, and have direct consequences for in vivo NOS activity as affected by continuous changes in local NO concentrations.
It is relevant that eNOS is a membrane-bound enzyme, whose activity is
considerably increased by shear stress due to blood flow. The
functional significance of eNOS association with plasma membrane is
poorly understood. Perhaps blood flow modulates eNOS activity by, among
other mechanisms, continuously depleting local NO concentrations. An
optimum regulatory role for eNOS, or any isoform of NOS, would seem to
require finely tuned affinity for NO.
In the much studied hemoglobins and myoglobins, there is a coordinate
covalent bond between the heme iron and a proximal histidine; and the
iron is exclusively Fe(II). In NOS, the bond is with the thiolate
function of a cysteine residue; and the heme iron cycles between its
ferric (+3) and ferrous (+2) oxidation states during catalytic cycles
(10). One might conjecture that changing the proximal ligand would have
significant effects. Proximal Cys favors the Fe(III) state and seems to
make the ligation kinetics of ferric and ferrous species more similar,
but heme proteins with either proximal base exhibit wide variation in
rate constants. Tuning of the reactivity in heme proteins is achieved
by varying the electrostatic and steric environment in the neighborhood
of the iron, both at the proximal bond and on the opposite, or distal, side.
Association rate constants of NO with heme proteins range from
~2 × 107 to ~200 M
1
s
1, and the dissociation rate constants range from 100 to
0.03 s
1 for the ferric species and at least 1000-fold
slower yet for the ferrous form (11, 12). The fastest association
rates, which are similar for a large number of heme proteins, are
limited by the rate of diffusion of the ligand through solvent to the protein and entry into the protein, after which bonding is very efficient. Apparently, entry of ligands into the protein and access to
the iron is quite similar for many proteins. The lower association rate
constants apply for proteins having substantial steric hindrance to
bonding or when there is a need to displace water or some other ligand.
The slow association in horse heart Cyt c is attributed to
the need to displace a methionine sulfur (11). Like other heme
proteins, NOS has charged or polar amino acid side chains in the distal
pocket, which might stabilize or destabilize coordination of a ligand,
such as H2O (13). Ligand dissociation rate constants are
also affected by factors that stabilize ligand bonding. For example, NO
dissociates very slowly in many Mb and Hb, because it is stabilized by
a particular distal His. In contrast, NO dissociates is much more
rapidly from Mb(EL) (Asian elephant), which lacks the distal histidine
(14). Effects of distal histidines have been examined recently in great
detail using site-directed mutations in Mb (15, 16). The overall
mechanisms proposed (10, 17, 18) for NOS-catalyzed conversion of
L-Arg to citrulline specify both an interaction of
L-Arg or N-hydroxyarginine bound to the enzyme
in the distal heme pocket with a ligand (a reduced oxygen species, in
this case) coordinated to the iron, as well as formation of hydroxyheme
(NOS+-H2O) just before release of NO in the
last step. Furthermore, it has been speculated that a coordinated water
molecule in the hydroxy intermediate of NOS may be responsible for
slowing down NO ligation long enough for the NO to diffuse away from
the binding site and, consequently, minimizes self-inactivation of
NOS.
For ferrous heme proteins, affinity of NO is usually extremely high, so
that NO binds more strongly than other ligands, such as O2,
and often acts as a "poison." One significant exception is Cyt
c, in which the affinity of the ferrous species for NO is
quite low and only 20-fold greater than that of its ferric counterpart
(11). This is due to an exceptionally small association rate, only 8.3 M
1 s
1 (11). In a different,
recently described case, that of soluble guanylate cyclase in the
presence of substrate GTP, low affinity is achieved in the opposite
manner, i.e. primarily by unusually rapid dissociation (19).
