(Received for publication, June 5, 1995; and in revised form, August 14, 1995)
From the
Nitric oxide synthase (NOS) catalyzes sequential NADPH- and
O-dependent mono-oxygenase reactions converting L-arginine to N
-hydroxy-L-arginine
and N
-hydroxy-L-arginine to citrulline and
nitric oxide. The homodimeric enzyme contains one heme/monomer, and
that cofactor is thought to mediate both partial reactions. Here we
show by electron paramagnetic resonance spectroscopy that binding of
substrate L-arginine to neuronal NOS perturbs the heme
cofactor binding pocket without directly interacting as a sixth axial
heme ligand; heme iron is exclusively high spin. In contrast, binding
of L-thiocitrulline, a NOS inhibitor, produces both high and
low spin iron spectra; L-thiocitrulline sulfur is a sixth
axial heme ligand in one, but not all, of the low spin forms. The high
spin forms of the L-thiocitrulline NOS complex display a
distortion in the opposite direction to that caused by L-arginine binding. The findings elucidate the binding
interactions of L-arginine and L-thiocitrulline to
neuronal NOS and demonstrate that each causes a unique perturbation to
the heme cofactor pocket of NOS.
Three isoforms of nitric oxide synthase (NOS) ()have
been identified in mammals; all catalyze the NADPH- and
O
-dependent oxidation of L-arginine to citrulline
and nitric oxide (NO) (1, 2, 3) . Neuronal
NOS (nNOS) and endothelial NOS are constitutive,
Ca
/calmodulin-dependent isoforms that, when
activated, produce NO in low (i.e. nanomolar) concentrations
as part of signal transduction pathways involved in neurotransmission (4) and blood pressure regulation(5, 6) ,
respectively. A distinct NOS isoform, not regulated by changes in
intracellular Ca
levels, is expressed in many cell
types in response to inflammatory cytokines. Nitric oxide produced by
this isoform can reach levels (10-100 µM) that are
disruptive to processes normally controlled by endothelial NOS or nNOS.
Such levels are also potentially toxic to inducible NOS-containing
cells and adjacent cells as well as to viruses and microbial pathogens
within those cells(7, 8) .
The overall NOS reaction
proceeds via two mono-oxygenations; the first forms
N-hydroxy-L-arginine (NOH-Arg) from L-arginine, and the second converts that tightly bound
intermediate to citrulline and NO(9) . Both mono-oxygenations
are reminiscent of reactions catalyzed by the cytochrome P-450 system.
Consistent with this view, all NOS isoforms contain a C-terminal
reductase domain having significant (
39%) sequence homology to
cytochrome P-450 reductase; putative binding sites for NADPH as well as
for FAD and FMN, tightly bound cofactors, are identified in this
domain(3, 10) . The NOS N-terminal oxygenase domain
shows little or no sequence homology to known cytochrome P-450 but does
contain a heme cofactor binding site in which a cysteine residue is
positioned to act as a fifth axial ligand in cytochrome P-450-like
fashion(11, 12, 13) . Recent studies with the
isolated oxygenase domain establish that this region also contains
binding sites for substrate L-arginine in addition to
tetrahydrobiopterin, a cofactor of poorly characterized
function(14, 15, 16) .
The oxygenase and
reductase domains are connected by a calmodulin-binding
region(10) . For nNOS and endothelial NOS, binding of
Ca/calmodulin to this region activates electron
transfer between the reductase and oxygenase domains and allows
electron flow from NADPH, through the flavins, to the heme
cofactor(17) . Dioxygen can then be bound to and activated by
the heme cofactor, and a guanidino nitrogen of L-arginine is
oxidized first to NOH-Arg and then to NO. In inducible NOS, calmodulin
is tightly and permanently bound, and the enzyme is fully active at
basal Ca
levels(18) .
