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INTRODUCTION |
Nitric-oxide synthase (EC 1.14.13.39;
NOS)1 catalyzes the oxidation
of L-arginine to L-citrulline and nitric oxide
(NO) in two reaction cycles. The first one leads to the formation of
N
-hydroxy-L-arginine
(NOHLA), and the second one leads to the formation of
L-citrulline and NO (1, 2). The latter reaction is not specific for NOS; the formation of L-citrulline and
nitrite, an oxidation product of NO, has also been reported for
cytochrome P450 (3). Similarly to cytochrome P450, NOS contains a
thiolate ligated heme, NADPH is the initial electron donor, and the
reaction requires molecular oxygen (O2), which has to be
activated and split for the oxidation of L-arginine and the
formation of water (4, 5). In contrast to P450, in NOS the entire
electron transfer chain is contained in a single molecule, consisting
of a flavin-containing reductase domain and a heme-containing oxygenase domain (6). Furthermore, it requires a cofactor,
(6R)-5,6,7,8-Tetrahydro-L-biopterin (tetrahydrobiopterin; BH4), the role of which is still
unclear but the presence of which is essential for NO synthesis (7, 8).
In addition, Ca2+/calmodulin is required for electron flow
from the reductase domain to the oxygenase domain (9).
NOS catalysis involves the interaction of the reduced enzyme with
molecular oxygen. Although little is known about this process for NOS,
a wealth of data is available about the analogous P450 system (10). As
shown in Scheme 1, reduced P450 is known
to react rapidly with oxygen to form a transient oxygen complex, which
has been stabilized at low temperature (11). Several biophysical studies have elucidated the electronic structure of this complex as a
Fe(II)O2
Fe(III)O2· equilibrium,
which is poised in favor of the ferric form (Scheme 1, 1)
(12-14). The subsequent reduction of this complex is thought to lead
first to a peroxy compound (2) and eventually, after two
protonation steps, to an iron(IV) porphyrin
-cation radical
(3). Both species 2 and 3 have been proposed to act as oxygenating agents (15). The breakdown of these
species results in product release and formation of ferriheme via still
unknown intermediates (4).

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Scheme 1.
Supposed reaction of cytochrome P450 with
oxygen within its catalytic cycle. The numbers refer to
species cited in the text. For simplicity the possible interaction of
2 with substrate R-H is not shown.
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The corresponding reactions for NOS have not been reported, except for
a very recent stopped-flow study, which demonstrated the transient
formation in the oxidation of ferrous NOS by O2 of a
compound absorbing at 427 nm (16). In this work, we used a different
approach to study the reaction between molecular oxygen and reduced
neuronal NOS (nNOS). In order to trap possible intermediates, most of
the experiments were carried out at subzero temperatures in the
presence of 50% ethylene glycol as anti-freeze solvent (17). The same
procedure was employed previously for the study of P450 oxygen
complexes (11, 18, 19). Special attention was paid to the role of
BH4 in the reduction of the oxygen complex in the presence
of L-arginine. Our results throw more light on the function
of BH4 in NOS catalysis.
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EXPERIMENTAL PROCEDURES |
Materials--
Enzymes used for molecular biological procedures
were from New England Biolabs (supplied by Biotrade, Vienna). The TA
cloning kit from Invitrogen was from the same vendor. The Sequenase kit was obtained from Amersham Pharmacia Biotech (supplied by MedPro, Vienna). Oligonucleotides were synthesized by the Institute of Microbiology and Genetics at the Biocenter of the University of Vienna.
Glutathione-agarose beads, thrombin, and all other chemicals were from
Sigma. BH4 was from Alexis Biochemicals (Switzerland); 7,8-dihydro-L-biopterin (BH2) was from Schircks
Laboratories (Switzerland); calmodulin, L-arginine, and
NOHLA were from Sigma; L-[2,3,4,5-3H]arginine
hydrochloride (57 Ci/mmol) was from Amersham Pharmacia Biotech and
purified by HPLC before use. All other reagents were purchased from
Merck except for CHAPS, which was from Boehringer Mannheim. Oxygen
(99.995% pure) was from Aga (Toulouse, France).