Since both guanylate cyclase and NOS are involved in the NO regulatory
cycle, it is appropriate that they both be regulated by NO but not
poisoned by very high affinity for NO. So, questions for NOS are as
follows. Is affinity for NO reasonably low? Does affinity differ
between ferric and ferrous oxidation states? Is low affinity achieved
by slow association or by fast dissociation? Are the kinetics or the
equilibria affected by cofactors or substrate? In the studies reported
here, we answer those questions by determining the kinetics of NO
ligation with both ferric and ferrous derivatives of nNOS holoenzyme
and its heme domain (residues 1-714). We investigated the effects of
tetrahydrobiopterin (BH4) and L-arginine. From
the bimolecular rate constants, we could derive an equilibrium constant
for NO binding to nNOS.
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MATERIALS AND METHODS |
Nitric oxide, high purity argon, and premixed NO diluted in
argon were all obtained from Matheson. Nitric oxide was further purified by passage through aqueous KOH solution. Buffers (20 mM Tris-HCl, pH 7.8, 100 µM EDTA, 100 mM NaCl) were prepared in a gas-tight syringe and
deoxygenated by bubbling with argon for at least 40 min. When
indicated, buffers were prepared with 250 µM
BH4 and/or 10 mM Arg.
Rat nNOS holoenzyme was expressed in Escherichia coli and
purified and reconstituted as described previously (20). The
amino-terminal heme-binding domain (residues 1-714) was also rat nNOS
expressed in E. coli and purified as reported previously
(21). Assay for activity and other characterization and controls were
as reported previously (9). Proteins were degassed very gently by
blowing argon over the solution in a cuvette with a large
surface-to-volume ratio. For studies of the ferrous oxidation state,
protein solutions were reduced by adding small amounts of 100 µM sodium dithionite. Sample solutions were prepared by
vigorously bubbling premixed NO diluted in argon through degassed
buffer to obtain the desired [NO] and then adding small amounts of
degassed concentrated protein solution. Measurements were made at
23 ± 0.5 °C.
Kinetic measurements were carried out as described previously (9). For
the laser flash photolysis, 3 mJ in 4 ns at 550 nm was used for
excitation over an area of 0.1 cm2 collinear with the probe
light. Protein concentrations were 10 µM for the heme
domain and 5 µM for holoenzyme, and the path length was 1 cm. The probe light bandwidth was 8 nm. Digitization was by a Lecroy
9361 oscilloscope. Typically, 200-1000 laser "shots" were averaged
for each run. Kinetics were always characterized at two different
wavelengths, as a check that they gave similar results, one near 445 nm
corresponding to bleaching of the original solution and the other near
405 nm corresponding to maximum transient absorption. Preliminary
measurements were made from 390 nm to 450 nm to search for any
anomalous behavior, but none was found. The isosbestic region near 420 nm was examined to confirm that only two states were involved, at least
within available resolution. Steady state absorption was monitored
before and after laser irradiation in order to verify that there was
very little photodegradation. Statistical noise was reduced to

Arms = 0.00002, typically. This was
necessary because the low quantum yield for escape of dissociated NO
from the protein resulted in small transient absorbance changes (at
best,
A(t = 0)
0.01). A more
extensive discussion of signal-to-noise issues has been given elsewhere
(9).
Kinetics of NO dissociation from ferric nNOS-NO were determined using a
Durrum stopped-flow instrument. NO dissociation was monitored at 412 nm
during reaction with oxymyoglobin, which is converted to metmyoglobin
by reaction with NO, a method we used recently for another protein
(22). Trapping of free NO by MbO2 is fast and irreversible.
Four different concentrations of scavenger were used in order to
establish that the absorption changes were independent of scavenger
concentration and, therefore, rate-limited by NO dissociation.
Dissociation from ferrous nNOS-NO is so slow that it could be measured
in an ordinary spectrophotometer, utilizing sodium dithionite (SDT) as
a scavenger, in the presence of CO, and monitoring absorbance change at
420 nm (23). Concentrations of some components are slightly different
for the dissociation measurements than for photolysis; they are listed
in figure captions.