Detailed chemical mechanisms for the NOS reaction have been proposed (1, 2, 19, 20, 21, 22) (e.g. see Fig. 5 in (1) ). Central to all of the proposed schemes is the close physical approximation of heme iron and the reactive guanidino nitrogen of substrate L-arginine. In previous studies, we have probed possible interactions between these groups using substrate and inhibitor perturbation optical difference spectroscopy(23, 24) . In the present studies, we have used electron paramagnetic resonance (EPR) spectra of the heme ferric iron to further elucidate the degree and nature of heme and heme pocket interaction with the substrate, L-arginine, and with the putative heme-binding inhibitory citrulline analog, L-thiocitrulline. EPR spectra of the ferriheme and flavosemiquinone groups of native NOS have previously been reported by Stuehr and Ikeda-Saito(25) .
The low spin
features associated with Zeeman field orientation in the heme plane are
the two numerically smallest values and correspond to g and g
; the largest value of g lies
approximately along the heme normal. The positions of these features
depend directly on the relative energies of the iron t
d orbitals (29, 30, 31) .
Simulations of EPR spectra for quantitation were performed using previously described programs(32) . Double integration of high spin spectra used the intensity factor of Aasa and Vaangard(33) . Ligand field splittings of low spin ferrihemes were calculated from g tensors as described by Peisach et al.(29) .
Fig. 1(line B) shows the X-band EPR spectrum at 11
°K of nNOS as isolated. Both the high and low spin components of
the heme spin state equilibrium are visible and are completely
resolved. Features from the high spin state of the ferric heme are
visible near g = 7.65, 4.04, and 1.89. The g = 4.04 feature is overlapped with the g =
4.3 signal from the middle Kramer's doublet of adventitious
rhombic high spin ferric iron. Because of the high transition
probability and nearly isotropic nature of this species, this intense
feature represents only a small concentration of contaminating iron in
the enzyme preparation or buffer. Partly saturated signals from the low
spin state of native nNOS can be observed near g =
2.43, 2.28, and 1.89; the sharp signal at g = 2 is
contributed by a flavin radical, probably FMNH(25) . The
broad signal near g = 2 is contributed by impurities in
the cavity and Dewar assembly.
Figure 1: EPR Spectroscopy of nitric oxide synthase. Samples (200 µl) of 20 µM NOS were prepared for spectroscopy as described under ``Experimental Procedures.'' The spectra were acquired at 11 °K using the described parameters. Spectrum A was obtained in the presence of 100 µML-arginine, and spectrum B was obtained with the unperturbed enzyme.
Addition of L-arginine to
nNOS alters the spin state equilibrium in favor of the high spin form;
no low spin form remains detectable either at 11 °K (Fig. 1, line A) or at 21 °K (not shown). The amplitude of the low
field peak of the high spin form (g = 7.56) increases
by 15% following L-arginine addition. Integration of this
slightly narrowed peak indicates that the high spin fraction has
increased by 10-12%, indicating that the high spin/low spin ratio
for nNOS in the absence of L-arginine is
90/10. A similar
conclusion has been reached through simulation studies comparing the
high spin component of nNOS measured without L-arginine and
the 100% low spin EPR signal seen when nNOS is saturated with
imidazole, a heme-binding ligand. (
)The temperature
dependence of the EPR signals could be fit by Curie Law behavior for
the low spin features up to 40 °K, where signal to noise problems
became significant. For high spin signals, an additional term
representing the distribution within the S = 5/2 sextet
with D =
3.8 cm
produced a good
fit. Although future studies might provide a more accurate measurement
of D, the current result indicates that there is no temperature
dependence in the spin state equilibrium below 25 °K.
It is apparent from results shown in Fig. 1that the high spin species observed in the presence and absence of L-arginine are similar but not identical. In Fig. 2, the region between 0.07 and 0.21 tesla is displayed for the purpose of more detailed comparison. The calculated g values of the high spin features of the enzyme as isolated are 7.65 and 4.04 (line B); the latter g value is difficult to measure precisely due to the overlap with the rhombic ferric iron contaminant. The small shoulder on the high field side of the g = 7.65 peak suggests that the enzyme as isolated also contains a minority high spin species. In the presence of L-arginine, the corresponding high spin g values are 7.56 and 4.09 (line A). The narrowing of the EPR signals on L-arginine binding is even more pronounced in the g = 4 region than near g = 7.6. Note also that comparison of line A and line B shows that virtually none (<10%) of the original high spin species remains after addition of L-arginine, a finding indicating complete conversion of the original high and low spin species to a distinct high spin enzyme-arginine complex (i.e.L-arginine binds to both the low and high spin forms of nNOS).