Preparation of Nitric-oxide Synthase--
Recombinant rat brain
nNOS was purified from baculovirus-infected insect cells as described
previously (20). The holoenzyme contained tightly bound BH4
in the ratio of 0.5 per heme. BH4-free nNOS was obtained
from Sf9 cells treated with 2,4-diamino-6-hydroxypyrimidine according to a published procedure (8). The nNOS oxygenase domain was
cloned and expressed in Escherichia coli by a procedure to
be published elsewhere. The molar concentrations of nNOS and its
oxygenase domain were determined from absorption coefficients of the
oxidized and the ferrous-CO complex according to Sono et al.
(21).
Preparation of Cytochrome P450--
Cytochrome P450 CYP2B4 from
rabbits, which had been under phenobarbital treatment for 1 week, was
prepared according to the procedure of Coon et al. (22). The
protein was kept frozen as a 69 µM solution in 10 mM potassium phosphate buffer, pH 6.8, containing 20%
glycerol and 1 mM EDTA. The purified enzyme was electrophoretically homogenous, and in the oxidized state it displayed an absorbance ratio of R417/277 = 1.06. Neither
NOS nor cytochrome P450 showed P420 characteristics.
Low Temperature Spectroscopy--
Base line-corrected absorbance
spectra in the 350-700-nm range were recorded with a Cary 3E (Varian)
spectrophotometer in the double beam mode, using a 1.5-nm slit width.
Data acquisition was in steps of 0.5 nm, with an acquisition time of
0.033-0.5 s/data point. The instrument was equipped with a home-built
double sample compartment, which allowed spectral recordings at low
temperatures (down to
50 °C). Sample and reference cuvettes were
placed in a bloc made of aluminum, which was thermostatted by
circulation of ethanol, thermoregulated in a Haake F3-Q bath. Inside
the cells, the temperature was monitored continuously with a
thermocouple connected to an AOIP voltmeter. For thermal insulation,
the sample compartment was surrounded by polyvinyl chloride walls,
equipped with double quartz windows. Formation of ice and condensation of water on the windows was prevented by a flow of dry nitrogen.
Formation of Intermediate Oxygen Complexes--
To prevent
freezing of the sample, the experiments were done in mixed organic
solvents. If not specified otherwise, we used ethylene glycol/water
(1:1, v/v). This solvent did not change significantly the spectral
properties of NOS, it did not induce a transition to the P420 state,
and in its presence the enzyme was still active in a standard assay
(23). The buffer was 50 mM potassium phosphate, pH 7.2, containing 1 mM CHAPS, 0.5 mM EDTA, and 1 mM 2-mercaptoethanol. It contained no calmodulin, and,
unless specified, no BH4 was added. For CYP2B4, the solvent was 50% glycerol, and the buffer was sodium phosphate, 50 mM, pH 7.4. The pH of these mixed solvents is known to vary
only little as a function of temperature (17). The sodium dithionite
stock solution (23 mM) was prepared in the same solvent.
Prior to use, argon was bubbled through the solutions for 30 min. The
enzymes were diluted in the oxygen-free buffers at final concentrations of 3 µM in Teflon closed cuvettes in total volumes of 2 ml. They were reduced at 15 °C by the addition of 20 µl of a
concentrated solution of sodium dithionite (final concentration, 230 µM) using a Hamilton syringe. When reduction was
complete, the temperature was decreased to the desired value (typically
30 °C). 2-5 ml of precooled oxygen were then bubbled in the
enzyme solution with a syringe. This procedure took about 5 s, and
thereafter the spectra were recorded in intervals of 2 min.