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RESULTS |
Definition of the Rate Constants--
Rate constants for
bimolecular association are defined in Reaction 1.
Photolysis perturbs the equilibrium and creates a temporary excess
population on the right. Flooding the sample with excess NO ensures
pseudo-first-order conditions under which relaxation back to
equilibrium is described by Equation 1.
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(Eq. 1)
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When kobs is plotted versus
[NO], the slope and intercept determine ka and
kd. We used 100% NO (2000 µM) and
certified mixtures of 10%, 0.5%, and 0.1% NO in Ar in order to
ensure known [NO]. In all cases, there was good linear behavior
extrapolating close to the origin. Since association was quite fast, we
needed 0.5% and 0.1% NO mixtures to establish the intercept. Repeated
measurements verified that the y intercept was measurably
above the origin for ferric nNOS+-NO. For ferrous nNOS-NO,
the intercept was indistinguishable from the origin and could be
measured only by a mixing experiment. The overall bimolecular rate
constants ka and kd for a
variety of conditions are collected in Tables
I and
II.
An observation that is only semiquantitative, but nonetheless
important, is that the net photodissociation yield for ligands to leave
the protein was quite low, roughly 1%. Even this low value is
significantly higher than for Mb-NO, but small compared with
photodissociation yields for Mb with most other ligands. Consistent
with the small photodissociation yield, we also observed geminate
recombination. It was identified by the fact that the process was
independent of NO concentrations and occurred on much faster time
scales than the bimolecular association process. The largest geminate
recombination efficiency seemed to occur for the conditions that yield
the fastest bimolecular association rates in the tables, as should be
expected (24).
Heterogeneous Behavior?--
An important question is whether the
kinetics are well described by single exponentials, as prescribed by
Reaction 1 and Equation 2. Fig. 1 shows
that they are, unless a very good measurement is made. In the past, a
noisier version of Fig. 1 would have been fit to a single exponential,
at least over two or three half-lives. With today's technology,
capable of better signal-to-noise ratios, it is not unusual to discover
that multiple processes over a narrow range of
ka are present, as is the case in Fig. 1. Data
there can be fit over many half-lives with two exponentials differing in rate by a small factor of only 2-4. Since the rates are so close
and the heterogeneity remains similar over all conditions reported
here, we characterize the bimolecular association by a single,
"effective" rate. It is calculated as the harmonic mean (reciprocal
of the weighted mean of the reciprocals) of the two rates used in
fitting (9). This gives a datum that is very close to what is obtained
for the best fit of a single exponential to the same record, as long as
the entire record is fit in both cases and not truncated by
instrumental limitations or human bias.

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Fig. 1.
Base-10 logarithms of absorbance changes over
seven half-lives following flash photolysis of solutions of nNOS-NO
holoenzyme in the presence of L-Arg and
BH4. Top panel, ferric form;
bottom panel, ferrous; both have the same time
scale. Dots are data points; lines are best fits;
fits to a double exponential are indistinguishable from the data,
except where visible in the tails; fits to single exponentials
intersect the data curve at two points, but fail in the tails and also
at early times, although the semilogarithmic plot obscures how bad they
are in that region. Still, the two rates used differ only by a factor
of just over 2 in the lower panel and under 2 in
the upper panel.
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The possibility that systematic distortions could be present in the
detection apparatus is rejected because we do not see the same sort of
heterogeneity for Mb. It could well be that the heme enzymes tolerate
or even require a range of different protein conformations that the
simple oxygen carriers do not, and this leads to a narrow distribution
of ka; however, at this point in time, it is
difficult to rule out absolutely the possibility that the more complex
isolation and purification procedures required for the enzymes might
lead to slightly damaged protein that is avoided in the much simpler
procedures used for the globins. The main point is that with precision
capable of detecting such inconsequential heterogeneity even in the
very difficult case of NO photolysis, with its low photodissociation
yield, we can be very confident that no major feature is obscured by
inadequate sensitivity.