Figure 2: Effects of L-arginine on the high spin components of NOS. Spectra were acquired at 22 °K using the same samples as in Fig. 1. Spectrum A was of the L-arginine-containing sample, and spectrum B was of the resting enzyme, with the difference (A-B) shown as the final spectrum of the figure.
Fig. 2(line A-B) shows the difference spectrum of
the L-arginine-saturated enzyme versus the enzyme as
isolated. The orientation of the sigmoidal feature at g = 7.7 confirms the upfield shift of the low field peak in
response to L-arginine binding. The larger size of the
positive upfield lobe is due to the increase in the percentage of high
spin heme noted earlier. The complex form of the difference spectrum
near g = 4 is due to the sharpening of this feature by L-arginine as it shifts downfield. The central positive and
negative lobes are contributed primarily by the sharp g feature of the L-arginine-saturated enzyme. The broad
positive lobe on the high field side (extending downfield from g = 4) is contributed by the enzyme as isolated.
As shown in Fig. 3(line A) nNOS in the presence of a saturating
concentration of L-thiocitrulline exhibits a complex high spin
ferriheme spectrum; for comparison, line C of Fig. 3reproduces from Fig. 2the EPR spectrum for nNOS as
isolated. The low field peak has a g value of 7.70; this
is a significant shift downfield from the position of the peak in
untreated enzyme. Note that the L-thiocitrulline-induced shift
is in the opposite direction from the shift produced by L-arginine binding. As in the untreated sample, a shoulder on
the high field side of this peak in the L-thiocitrulline-treated sample represents a distinct minority
high spin component, in this case with a g value of 7.33.
Figure 3: Effects of L-thiocitrulline and L-homothiocitrulline on the EPR properties of NOS. Spectra were acquired at 11 °K in the presence of 100 µML-thiocitrulline (spectrum A) or 500 µML-homothiocitrulline (spectrum B) and are shown in comparison to the resting enzyme (spectrum C). Difference spectra for L-thiocitrulline and L-homothiocitrulline are represented by spectra A-C and B-C, respectively.
The high spin features of the nNOS-thiocitrulline complex in the g = 4 region are more complex. Two components of
approximately equal amplitude can be resolved at g values of
about 3.98 and 3.83. Neither of these features is associated with the
species responsible for the minority line at g = 7.33;
the amplitudes are mismatched, and the position of the peaks in the g = 4 region is inappropriate (see
``Discussion''). They must therefore be the g features of two high spin components, which are unresolved at g
= 7.7. The difference spectrum for L-thiocitrulline binding is shown as line A-C in Fig. 3. The relative positions of the positive and negative
lobes illustrate that the general effect of L-thiocitrulline
binding is an increase in the rhombicity of the high spin state.
The low spin state species associated with nNOS as isolated and nNOS in the presence of L-thiocitrulline are compared in Fig. 4. Whereas the native enzyme shows a low spin species with features at 2.43, 2.28, and 1.89 (line B), the nNOS-thiocitrulline complex shows two new low spin species with principal g values of 2.47, 2.27, and 1.89, and 2.39, 2.27, and 1.90 (line A). Assignment of the last g value in each set was made on the basis of crystal field theory; the assignment given produced an orbital reduction factor closer to 1 for both species than the reverse assignment.
Figure 4: Effects of L-thiocitrulline on the low spin components of NOS. The spectra were acquired at 22 °K using the sample employed for the spectrum in Fig. 3.
It is notable that the percentage of ferriheme in the high spin state in the presence of L-thiocitrulline as measured by EPR at 11 °K increases slightly in comparison with the enzyme as isolated. This can be appreciated from the decrease in the low spin signals shown in Fig. 4and also from the slight increase in the high spin signals seen following L-thiocitrulline binding in Fig. 3(compare line A with line C or note the relatively larger size of the positive lobes in the difference spectrum, line A-C). This finding is in contrast to the increase in low spin form following L-thiocitrulline binding at room temperature deduced from changes in the optical spectrum in the Soret region (24) and is reminiscent of other temperature-sensitive spin state equilibria (34) .