Product Analysis--
Reduction and oxygenation of NOS and its
heme domain were carried out as described above, except for the
presence of 500,000 cpm of 200 µM
L-[2,3,4,5-3H]arginine. After incubation for
5-60 min at the specified temperature, the reaction was stopped by the
addition of 75 µl HCl (1 M) to the reaction mixture (1 ml). After centrifugation with a bench-top centrifuge, 100 µl of the
supernatant were injected into a 25-cm Nucleosil SA 100-5 HPLC column
from Macherey & Nagel. The arginine derivatives were separated at room
temperature in 50 mM sodium acetate, pH 6.5, at a flow rate
of 1.5 ml/min. Fractions of 750 µl were collected for 30 min, and
radioactivity was determined in each fraction by liquid scintillation
counting. NOHLA was identified by comparison with an authentic standard
(24). Its formation was quantified from its integrated elution peak
relative to the total radioactivity. In one control experiment, we used
NADPH and not dithionite to reduce nNOS. In that case, the phosphate buffer was replaced by triethanolamine, 50 mM, pH 7.4, and
a procedure similar to that described by Abu-Soud et al.
(25) was used. First, the heme iron of nNOS was reduced by 200 µM NADPH in the presence of 14 µM
calmodulin and 1 mM calcium chloride in aqueous buffer at
15 °C under the experimental conditions we had employed for
reduction by dithionite. After dissociation of calmodulin by excess
EDTA (1.8 mM final concentration), 200 µM
radiolabeled L-arginine, was added, and oxygen was bubbled
through the solution. The production of NOHLA was then quantified as
described above.
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RESULTS |
Oxygen Binding to Reduced nNOS in the Absence of
Substrate--
Under anaerobic conditions, 230 µM
dithionite reduced the NOS sample within 10-15 min at 15 °C.
Spectrally, the reduction was characterized by a loss of the flavin
absorbance, a shift of the Soret band from 400 to 410 nm, a shoulder at
455 nm, and a maximum at 554 nm. During the subsequent temperature
decrease to
30 °C, the spectrum of Fe(II) nNOS remained
essentially the same, except for a slight sharpening of the Soret band.
After the addition of oxygen at
30 °C, the spectrum changed within 1 min. As shown in Fig. 1, the amplitude
of the Soret band was slightly decreased, and its maximum was
red-shifted to 416.5 nm. In the visible region, the main effect was a
broadening of the absorption band. As shown in Fig.
2A, the oxygenated minus
reduced difference spectrum showed a strong maximum at 426 nm, two
sharp minima at 401 and 458 nm, and an isosbestic point at 415 nm.
Furthermore, two broad maxima appeared around 500 and 600 nm, as well
as a minimum at about 550 nm.

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Fig. 1.
Spectral changes of reduced NOS
(Fe2+) after the addition of oxygen at 30 °C.
Reduced full-length nNOS (3 µM) was cooled to 30 °C,
and then precooled oxygen was bubbled through the solution. The buffer,
50 mM potassium phosphate, pH 7.2, contained 1 mM CHAPS, 0.5 mM EDTA, 1 mM
2-mercaptoethanol, and 50% ethylene glycol. Spectra immediately after
oxygen addition (Fe2+ +
O2) and 45 min later (reoxidized) are
shown.
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Fig. 2.
Comparison with the oxyferrous complex of
cytochrome P450. The P450 LM2 (···) difference spectra were
normalized with respect to those of nNOS ( ). A,
formation of the oxygen complex; difference spectra between the oxygen
complex minus the reduced state. The experimental conditions of nNOS
were those of Fig. 1. For P450, 2 µM, the buffer was
sodium phosphate, 50 mM, pH 7.4, containing 50% glycerol.
B, decomposition of the oxygen complex; difference spectra
in the course of reoxidation relative to the oxygen complex, 17 min
(NOS) and 25 min (P450) after addition of oxygen.
