(Received for publication, October 16, 1995; and in revised form, January 12, 1996)
From the
Neuronal nitric-oxide (NO) synthase contains FAD, FMN, heme, and
tetrahydrobiopterin as prosthetic groups and represents a
multifunctional oxidoreductase catalyzing oxidation of L-arginine to L-citrulline and NO, reduction of
molecular oxygen to superoxide, and electron transfer to cytochromes.
To investigate how binding of the prosthetic heme moiety is related to
enzyme activities, cofactor, and L-arginine binding, as well
as to secondary and quaternary protein structure, we have purified and
characterized heme-deficient neuronal NO synthase. The heme-deficient
enzyme, which had preserved its cytochrome c reductase
activity, contained FAD and FMN, but virtually no tetrahydrobiopterin,
and exhibited only marginal NO synthase activity. By means of gel
filtration and static light scattering, we demonstrate that the
heme-deficient enzyme is a monomer and provide evidence that heme is
the sole prosthetic group controlling the quaternary structure of
neuronal NO synthase. CD spectroscopy showed that most of the
structural elements found in the dimeric holoenzyme were conserved in
heme-deficient monomeric NO synthase. However, in spite of being
properly folded, the heme-deficient enzyme did bind neither
tetrahydrobiopterin nor the substrate analog N-nitro-L-arginine. Our results
demonstrate that the prosthetic heme group of neuronal NO synthase is
requisite for dimerization of enzyme subunits and for the binding of
amino acid substrate and tetrahydrobiopterin.
Nitric oxide, an important effector and signaling molecule in
the nervous, immune, and cardiovascular
systems(1, 2, 3, 4) , is
enzymatically generated from L-arginine and molecular oxygen
by different nitric-oxide synthases (NOS, ()EC 1.14.13.39).
To date, two constitutively expressed, Ca
-dependent
NOS isoforms as well as a cytokine-inducible,
Ca
-independent protein have been
characterized(5) . The isozyme purified from brain (5, 6, 7) termed neuronal NOS (nNOS), was
identified as a 320-kDa homodimer, containing close to stoichiometric
amounts of heme, FAD, and FMN as well as variable amounts of
tetrahydrobiopterin (H
biopterin) (8, 9, 10, 11, 12, 13, 14) .
nNOS exhibits sequence similarities to cytochrome P450 reductase (15) and to the heme-binding sequence in P450
proteins(16) , suggesting that the enzyme represents a fusion
protein of a cytochrome P450-like protein and a P450
reductase(17) . In support of this hypothesis, nNOS was shown
to catalyze the reduction of cytochrome c(18) and to
possess a cysteine-ligated heme-iron(19, 20) with
spectral properties characteristic of a P450
protein(9, 12, 21) . Further studies have
confirmed the bidomain structure of
nNOS(20, 22, 23) , with the reductase domain
shuttling NADPH-derived electrons in a calmodulin-triggered fashion
from the flavins to the heme moiety(24, 25) , which is
located in close proximity to the amino acid substrate and pteridine
binding sites (26) and catalyzes a two-step mono-oxygenation of L-arginine to L-citrulline and NO with N
-hydroxy-L-arginine as
intermediate(27, 28) .
Recent work has focused on
the allosteric regulation of NOS by L-arginine,
Hbiopterin, and heme. The cytokine-inducible NOS dimer from
macrophages was shown to form catalytically inactive monomers in the
absence of H
biopterin or L-arginine and, notably,
to lose its prosthetic heme group in the absence of these ligands.
Reassociation of the monomers to catalytically active dimers required
the coincident presence of heme, L-arginine, and pteridine,
pointing to a role of these compounds in the post-translational
processing of inducible NOS(29) . However, in contrast to the
inducible isozyme, native nNOS maintained its catalytically active,
dimeric conformation even in the absence of pteridine and L-arginine(14) . Thus, it is currently unclear how
binding of heme to nNOS is related to subunit assembly, catalytic
activity, and pteridine binding. To address this issue, we have
purified heme-deficient nNOS (nNOS(h-)) from a baculovirus
overexpression system and analyzed the virtually heme-free protein for
catalytic activity, cofactor content, amino acid substrate, and
H
biopterin binding and determined the secondary and
quaternary structural features of the enzyme.