Direct
comparison of the spectral features of the nNOS-thiocitrulline and
nNOS-homothiocitrulline spectra (Fig. 3, lines A and B) shows that the majority L-homothiocitrulline
complex is intermediate in character between the two unresolved major L-thiocitrulline complexes. This is most readily apparent from
the g feature of the majority L-homothiocitrulline complex (g = 3.9), which
lies between the two L-thiocitrulline-induced g
features (g = 3.98 and 3.83). The shifts in the
positions of these high spin peaks and the shift in the spin state
equilibrium toward the high spin state upon L-homothiocitrulline binding to nNOS are confirmed in the
difference spectrum (Fig. 3, line B-C). Only weak low
spin ferriheme features could be observed for L-homothiocitrulline at temperatures between 7 and 25 °K,
consistent with the type I spectral changes induced at room temperature
(data not shown).
In previous studies, we have used substrate- or
inhibitor-perturbation optical difference spectroscopy to establish
that L-arginine, NOH-Arg(23) , L-thiocitrulline, and L-homothiocitrulline (24) alter the spin state equilibria of the heme cofactor of
nNOS. Such studies were not able, however, to distinguish or
structurally characterize specific high or low spin enzyme-ligand
complexes. In the present studies, we show that EPR spectroscopy can be
used not only to confirm the central findings of the optical studies
but also to provide for each substrate or inhibitor a spectroscopic
``fingerprint'' characteristic of its interaction with the
heme and heme-binding pocket. Table 1summarizes the findings
with respect to the species detected. In addition, the sensitivity of
EPR spectra to ligand structure provides structural information on the
binding of heme to nNOS and establishes a powerful probe of the
interactions between the O-binding position of heme and the L-arginine-binding site.
The power of EPR spectroscopy for
these purposes derives from the exquisite sensitivity of the iron d orbitals in ferriheme to both the nature and number of axial
ligands (1 or 2) and to subtle, heme-binding pocket-induced
perturbations of the iron ligation geometry. More specifically, the
features of the high spin nNOS EPR spectra near g =
7.6, 4, and 1.8 are associated with transitions within the lowest
Kramer's doublet of the S = 5/2 sextet of high
spin ferriheme. The g = 7.6 and g = 4
features derive from enzyme molecules in which the plane of the heme
lies in the x and y direction of the Zeeman magnetic
field; the z direction of the field (normal to the heme) gives
rise to a transition near g = 1.8 (Table 1). As long as intermixing of the Kramer's doublets
by Zeeman terms (
0.3 cm
) is small, the spectra
are controlled by the ratio of E/D, where E is the rhombic zero field splitting parameter and D is
the axial zero field splitting parameter(27, 28) .
Terms in E mix the three S = 5/2
Kramer's doublets, splitting g
and g
about an axial value of
6.(
)
These considerations were used to help assign the features shown in Fig. 1Fig. 2Fig. 3Fig. 4(e.g. in Fig. 3, line B the g
7.33
peak could only be matched by a corresponding g
feature near 4.35).
Because E and D are determined by the ligand-induced splittings of the iron d orbitals, they reflect both the identity of the axial ligands and the geometry of the heme site. For high spin nNOS, the only axial heme ligand is provided by an nNOS amino acid residue; in all cases E/D (Table 1) was in the range characteristic of enzymes and model complexes having thiolate ligands(29, 35) . This finding directly confirms predictions of cysteine residue participation based on sequence analysis(10, 12) , spectral studies(11, 36) , and work using mutational analysis(13, 16) .
Our data indicate that an enzyme
thiolate-heme axial ligand bond similar to that of native nNOS persists
in the complexes of nNOS with L-arginine, L-thiocitrulline, and L-homothiocitrulline, but it is
notable that each of these ligands causes a characteristic shift in the
EPR spectrum reflecting changes in E/D. ()Such changes indicate that binding of substrate or
inhibitors induces a (probably local) conformational change in the
enzyme that is reflected in a change in the geometry of the heme site.
Note that none of the changes seen in the high spin spectral features
are attributable to ligand-iron bond formation; if L-arginine
contributed a sixth axial heme ligand, it would drive the heme low
spin. This is not seen with the nNOS-arginine complex, a finding
consistent with the fact that CO or O
can still bind to the
heme of such a complex in the ferrous state and that CN
can bind to the ferric complexes.