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At
30 °C, the slow decomposition of the oxygen complex led to an
oxidized heme with reduced flavins (Fig. 1). As shown in Fig.
2A, the oxidized minus oxygen complex difference spectrum grew with maxima at 387.5, 418.5, 516.5, and 650 nm, minima at 442.5 and 558 nm, and an isosbestic point at 427 nm. The observed changes and
the resulting spectrum are indicative of a mixture of high and low spin
ferriheme but with a much higher fraction in the low spin state than
before the experiment at 15 °C. Finally, when the temperature was
raised above
20 °C, the flavins became also oxidized, as evidenced
by a broad absorbance increase between 350 and 420, and between 470 and
550 nm (not shown). The latter reaction is probably due to a direct
(nonphysiological) reaction of the reduced flavins with oxygen or with
hydrogen peroxide resulting from the reaction of oxygen with
dithionite.
As shown in Fig. 2, the spectral characteristics of formation and
decomposition of the oxygen complex were similar to those observed with
cytochrome P450. An exception was the trough at 458 nm in the
difference spectrum (intermediate
reduced state, Fig.
2A), which was absent in P450. However, a comparison of the spectra of the reduced states shows that this spectral trough originates from the properties of the reduced state of NOS rather than
from its oxygen complex. Indeed, the spectrum of reduced NOS, but not
reduced P450, exhibits a shoulder at this wavelength. The differences
observed during the autoxidation of the oxygen complex as compared with
the analogous reaction in P450 (Fig. 2B), i.e.
additional absorbance increases at 387.5, 516.5, and 650 nm, can be
ascribed to the formation of a significant fraction high spin
ferriheme.
The kinetics of oxygen binding and autoxidation were followed at 442.5 nm. Oxygen binding to reduced NOS was very rapid even at
30 °C.
Subsequent oxidation of the heme was monoexponential (
= 6.5 min at
30 °C). Oxidation of the flavins could only be observed at
considerably higher temperatures (
= 15.6 min at
11 °C).
Oxygen Binding in the Presence of Substrate--
The addition of
L-arginine (200 µM) resulted in the well
known shift of the heme iron spin equilibrium toward more high spin. This effect persisted in the presence of ethylene glycol and at low
temperatures. Reduction gave rise to a spectrum very similar to that of
substrate-free reduced nNOS (Fig.
3A). In this case, too, mixing
of reduced nNOS with oxygen at
30 °C produced a detectable intermediate, but its Soret band was blue-shifted (
max = 404.5 nm) (Fig. 3B). In the visible region, the intermediate
spectrum was similar to that obtained in the absence of
L-arginine. At
30 °C, its decay kinetics resulted
within 37 min in an oxidized type high spin enzyme. The difference
spectra reflecting formation (intermediate
reduced state, Fig.
3C) and decomposition (reoxidized
intermediate) were
strikingly different from those obtained in the absence of
L-arginine (see above). The formation was characterized by
a strong maximum at 375 nm and a double minimum at 417 and 453 nm.
During the decomposition of the intermediate, a broad maximum appeared
at 370 nm, with a shoulder at 420 nm and a minimum at 444 nm.

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Fig. 3.
The oxygen complex in the presence of
L-arginine. A, reduced states of nNOS and P450
LM2 at 30 °C. nNOS in the absence ( ) and in the presence (- - -) of 200 µM L-arginine, P450 LM2
(···). B, spectral evolution of nNOS in the presence
of L-arginine after the addition of oxygen at 30 °C:
reduced state (Fe2+), immediately after oxygen
addition (intermediate), and 37 min later
(reoxidized). C, difference spectra of NOS
reflecting formation (intermediate Fe2+) and
reoxidation (reoxidized intermediate) of the intermediate. The
experimental conditions were those of Fig. 1.