To investigate the role of heme in nNOS structure and
function, we attempted to purify recombinant heme-deficient nNOS from a
baculovirus overexpression system. From 4.5 10
cells, which had been infected with the rat brain NOS-recombinant
baculovirus for 48 h in the absence of hemin chloride, we obtained
80 mg of nNOS with a heme content of 0.29 ± 0.03 eq per
monomer (n = 3). The amount of heme bound to the
recombinant protein was not significantly reduced by preincubation
(24-48 h) and infection (48 h) of Sf9 cells in the presence of
0.25 and 0.50 mM succinyl acetone, an inhibitor of heme
biosynthesis(47) , although cytosolic heme levels were reduced
down to 10% of untreated control cells under these conditions (not
shown). Partially heme-deficient nNOS was further analyzed for
H
biopterin, FAD, and FMN, revealing that substoichiometric
amounts of these cofactors were incorporated into the protein (0.12
± 0.01, 0.17 ± 0.01, and 0.11 ± 0.01 eq per
monomer, respectively; n = 3). In the presence of
saturating concentrations of exogenous H
biopterin, FAD, and
FMN, the partially heme-deficient enzyme exhibited only low specific
activity (0.30 ± 0.03 µmol of L-citrulline
min
mg
, n = 3) as compared with the holoenzyme (1.08 ± 0.14
µmol
min
mg
, n = 3).
To investigate whether partially
heme-deficient nNOS can be reconstituted with flavins, we have
preincubated the protein with a 2-fold molar excess of FAD and FMN for
5 min at room temperature and subsequently removed free flavins by gel
filtration chromatography. Under these conditions, the amount of
protein-bound FAD and FMN was increased 6-fold to 0.98 ±
0.06 and 0.65 ± 0.04 eq per monomer, respectively (n = 3). Increasing the incubation time to 60 min did not
further enhance FMN binding. To confirm that the flavins were
incorporated into the nNOS protein in a catalytically active form, we
have determined cytochrome c reductase activities of the
reconstituted protein in the absence of exogenous flavins. Enzyme
kinetic analysis revealed that reconstitution of nNOS with flavins had
no effect on the affinity of cytochrome c for nNOS but was
accompanied by a
5-fold increase of cytochrome c reductase activity as compared to controls (K
, 14 ± 3 versus 15 ± 1
µM; V
, 2.6 ± 0.6 versus 15 ± 2 µmol
min
mg
; n = 3). Maximal rates of L-citrulline formation as well as L-arginine- and
H
biopterin-independent NADPH oxidation were not affected
(not shown), demonstrating that the amount of enzyme-bound heme was
limiting for NOS activity of the protein.
Gel filtration
chromatography of the partially heme-deficient enzyme preparations,
which had been reconstituted with FAD and FMN, revealed that NOS
activity eluted in a single and well defined peak centered at an
elution volume of 11.8 ml (Fig. 1, upper panel, filled circles). nNOS eluting in this peak fraction contained
0.56 ± 0.06 eq of heme per monomer (n = 3) and
had a specific activity of 0.88 ± 0.09 µmol
min
mg
(n = 3) when assayed in the presence of saturating
concentrations of exogenous H
biopterin and flavins.
Cytochrome c reductase activity eluted in a broad peak with
two maxima at 11.8 and 13.0 ml, and
40% of the total reductase
activity were found in fractions which did not exhibit detectable
citrulline formation (Fig. 1, upper panel, open
circles), suggesting the separation of two protein species. As
shown in the lower panel of Fig. 1, comparable elution
profiles were found when the eluate was assayed for protein (filled
circles), FAD (open circles), and FMN (filled
squares), demonstrating that both protein species contained
virtually stoichiometric amounts of FAD and
0.6 eq of FMN per
monomer. Substoichiometric binding of FMN as well as a rightward shift
of the FMN elution profile points to dissociation of the flavin during
gel filtration. In contrast to FAD and FMN, which co-eluted with the
reductase activity of nNOS, heme and H
biopterin strictly
co-eluted with L-citrulline forming activity, demonstrating
that heme-deficient nNOS was separated from the active heme-containing
enzyme.