Variation of ka--
All the association rates for
both ferrous and ferric nNOS are quite fast, in the neighborhood of 10 µM
1 s
1. In ferric
nNOS+, the NO association rate for the heme domain alone is
consistently 2-3 times faster than that for the holoenzyme. Ferrous
nNOS does not show this variation, and its ka
have an intermediate value. For ferric nNOS+, we were able
to prepare protein with and without substrate, L-Arg, and
cofactor, BH4. Any differences are very small. All of the
holoenzyme cases lie within statistical scatter. For the heme domain,
the difference with and without L-Arg is probably real, but
just barely measurable. For ferrous nNOS-NO, solutions without arginine
are unstable, a point of considerable significance in itself, and could
not be characterized. Instability in nNOS has been discussed by others
at some length (25), although that study put more emphasis on the role
of BH4.
Once BH4 is present, it is difficult to remove completely,
so it is important that we started with protein prepared (21) so that
BH4 was never present, by employing recombination in
E. coli that itself does not make BH4. It is
also difficult to exclude arginine completely. The cases listed as
lacking it may have trace amounts.
For comparison, we note that the common Hb and Mb proteins have
ka that differ substantially between ferrous and
ferric forms. Ferrous globins usually have ka
roughly similar to what we find for nNOS; but the ferric globins have
much smaller association rate constants.
Variation of kd--
For ferric nNOS, the values
derived from the intercept of kobs in the flash
photolysis experiments are near 50 s
1, with a
significantly higher rate constant only for the holoenzyme in the
presence of L-Arg. There was scatter in measurements made during several repetitions on different days, as much as a factor of 2, but no observations anywhere near 10 s
1, which would have
been easy to detect. This presented a conundrum, because such a fast
kd in conjunction with the
ka already determined implies that very little
NO should be bound at 0.1% (2 µM) NO. Nonetheless, we
were able to prepare and characterize such samples. Titration of nNOS
by NO to determine Ka predicted a
kd that was a factor of 10 smaller than that
determined in the photolysis experiments. Stopped-flow mixing
experiments were then undertaken. They gave good signals, as
illustrated in Figs. 2 and
3, that yielded values for
kd that are smaller by a factor of 10-20 than the flash photolysis results, but consistent with estimates from equilibrium titration.

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Fig. 2.
Reaction time course for NO dissociation from
ferric nitrosyl heme domain of nNOS+, measured using
MbO2 as scavenger for NO. = 409 nm; T, 20 °C;
pH, 7.8; Tris, 20 mM; EDTA, 100 µM; NaCl, 100 mM; BH4, 250 µM; protein, 0.5 µM; MbO2, 6 µM; NO, 2.5 µM. Symbols are measured absorbances;
continuous line is a best fit to a single
exponential with parameters as in Table I.
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Fig. 3.
Reaction time course for NO dissociation from
ferrous nitrosyl heme domain of nNOS, measured using sodium dithionite
as scavenger for NO in the presence of CO. = 420 nm; T,
20 °C; pH, 7.8; Tris, 20 mM; EDTA, 100 µM;
NaCl, 100 mM; l-Arg, 10 mM; protein, 2 µM; SDT, 30 mM; NO, 20 µM; CO,
945 µM. Symbols are measured absorbances;
continuous line is a best fit to a single
exponential with parameters as in Table II.
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For ferrous nNOS the flash data extrapolated close to the origin,
implying slower kd and larger
Ka. The equilibrium association constant
Ka was, in fact, very large, beyond our ability to
measure by titration, since we worry that NO is sufficiently reactive
to cast some doubt on efforts to prepare very low [NO] with accuracy.
We determined kd by trapping spontaneously released NO and found the small kd reported in
the table. We have no way to guess whether the intercept in flash
photolysis might actually be a factor of 10 higher than the
kd obtained by scavenging, as there is no way to
measure by extrapolation such a small departure from the origin.