The fact that native nNOS
shows none of the ferriheme EPR spectral features of the nNOS-arginine
complex indicates that nNOS as isolated does not contain L-arginine or any intermediates or substrate analogs that
would distort the heme-binding pocket as L-arginine does. On
the other hand, the finding that nNOS as isolated has at least 10% low
spin heme suggests that in this fraction of the enzyme water or an
adjacent residue in the active site is serving as a sixth axial heme
ligand. The g values of the native nNOS low spin species
indicate that the axial ligand opposite the thiol is
oxygen(35) . A small amount of species with g =
2.5 is usually present; this species may have a
nitrogenous sixth ligand, probably derived from an amino acid side
chain.
The results shown in Fig. 3demonstrate that both L-thiocitrulline and L-homothiocitrulline produce
changes in the geometry of the heme site detectable by EPR
spectroscopy. As indicated by E/D values of 0.081 and
0.087 (Table 1), the two major high spin components seen in the
presence of L-thiocitrulline are both more rhombic than the
high spin form of nNOS as isolated (E/D =
0.079). That is, although the energies of the d and d
orbitals are more nearly equivalent
in the arginine complex than in the enzyme as isolated, the inhibitors
both appear to distort the heme iron ligation geometry in a way that
increases the d
- d
splitting. We do not believe the E/D = 0.075
species of the nNOS-thiocitrulline complex is a remnant of the
unliganded native state, which it closely resembles, for two reasons.
First, the concentration of L-thiocitrulline used (100
µM) is >1000-fold the K
(24) and presumably saturating. Second, the g
peak is shifted far enough to low field by L-thiocitrulline that there does not appear to be enough
intensity at g values below 7.6 to account for the large
component visible at g =
4. The improved
resolution of the g =
4 feature may result in part
from a small upfield shift, separating it somewhat from the rhombic
iron signal.
The minority component of the high spin L-thiocitrulline spectra with g =
7.33 has a significantly smaller rhombicity than even the L-arginine complex; E/D is about 0.06 (not
listed in Table 1). Although the E/D value is
low, the enzymatic thiolate ligand probably remains in place even in
this complex. In the presence of L-homothiocitrulline, a
similar complex (E/D = 0.063) is easily
detected and is in sufficient concentration to resolve both the g
and g
signals ( Fig. 3and Table 1). Detection of multiple species suggests
that in contrast to L-arginine, L-thiocitrulline and L-homothiocitrulline share an ability to bind in several
different modes giving distinct high spin species. Whether these
species represent alternative ligand structures (e.g. thione versus thiol tautomers) or alternative conformational
adjustments in the binding site is unclear. We note that in normal
catalysis, heme-bound oxygen first reacts with a guanidino nitrogen of L-arginine and that a second heme-bound oxygen then reacts
with the guanidino carbon of NOH-Arg. In order to do this, the
substrate-binding site may accommodate some slippage. The enzyme may
have evolved to bind its normal substrate, L-arginine,
uniquely, but L-thiocitrulline and L-homothiocitrulline may bind in either an L-arginine
or a NOH-Arg mode. It is worth noting that although binding of L-thiocitrulline leads to the formation of two high spin
species with increased rhombicity (compared with the enzyme as isolated
and the arginine complex), the longer side chain of L-homothiocitrulline permits the formation of only one such
species. The increased formation of the lower rhombicity (g
= 7.33) species suggests that this represents a binding
mode/conformational state in which the extra length of the side chain
is less energetically unfavorable.
Whereas the nNOS-arginine (23) and nNOS-homothiocitrulline (24) complexes are
exclusively high spin, nNOS forms two distinct minority low spin
complexes with L-thiocitrulline(24) . As shown in Fig. 4and summarized in Table 1, neither of the
nNOS-thiocitrulline low spin species can be attributed to the minority
low spin species of nNOS as isolated; in all cases the g values are distinct. Furthermore, crystal field calculations
indicate that the values of the axial and rhombic ligand field
splittings and V of the t
set of
iron d orbitals for the low spin state of nNOS as isolated
are, respectively, about 5.66 and 1.9 (in units of spin orbit coupling)
using the axis system introduced originally by Peisach et
al.(29) . The corresponding parameters for the L-thiocitrulline complexes are about 5.46 and 2.02 for the
species with g
= 2.47 and about 5.96 and
1.83 for the species with g
= 2.39. If we
refer these values to the Blumberg and Peisach crystal field
diagrams(29) , we find the species fall in region P. This
region contains low spin species with thiolate ligands;
and V for nNOS low spin species are comparable with ferriheme model
complexes with a thiolate ligand described by several groups.