Figure 1:
Gel filtration chromatography of
partially heme-deficient nNOS. Aliquots of flavin-reconstituted,
partially heme-deficient nNOS (200 µl; 0.10 mM nNOS,
containing 0.3 eq of heme per monomer) were injected onto a Superose 6
gel filtration column and eluted as described under ``Experimental
Procedures.'' Upper panel, fractions of 0.30 ml were
collected and assayed for NO synthase (filled circles) and
cytochrome c reductase activity (open circles) as
described under ``Experimental Procedures.'' Lower
panel, column fractions were analyzed for protein (filled
circles), FAD (open circles), FMN (filled
squares), heme (open squares), and Hbiopterin (filled triangles) as described under ``Experimental
Procedures.'' Data are expressed as the total amount of enzyme
activity, protein, or prosthetic group per 0.30-ml fraction and
represent means of three separate series of
experiments.
Virtually pure heme-deficient nNOS (nNOS(h-), 0.04
eq of heme per monomer) was obtained by pooling gel filtration column
fractions which contained >0.4 nmol nNOS and <0.1 mol eq of heme
per monomer. nNOS(h-) was found to contain only marginal amounts
of heme and H
biopterin (0.030 ± 0.006 and 0.007
± 0.001 eq per monomer, respectively; n = 3).
The amount of flavins bound to nNOS(h-) was almost identical with
that of the starting material used for the isolation of the
heme-deficient protein (0.87 ± 0.14 versus 0.98
± 0.06 and 0.61 ± 0.08 versus 0.65 ± 0.04
eq per monomer, respectively; n = 3), indicating that
heme deficiency does not affect the affinity of nNOS for FAD and FMN.
Enzyme kinetic parameters for nNOS(h+) and nNOS(h-) were
calculated from weighted Lineweaver-Burk plots obtained by determining
rates of L-citrulline formation in the presence of saturating
concentrations of Hbiopterin and flavins and increasing
concentrations of L-arginine (1-50 µM).
Under these conditions, nNOS(h-) exhibited only very little NOS
activity (V
= 26 ± 4 nmol L-citrulline
min
mg
, n = 3) as compared with
nNOS(h+) (V
= 1.2 ± 0.1
µmol of L-citrulline
min
mg
, n = 3), whereas
the affinity of both protein species for L-arginine was
comparable (K
= 7.2 ± 0.6 µM (nNOS(h-)) versus 5.5 ± 0.4 µM (nNOS(h+)); n = 3), indicating that residual
nNOS(h+) present in preparations of the heme-deficient enzyme
accounts for the observed NOS activity. In support of this, we found
that turnover numbers (k
), which had been
corrected for the heme content of the protein preparations, did not
significantly (p = 0.09, unpaired t test)
differ (nNOS(h-): 0.030 ± 0.006 eq of heme per monomer, k
= 139 ± 35 min
(n = 3); nNOS(h+): 0.93 ± 0.05 eq of
heme per monomer, k
= 206 ± 20
min
(n = 3)).
We have recently
shown that nNOS purified from porcine brain is converted to an
SDS-resistant dimer upon binding of Hbiopterin and L-arginine(14) . Heme saturation of the porcine enzyme (13) precluded, however, investigation of the contribution of
the prosthetic heme group to this tight interaction of nNOS subunits.
To address this issue, we have subjected nNOS(h-) and
nNOS(h+), which had been preincubated with 2% SDS in the absence
and presence of H
biopterin (0.1 mM) and L-arginine (1 mM), to low temperature SDS-PAGE. In
the absence of the added ligands (Fig. 2, upper panel, lane A), the ratio of SDS-resistant nNOS(h+) dimers
(
300 kDa) to monomers (
150 kDa) was
15:85. Preincubation
of nNOS(h+) with exogenous H
biopterin (0.1
mM) and L-arginine (1 mM) increased the
relative amount of dimeric nNOS to
50%. With nNOS(h-), we
did not detect any SDS-resistant protein dimers both in the absence (lane C, upper panel) and presence (lane D, upper panel) of saturating amino acid substrate and cofactor
concentrations. Heme staining of the gels by means of the
dimethoxybenzidine/H
O
method (Fig. 2, lower panel) shows that the ratio of heme-containing
nNOS(h+) dimers to monomers was
90:10 in the absence of
H
biopterin and L-arginine (lane A, lower panel) and, thus, markedly higher than that determined
by means of protein staining (see lane A, upper
panel). Preincubation in the presence of exogenous pteridine and L-arginine led to virtually complete dimerization of the
heme-containing protein (lane B, lower panel),
demonstrating that
50% of nNOS(h+) had lost their prosthetic
heme group during electrophoresis. As expected, nNOS(h-) monomers
did not stain for heme (lanes C and D, Fig. 2, lower panel).