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DISCUSSION |
A small photodissociation yield, as NOS-NO has, entails very
efficient geminate recombination. The corollary is that almost every
ligand that enters the protein will bond to the iron, so that
ka is controlled by the time needed to reach the
protein and pass through whatever "gate" exists at entry. This
means that steric and electronic effects exercised at the heme iron
binding site are not very effective in modulating bimolecular
association ka. In contrast, the net ligand
dissociation rate kd remains directly proportional to the rate of breaking the Fe-NO bond, albeit reduced by
a constant factor dependent on the geminate recombination probability. Protein control of ligand affinity in the case of NO can be exerted effectively through the dissociation constant, utilizing steric and
electronic variation on either proximal or distal sides of the iron.
The situation is different for CO and any other situation with large
photodissociation yield, in which variation near the iron immediately
affects both ka and kd
and, consequently, Ka.
Bimolecular Rate Constants ka and
kd--
According to Tables I and II, rate constants for
ligand association of NO are very fast for both ferric and ferrous nNOS derivatives and quite similar in magnitude. All of these values for
ka are among the fastest association rate
constants known for binding to heme iron in a protein, equal to or
faster than rates constants common to many ferrous heme proteins and
faster than all but a few known for ferric species. The fast
association is a reflection of efficient geminate recombination, as
discussed above. It also suggests that there is no water molecule nor
any other ligand able to coordinate to the distal side of heme prior to
NO addition after flash photolysis, which would have to be displaced
before NO could return. The values of ka are
close to those for Hb opossum
-chains or Mb elephant, neither of
which have distal histidine nor a water molecule at the ligand binding site (14). The lack of interfering water is not due to lack of access
for an H2O, which is smaller than O2 or NO.
Elephant Mb seems to allow access to the iron. The issue, rather, is
stabilization of H2O by hydrogen bonding, which is not
available in elephant Mb, which has the double substitution H64Q/L29F.
Effects of distal histidines have been explored recently in great
detail using site-directed mutations in Mb (15, 16). The observed NO
association and dissociation rate constants for H64A, H64V, H64T, and
H64F metMb are all near 100 µM
1
s
1 and 100 s
1, respectively. These values
are close to those for the ferrous forms of the same mutants. Eich
et al. (15) also showed that the single H64Q mutation
increases ka for NO binding to metMb from 0.07 to 8.2 µM
1 s
1. The fact that
stabilization is also lacking in nNOS is interesting in that there are
a number of charged residues in the substrate binding pocket (26, 27),
which might possibly have stabilized a water ligand, but apparently do
not. All this may seem to be inconsistent with some indications of low
spin hexacoordinated heme in the equilibrium deliganded state, which
suggests a sixth coordinated ligand (28). A likely resolution could be
slow incorporation of water, requiring times longer than the few
seconds or less needed for ligand association after photolysis. Perhaps
a slow conformational change is needed to stabilize water bonding. This would explain indications of hexacoordination at equilibrium. It could
also explain the different results we obtained for two different
methods of measuring kd.
Rate constants in Table I for ka of the heme
domain are consistently higher than for the holoenzyme by a factor of
3-4, which demonstrates that the active site is influenced in some
manner by more distant parts of the protein.
There is a striking qualitative difference between association of NOS
with CO and with NO, aside from any quantitative differences. Two
independent studies (9, 29) demonstrated that CO association exhibits
two distinct phases separated in rate by a factor of about 100. Reaction with NO either has none of the slower phase or so little that
we have not been able to resolve it. The major effect of cofactor,
BH4, or substrate, L-Arg, on CO reactivity is
to shift the relative contributions of the two phases. Those two agents
have very little effect on NO reactivity, which is reasonable since
there is only one phase present.