All
three low spin species appear to retain the endogenous axial thiolate
ligand. The g = 2.39 nNOS-thiocitrulline species is
similar to model complexes with g between 2.33 and
2.42 in which both axial ligands are thiolate sulfurs(35) .
This supports a model for L-thiocitrulline binding in which
the inhibitor is close enough to the ferriheme to form a true
coordination complex. Note, however, that this is a minority species;
as shown previously in optical studies(24) , most L-thiocitrulline is bound without direct sulfur-ferriheme
coordination. The identity of the sixth heme ligand in the other two
low spin complexes is less certain. Comparison with model complexes
suggests that the sixth ligand may be oxygen in both cases. This is
almost certainly the case for the native nNOS species with g
=
2.43, but whether the sixth ligand
is derived from solvent or the side chain of an nNOS amino acid is
unclear. The difference between that low spin complex and the g
= 2.47 complex of L-thiocitrulline could be the result of a small distortion of
the original ligand geometry rather than ligand replacement. We note
that the g
= 2.47 peak seen with L-thiocitrulline is also consistent with a nitrogen ligand as
might be expected if the inhibitor bound with the terminal thioureido
nitrogen rather than the sulfur near heme. L-Citrulline, a
product of NOS, does not inhibit (K
10
mM), although it could presumably bind similarly (i.e. with the thioureido nitrogen in the heme pocket).
The present
results, taken with previous kinetic and optical spectroscopy
studies(24) , demonstrate that L-arginine, L-thiocitrulline, and L-homothiocitrulline all bind
tightly (K and K
<
0.06-10 µM) but distinctly to nNOS. Because binding
is in all cases enantiomer-specific, the enzyme must associate
similarly with the
-amino and carboxylate groups of the three
species. On the other hand, the L-arginine side chain clearly
adopts a unique high spin binding mode that is distinct from the
multiple high spin binding modes seen with L-thiocitrulline
and L-homothiocitrulline. The guanidino group of L-arginine does not contribute a sixth axial ligand to the
heme cofactor or cause dissociation of the thiolate ligand but
nonetheless converts the high spin state associated with nNOS as
isolated to a high spin state with different properties.
Because E and D, and hence the EPR spectra, are primarily a
function of the ligand field splittings of the iron d orbitals, binding of arginine (and arginine analogs) must perturb
the ligation geometry by more subtle interactions in the heme pocket.
It does this presumably by directly interacting with and perturbing the
heme porphyrin ring or by interacting with nNOS residues forming the
heme pocket. This could transmit strain to the iron through
interactions between the polypeptide and the porphyrin periphery or
through the axial thiolate ligand. Whereas the high spin nNOS-arginine
complex is less rhombic (lower g) than the high
spin form of nNOS as isolated, the majority high spin forms of
nNOS-thiocitrulline and nNOS-homothiocitrulline are more rhombic
(higher g
). These species, in which the ligand
does not directly coordinate heme iron, must perturb the heme porphyrin
or its binding pocket differently than does L-arginine.
Although nNOS may have evolved to bind L-arginine uniquely, these synthetic inhibitors are bound in multiple modes with at least three high spin and two low spin states for L-thiocitrulline. It seems clear that arginine analogs with various substituents binding in the site normally occupied by the reactive guanidino nitrogen produce characteristic modifications of ligand-heme pocket interactions, which are reflected in characteristic spectroscopic signatures for each complex. Because this group is the initial hydroxylation site, it is apparent that only short range interactions need be invoked and that this part of the substrate-binding site interacts intimately with the heme. It will be interesting to explore the extent to which various EPR-detectable heme pocket perturbations are induced by ligand binding as specific structural changes are made to substrates and to inhibitors such as L-thiocitrulline.