Figure 2:
Low-temperature SDS-PAGE of nNOS(h+)
and nNOS(h-). nNOS(h+) (lanes A and B) and
nNOS(h-) (lanes C and D) were incubated at 37
°C in 50 µl of 50 mM triethanolamine/HCl buffer (pH
7.0) in the absence (lanes A and C) or presence of
0.1 mM Hbiopterin and 1 mML-arginine (lanes B and D). After 10
min, 50 µl of chilled Laemmli buffer (0.125 M Tris-HCl, pH
6.8, 4% (w/v) SDS, 20% (w/v) glycerol, 0.02% (w/v) bromphenol blue)
were added. Subsequently, samples were subjected to low-temperature
SDS-PAGE (30 pmol of protein per lane) on 6% slab gels and stained for
protein with Coomassie blue (upper panel) or for heme with
3,3`-dimethoxybenzidine/H
O
(lower
panel) as described under ``Experimental Procedures.'' NOS and Di-NOS refer to nNOS monomers and homodimers,
respectively. The gel shown is representative of
three.
To investigate the involvement of the
prosthetic heme group in the dimerization of nNOS under native
conditions, we have analyzed nNOS(h+) and nNOS(h-) by means
of gel filtration chromatography and static light scattering. In the
course of gel permeation chromatography on Superose 6, heme-saturated
(>0.90 eq of heme per monomer) and -deficient (<0.05 eq of heme
per monomer) nNOS eluted at 11.6 and 13.1 ml, respectively (Fig. 3A). From these data, we calculated Stokes radii
of 6.3 ± 0.3 and 8.1 ± 0.1 nm (n = 3) for
nNOS(h-) and nNOS(h+), respectively (Fig. 3B), showing that the hydrodynamic volume of
nNOS(h-) (1.05 ± 0.05 10
m
) is 2.1 ± 0.1-fold smaller than that of the
holoenzyme (2.23 ± 0.03
10
m
). Together with the observation that the native,
heme-saturated holoenzyme forms a 320-kDa
homodimer(11, 14) , our results suggest that
nNOS(h-) is a 160-kDa monomer. Alternatively, the smaller Stokes
radius of nNOS(h-) may result from a more compact, globular
conformation compared to the elongated holoenzyme, which was shown to
exhibit an axial ratio of
20:1(14) . This was clarified by
means of static light scattering, a technique which allows us to
determine the molecular mass ratio of related macromolecules in
solution. The specific mean scattering intensities of equally
concentrated solutions (5.4 ± 0.4 µM) of
nNOS(h+) and nNOS(h-) were 16 ± 0.2
10
and 8.3 ± 1.0
10
counts s
M
(n = 2, 20 scans
each), respectively (Fig. 3C), demonstrating that the
molecular masses of nNOS(h-) and nNOS(h+) differed by a
factor of 1.9 ± 0.3.