Several considerations: the proposed overall reaction mechanism, the
recently determined (26) crystal structure for part of iNOS (residues
66-498 of the oxygenase domain), as well as ENDOR studies of intact
nNOS (30), all entail interaction between activated oxygen coordinated
to heme and the guanidino group of L-Arg or
N-hydroxy-Arg. The NO association rates do not reflect such
interactions. The lack of sensitivity of association rates to
L-Arg and BH4 has consequences given our
finding that the rate-limiting step for ka is
diffusion to and entry into the protein. Diffusion should not be
affected, and protein entry apparently is not affected by
L-Arg or BH4.
Ligand dissociation rate constants are also unaffected by
L-Arg or BH4. The kd for
ferric nNOS+, as determined by extrapolation of flash
photolysis transient kinetics, all lie within a factor of 3 of each
other and are very fast, suggesting that NO coordination has little
stabilization by hydrogen bonding with distal residues. This was
discussed above in the context of ka for
different species and mutations of Mb. It has also been observed
previously for catalase (11) and cytochrome c peroxidase
(11), as confirmed in our own studies (12). However, kd for the ferric species are lower by a factor
of 10 or more when determined by extrapolation by stopped-flow mixing
with a scavenger. The latter values are closer to the value for ferric HbA+-NO, in which coordinated NO is stabilized
somewhat by hydrogen bonding with distal histidine. A plausible
explanation of the difference between flash photolysis and scavenging
is the same one invoked above for another purpose: that following
bimolecular addition of NO there is a structural change on a slow time
scale that stabilizes NO binding. The hydrophilic residue nearest to the heme is Glu592. The analogous residue in iNOS,
Glu371, although a little removed from the ligand binding
site, is known to interact with L-Arg in the heme pocket
(26). It has been found that L-Arg and the mechanism-based
inactivator aminoguanidine bind in the distal pocket adjacent to the
heme and interact directly with Glu592. We speculate that
either substrate arginine or, in its absence, Glu592 may be
stabilizing NO binding via a hydrogen-bonded water molecule drawn into
the pocket after initial NO ligation.
In the case of ferrous nNOS, extrapolation of observed association
rates to zero nitric oxide concentration did not yield an intercept
significantly different from zero, but that only means <10
s
1. The kd measured by scavenging
NO from ferrous nNOS-NO is near 10
4 s
1, and
comparable to that in Mb-NO. It too is minimally affected by Arg or
BH4. This suggests that there is no serious steric
interaction between coordinated NO and arginine. The fact that electron
transfer occurs between these centers does not present a contradiction. Tuning donors and acceptors to facilitate efficient electron transfer is not just a matter of having them as close as possible; rather, they
are optimized carefully to control both forward and reverse transfer,
and that frequently involves less than closest packing, in order to
reduce back transfer.
Hurshman and Marletta (28) have reported that L-Arg
decreases affinity of NO for ferric iNOS+ but not for
ferrous iNOS. They also measured the inverse dependence, that the
apparent Kd for arginine binding to ferric
iNOS+ varies over the range 60-200 µM,
depending on NO concentration. For the reactant concentrations used in
the present study, the kinetics of NO ligation to ferric
nNOS+ showed no dependence on L-Arg. The
affinity of NO for ferrous nNOS is so high that any affect of arginine
is inconsequential.
Equilibrium Ka--
From the tables, we conclude that
Ka(NO-nNOS+)
105
M
1 s
1 within seconds after
photolysis; Ka(NO-nNOS+)
2 × 106 for "real" equilibrium after some relaxation;
Ka(NO-nNOS)
4 × 1010 for
"real" equilibrium after any possible relaxation. The fourth case,
if it exists, was not measurable.