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BH4-free NOS--
In the absence of
L-arginine, binding of oxygen to BH4-free NOS
at
30 °C resulted in similar spectral changes as those observed with BH4-bound NOS (cf. "Discussion" and
Table II); the autoxidation of the oxygen complex (
max = 415 nm), resulted in low spin ferric heme. In contrast, the presence of
L-arginine did not induce the blue-shifted oxygen complex
observed in BH4-bound NOS, and the spectral characteristics
of the oxygen complex were close to those observed in the
BH4-free NOS in the absence of L-arginine. As for the BH4-bound NOS in the presence of
L-arginine, the autoxidation of this complex resulted in
high spin ferric heme.
The Oxygenase Domain--
The spectral changes upon oxygen
addition to the reduced oxygenase domain were recorded at
30 °C as
described above, in the absence and presence of BH4 (20 µM) and L-arginine (200 µM). In all cases, a direct transition from the reduced to the oxidized state
was observed, and no intermediate state could be detected. Whereas the
cofactors did not significantly affect the spectrum of the reduced
state, the final oxidized state was primarily low spin in the absence
of substrate and high spin in the presence of L-arginine
and/or BH4. As an example, the spectral changes in the
presence of BH4 and L-arginine are shown in
Fig. 4. Clear isosbestic points were
observed at 406, 483, 534, and 595 nm. At
30 °C, the oxidation
kinetics were relatively slow (
= 12.4 min) without being affected
noticeably by L-arginine and/or BH4.

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Fig. 4.
Time-dependent spectral evolution
of reduced nNOS oxygenase domain, 4.5 µM, after the
addition of oxygen at 30 °C in the presence of 200 µM L-arginine and 20 µM
BH4. Other experimental conditions were those of Fig.
1. The reaction times are indicated in the figure. The blue shift of
the Soret band is indicated by an arrow.
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Oxygen Complex Decomposition Products--
One explanation for the
blue-shifted oxygen complex of BH4-bound full-length NOS in
the presence of L-arginine might be that it represents a
later intermediate in the activation of oxygen, one electron reduced
beyond the ferrous oxygenated species. Since such a state could in
principal furnish the two electrons required for the oxidation of
L-arginine to NOHLA and the concomitant reduction of oxygen
to water, we decided to analyze the decomposition products of the
reaction with radiolabeled L-arginine. Fig.
5 shows a typical HPLC profile of the
reaction products. Only one radioactive reaction product was formed,
and this had the same retention time as authentic NOHLA. Control
experiments in the absence of enzyme with or without added
BH4 showed no product formation, and no NOHLA was detected unless the enzyme was reduced prior to the addition of oxygen. As shown
in Table I, NOHLA formation was
proportional to the NOS concentration in a stoichiometry between 0.3 and 0.5 per nNOS heme. NOHLA formation took place in both aqueous and
mixed organic solvent. Moreover, the production of NOHLA was not
specific to the presence of dithionite as the source of the first
electron. When dithionite was replaced by NADPH (see "Experimental
Procedures"), the yield of NOHLA production in aqueous buffer was
0.47 ± 0.03 per heme, which is the same value as that obtained
with an initial reduction by dithionite. It must be mentioned here that
under similar conditions (reduction with NADPH, but with excess
BH4) Abu-Soud et al. (25) did not detect the
formation of NOHLA. The reason for this difference is not clear.
However, we are confident in our result; the experiment was carried out
under argon atmosphere, and the result was reproducible.

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Fig. 5.
Effect of BH4 on NOHLA formation
after the addition of oxygen to reduced nNOS oxygenase domain, 4.5 µM, in the presence of 200 µM
L-[3H]arginine. The buffer composition
was that used in Fig. 1. After incubation at 15 °C for 5 min in the
absence (full circles) and presence (open
circles) of 20 µM BH4, the reaction was
stopped by the addition of HCl (see "Experimental Procedures").
Radiolabeled L-arginine derivatives were separated by
cation exchange HPLC, and fractions were analyzed by liquid
scintillation counting (right scale). Left scale,
background subtracted profiles; the background was the HPLC profile of
L-[3H]arginine.