Figure 3:
Gel
filtration chromatography and static light scattering of nNOS(h-)
and nNOS(h+). A, aliquots of nNOS(h-) and
nNOS(h+) (100 µl; 5 µM nNOS) were injected
separately onto a Superose 6 gel filtration column, and protein in the
eluate was monitored at 280 nm (see ``Experimental
Procedures''). The chromatogram shown is representative of six. B, the Stokes radii of nNOS(h-) and nNOS(h+) were
calculated from calibration of the Superose 6 gel filtration column
with a set of standard proteins including thyroglobulin (Stokes radius
= 8.50 nm), ferritin (6.10 nm), catalase (5.22 nm), aldolase
(4.81 nm), and bovine serum albumin (3.55 nm) as described under
``Experimental Procedures.'' K
= (V
- V
)/(V
- V
) with V
, V
, and V
denoting the elution volume of the
protein, the column void volume, and the total bed volume of the
column, respectively. C, nNOS(h-) and nNOS(h+) were
adjusted to a protein concentration of 5.4 µM each and
analyzed by static light scattering as described under
``Experimental Procedures.'' Scattering intensities are means
± S.E. of two separate series of 20 scans performed with two
protein preparations.
To determine whether reconstitution of
nNOS(h-) monomers with heme results in the reassociation of
enzyme subunits into a homodimer, we have incubated heme-deficient
monomers (4 µM) with a 2-fold molar excess of hemin
chloride for 30 min at ambient temperature prior to removal of unbound
heme by gel filtration chromatography (Fig. 4). As estimated
from the peak areas, the relative amount of dimeric nNOS was increased
10-fold from
5% to
55% upon incubation with hemin. The inset to Fig. 4shows that dimerization of nNOS
subunits was accompanied by an
10-fold increase in protein-bound
heme from 20 ± 9 pmol to 195 ± 35 pmol (n = 3) which corresponded to a heme content of 0.5 eq of per
monomer. Increasing the incubation time to 60 min, varying the
protein:heme ratio from 1:1 to 1:5, or co-incubation with L-arginine and H
biopterin (8 µM each)
gave essentially the same results (data not shown). Reconstitution with
heme in the presence or absence of
H
biopterin/L-arginine did not restore enzyme
activity as determined by L-arginine-independent NADPH
oxidation and formation of L-citrulline from L-arginine (not shown).
Figure 4: Reconstitution of nNOS(h-) with heme. nNOS(h-) (4 µM) was incubated in the absence and presence of 8 µM hemin chloride for 30 min at ambient temperature in a 50 mM triethanolamine/HCl buffer, pH 7.0, containing 12 mM mercaptoethanol. Aliquots (100 µl, 0.4 nmol of nNOS) were injected onto a Superose 6 gel filtration column and eluted as described under ``Experimental Procedures.'' Protein in the eluate was monitored at 280 nm. The chromatogram shown is representative of three. Inset, column fractions of 0.30 ml were assayed for heme by HPLC as described under ``Experimental Procedures.'' Data are the total amount of heme co-eluting with nNOS and represent means ± S.E. of three separate experiments performed with two protein preparations.
Interestingly, the amount of
Hbiopterin bound to nNOS(h-) (0.007 ± 0.001 eq
per monomer) was markedly reduced as compared with the heme-saturated
enzyme (0.45 ± 0.03 eq per monomer). To find out whether
enzyme-bound heme is requisite for pteridine binding, we have performed
binding studies with nNOS(h+) and nNOS(h-) using
[
H]H
biopterin as radioligand (Fig. 5A). In accordance with previous studies
performed with nNOS from porcine brain(36, 38) ,
nNOS(h+) bound [
H]H
biopterin
with K
and B
values of 0.26
± 0.01 µM and 129 ± 3 pmol per nmol of
monomer (n = 3), respectively (panel A, solid symbols), whereas only marginal amounts of the pteridine
bound to the heme-deficient enzyme (panel A, open
symbols; B
< 10 pmol per nmol of
monomer, n = 3). Since the amino acid substrate site of
nNOS appears to interact with the heme domain of the
enzyme(48, 49) , we further investigated the role of
the prosthetic heme group in binding of the high affinity amino acid
substrate analog L-[
H]NNA(39) .
As shown in panel B of Fig. 5, nNOS(h+) bound L-[
H]NNA with an efficacy closely
similar to that observed with
[
H]H
biopterin (closed
symbols; B
= 133 ± 2 pmol per
nmol of monomer, K
= 0.17 ± 0.02
µM; n = 3), whereas binding to
nNOS(h-) was negligible (open symbols; B
< 10 pmol per nmol of monomer, n = 3). It should be pointed out that the binding assay is
not quantitative and does not allow, therefore, to determine the
absolute amount of bound H
biopterin (see
``Experimental Procedures'').