The answer to a question posed in the introduction is now clear. The
affinity of NO for ferric nNOS is modest, due exclusively to rapid
dissociation able to offset rapid association, which is actually quite
fast. This keeps nNOS from being poisoned, but also allows rapid
kinetic changes. The remaining question is how the enzyme escapes being
inactivated by irreversible binding of NO to ferrous nNOS. The
explanation probably lies in the observation that the ferrous nNOS-NO
complex is stable only in the presence of L-Arg. In a
sense, the protein shifts to its ferric form to offload NO. Due to the
anionic nature of the proximal ligand (Cys415), the higher
oxidation state is favored in NOS. Although the presence of the
positively charged guanidino group would tend to stabilize the ferrous
oxidation state, if the rate of heme oxidation is faster than the
second order combination of NO with ferrous NOS, then only very small
amounts of ferrous NO will be formed during the enzyme turnover.
The affinity of NO with ferric nNOS+ is sufficiently high
that in unstirred solutions high local concentrations of NO may cause self-inactivation, but not so high as to render the enzyme unresponsive to changes in physiologically plausible local concentrations of nitric
oxide. Since the ferrous form of the enzyme is present only in very
small amounts, and NO is produced while the enzyme is in the ferric
state, one may appreciate the observation by Griscavage et
al. that NO inactivates by binding to the ferric state of NOS
(8).
Kinetics of NO (and CO) binding to iNOS were reported very recently by
Abu-Soud et al. (31), after the experiments described here
were complete. In some ways, their study was similar to ours: both
compared the Fe(II) and Fe(III) species, and both investigated the
effects of L-Arg and BH4. In other ways, the
two studies were complementary: we studied nNOS, they studied iNOS; we
compare the heme domain with the holoenzyme, they studied only the heme (oxygenase) domain; we worked at 23 °C, they at 10 °C; we used flash photolysis, they used stopped-flow mixing; we used both extrapolation and scavenging to measure dissociation, they report only
association measurements; we began a characterization of geminate
recombination, their mixing experiments will never address geminate
recombination; we merely mention protein instability for certain
conditions, while they pursue that issue at considerable length. The
differences in methods and conditions suggest that a number of general
conclusions reached in both studies are robust and probably apply to
all NOS isoforms; our association rate constants are generally about an
order of magnitude faster, but after a plausible correction for
different temperatures, this implies that the association rate
constants are fairly similar in nNOS and iNOS. Both studies agree that
NO association is similar for Fe(II) and Fe(III) and that the effects
of L-Arg and BH4 are minimal. Both studies
agree that the dissociation rate for Fe(III) as measured by
extrapolating association kinetics is very high. The
kd are numerically very close in the two
studies, but this means either that they will diverge slightly after a
temperature correction is applied (which is entirely possible since
nNOS and iNOS are, after all, different), or that the activation energy
for dissociation is surprisingly small. Abu-Soud et al. (31)
were able to extrapolate association data for ferrous iNOS-NO and
determine intercepts that were as fast for Fe(II) as for Fe(III). Our
best estimates place our intercepts lower and close to zero, and our
scavenging experiments determined a very small
kd for ferrous nNOS-NO.
The general similarity between our nNOS and their iNOS is particularly
intriguing in light of a study by Raman spectroscopy (32), whose
authors found similarities between iNOS and eNOS but differences
between the two constitutive isoforms eNOS and nNOS, which might have
been expected to be the more similar pair. They explain the difference
by postulating a more open pocket in nNOS. That should have
implications for ligand binding kinetics, although they might be
subtle. Since ka is rate-limited by protein entry, one might expect an "open pocket" to affect
ka, but it does not. The situation with
kd is unclear because of the lack of scavenging
measurements on iNOS, but the only difference that seems like it may
exist (faster kd in iNOS) is in the wrong
direction; it should be nNOS that is faster, if it has the open pocket.
To summarize, we have shown that fast ligand association in
nNOS+ combines with fast dissociation to keep affinities
reasonable in the ferric form, but an additional mechanism (cycling
between oxidation states) plays an important role in avoiding
inactivation by strong NO binding to the ferrous state of nNOS.