Figure 5:
Binding of
[H]H
biopterin and L-[
H]NNA to nNOS(h+) and
nNOS(h-). For saturation binding of
[
H]H
biopterin (A) and L-[
H]NNA (B) to nNOS(h+) (filled circles) and nNOS(h-) (open circles),
0.02-0.05 nmol of nNOS were incubated for 10 min at 37 °C
with a fixed concentration (12 nM) of
[
H]H
biopterin (
17 nCi) or L-[
H]NNA (
70 nCi) and increasing
concentrations of the respective unlabeled ligand (10 nM-10
µM) in 0.1 ml of a 50 mM triethanolamine/HCl
buffer, pH 7.0. The amount of specifically bound radioligand was
determined as described under ``Experimental Procedures.''
Data are means ± S.E. of three separate experiments performed
with three nNOS preparations.
The apparent loss of amino
acid substrate and pteridine binding sites in nNOS(h-) suggests
that heme deficiency may be accompanied by unfolding of the protein. To
address this issue, we have analyzed nNOS(h+) and nNOS(h-)
by CD spectroscopy. As shown in Fig. 6, nNOS(h+) displayed
a well defined far-UV CD spectrum with minima at 208 and 220 nm, a
maximum at 192 nm, and base line crossovers at 200 nm and 180 nm (solid line). Similar spectra were obtained for the
heme-deficient enzyme (dashed line), and secondary structure
analysis revealed that both nNOS species were virtually identical with
regard to their content of parallel
-sheet, turns and other
structures (Table 1). However, nNOS(h+) and nNOS(h-)
differed slightly in their content of
-helical structures (0.27 versus 0.34) and antiparallel
-sheet (0.15 versus 0.11), pointing to subtle secondary structure changes occurring
upon heme binding.
Figure 6: CD spectra of nNOS(h-) and nNOS(h+). CD spectra of nNOS(h-) (solid line) and nNOS(h+) (dashed line) were recorded at 20 °C with 12 µM protein in 50 mM sodium phosphate buffer, pH 7.4, as described under ``Experimental Procedures.'' The spectra shown are representative of two.
It was the objective of the present study to isolate and
characterize heme-free nNOS in order to find out how binding of the
prosthetic heme group affects the catalytic and structural features of
the enzyme. Infection of Sf9 cells with rat nNOS-recombinant
baculovirus in the absence of added hemin yielded a partially
heme-deficient nNOS which was purified and subjected to gel filtration
chromatography for separation of the heme-free protein from the
holoenzyme. Consistent with the essential role of heme in NOS
catalysis, nNOS(h-) exhibited only marginal NOS activity, which
was apparently due to contamination with holoenzyme (7% of total
protein). Heme deficiency neither affected the kinetic parameters for
cytochrome c reduction nor the ability of the enzyme to bind
FAD and FMN, showing that nNOS(h-) contains a fully intact
reductase domain. These data are in agreement with previous reports
suggesting a bidomain structure of
NOS(20, 22, 23, 50) .
Results
from gel filtration chromatography and static light scattering revealed
that the hydrodynamic volume and accordingly the molecular mass of
nNOS(h-) was about half of the respective values calculated for
nNOS(h+). Based on the identification of latter species as 320-kDa
homodimer(7, 14) , these data demonstrate that
nNOS(h-) is monomeric. Reconstitution of nNOS(h-) with
hemin resulted in pronounced incorporation of heme and consequent
enzyme dimerization. However, the reconstituted heme-containing dimers
did not exhibit detectable NOS activity, indicating that
co-translational heme binding is essential for expression of
catalytically active NOS. In striking contrast with inducible NOS
dimerization, which was reported to require the coincident presence of
heme, Hbiopterin, and L-arginine(29) , the
heme-induced dimerization of nNOS(h-) occurred in a pteridine-
and L-arginine-independent fashion. Thus, in accordance with a
recent report showing that porcine nNOS homodimers are stable in the
absence of H
biopterin (14) , our data suggest that
heme is the sole cofactor controlling the assembly of nNOS subunits.
As previously shown for the enzyme purified from porcine
brain(14) , recombinant rat nNOS(h+) homodimers
dissociated in the course of low temperature SDS-PAGE, unless
Hbiopterin was present to induce formation of superstable
SDS-resistant dimers. However, while this effect was virtually complete
with the pig enzyme, we observed only
50% of rat nNOS(h+)
dimers under comparable experimental conditions, i.e. upon
preincubation with saturating concentrations of both
H
biopterin and L-arginine. As revealed by the
heme-iron staining experiments, this incomplete dimerization was
apparently due to loss of heme during SDS-PAGE of nNOS(h+). Thus,
the enzyme obtained from porcine brain seems to bind the prosthetic
heme group more tightly than the recombinant rat brain NOS. We are
currently investigating whether this reflects species differences in
the heme environment of NOS or results from a specific feature of the
baculovirus overexpression system.
There is experimental evidence
that the prosthetic heme group is part of the amino acid substrate site
and interacts with the pteridine binding domain. Thus, L-arginine and Hbiopterin induce perturbations of
the heme spectrum(20, 23, 48, 51) ,
sequences located near the axial cysteine ligand of the heme are
apparently involved in L-arginine and pteridine
binding(26) , and the heme site inhibitor 7-nitroindazole was
found to antagonize L-arginine and H
biopterin
binding to NOS(36) . In the present study, we demonstrate that
nNOS(h+) binds H
biopterin and the amino acid substrate
analog L-NNA with high affinity, whereas binding of these
ligands to the heme-deficient protein was negligible. The minor
alterations in the CD spectrum point to an effect of heme deficiency on
the secondary structure of the protein. Based on the CD spectra, it
cannot be ruled out that the lack of binding activity of nNOS(h-)
is due to a loss of secondary structure, but this appears to be
unlikely because most of the structural elements found in dimeric
nNOS(h+) were conserved in the heme-deficient monomer. As compared
with the holoenzyme, nNOS(h-) contained slightly more
-helical structures accompanied by some loss of antiparallel
-strands, indicating that heme binding and concomitant subunit
dimerization involve a subtle rearrangement of secondary structure
necessary for formation of appropriately folded amino acid substrate
and cofactor binding sites.
In conclusion, our data support a model
for dimeric nNOS assembly as shown in Fig. 7. Heme-free NOS is
monomeric, contains the flavins FAD and FMN, and exhibits only
cytochrome c reductase activity. Depending on the
intracellular availibility of free heme, nNOS is expressed as a loosely
associated homodimeric hemeprotein, which readily dissociates in the
presence of SDS. In this conformation, nNOS acts as NADPH oxidase and
catalyzes the formation of superoxide and
HO
(52, 53) . Binding of
H
biopterin to nNOS(h+) further modifies the enzyme
structure in that the subunits adopt a much tighter conformation
resulting in the formation of superstable SDS-resistant
dimers(14) . This pteridine-induced conformational change,
which may occur either co- or post-translationally, is requisite for
the coupling of reductive oxygen activation to L-arginine
oxidation and, thus, for the conversion of the enzyme from an NADPH
oxidase to a fully active NOS. Accordingly, the role of the prosthetic
heme group is not confined to NOS catalysis but extends to the
modulation of protein structure, controlling the interaction of nNOS
subunits as well as the formation of pteridine and amino acid substrate
binding sites. It remains to be seen whether the expression of
catalytically active nNOS dimers is limited by heme availability in
certain (patho)physiological situations. If so, co-translational
processing of nNOS may represent a novel mechanism for the regulation
of neuronal NO biosynthesis in vivo.
Figure 7:
Role of heme in nNOS structure and
function. nNOS(h-) represents a monomeric flavoprotein,
catalyzing the reduction of cytochrome c and cytochrome P450 (18) . The heme-deficient protein does not exhibit binding
sites for L-arginine and Hbiopterin. Upon heme
binding, the H
biopterin-deficient enzyme forms a loosely
associated homodimer, which functions as an NADPH oxidase, catalyzing
the formation of superoxide and
H
O
(52, 53) . Finally, this
protein species is converted by H
biopterin (H
B) to a tight dimer exhibiting full NOS
activity.