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Table I
Formation of NOHLA as the decomposition product of oxygenated reduced
nNOS or its oxygenase domain
Full-length nNOS contains BH4 in a stoichiometry of
approximately one per nNOS dimer. If not otherwise stated, the buffer
contained 50% ethylene glycol; for experimental conditions see
"Experimental Procedures."
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As shown in Table I, the oxygenase domain alone was sufficient for this
reaction. The presence of BH4, however, was essential. The
oxidation state of the biopterin is also important; with the oxygenase
domain, no NOHLA was produced when BH4 was replaced by
BH2. The low residual NOHLA production in the full-length
holoenzyme in the presence of BH2 is due to endogenous
BH4, which is not readily displaced by added
BH2 (26).
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DISCUSSION |
The Oxygen Complex of Reduced nNOS in the Absence of
Substrate--
By the use of subzero temperature spectroscopy, it was
possible to detect an intermediate complex between reduced nNOS and molecular oxygen. This technique was employed previously to trap the
analogous complex of various cytochrome P450 forms. The spectral characteristics of the complexes of both enzyme classes are quite similar, and the differences observed during complex formation (a
trough at 458 nm) are due to a spectral particularity of deoxyferrous nNOS. The shoulder at 458 nm persists in reduced nNOS in the absence of
the reductase domain and is not affected by BH4. The
shoulder, which was noted before by others (21), may be due to the
presence of a sixth ligand in a fraction of the enzyme. The additional absorbance increase at 380-390 nm suggests a hyperporphyrin spectrum, as has been reported for many ferrous complexes of cytochrome P450 (27,
28). The identity of the putative ligand cannot be established on the
basis of the spectral changes, since most exogenous ligands other than
O2 induce a red shift upon binding to ferrous P450 type
heme.
The spectral similarity of nNOS and P450 oxygen complexes suggests that
both complexes have a similar electronic configuration. Most
evidence in P450 points to a structure of
Fe(III)O2·, and this configuration is also expected
for NOS (29). Since the same intermediate spectrum was obtained also in
the absence of BH4, it may be concluded that, in
substrate-free nNOS, BH4 does not perturb the electronic
structure of the oxygen complex.
Stability of the Oxygen Complex--
The oxygen complex of nNOS is
rather unstable, and it was necessary to lower the temperature to
30 °C to prevent its immediate autoxidation. In that aspect, nNOS
resembles microsomal cytochrome P450 (18). The oxygen complex of
mitochondrial P450scc is more stable (several hours at
30 °C (19)). The most stable one, the complex of soluble
P450cam, can be detected without difficulty above 0 °C
(at 25 °C,
= 90 s (14)). It appears thus that the protein
environment plays a significant role in the stability of the oxygen
complex. This idea is further supported by the fact that with the nNOS
oxygenase domain the complex could not be detected, indicating that in
this case autoxidation of the complex was much faster than its
formation. Since reoxidation of the oxygenase domain was actually
slower than that of the full-length enzyme, formation of the
oxyferrocomplex must be considerably slower for the oxygenase domain.
The origin of the effect remains to be established. One possibility
might be that, in the full-length enzyme, the reductase domain enhances
the oxygen association rate. Alternatively, the differences may somehow
originate from the different expression systems used, since the
full-length enzyme and the oxygenase domain were obtained from
baculovirus-infected cells and from overexpression in E. coli, respectively.
The Oxygen Complex in the Presence of L-Arginine and
BH4--
The spectral shift of the nNOS oxygen complex in
the presence of L-arginine and BH4 may be
interpreted in two ways. One possibility is that the substrate and
BH4 interact strongly with the
Fe(III)O2· complex and modify sufficiently the
energy of its electronic orbitals to induce the blue shift. Examples
for an effect of substrates on the Fe(III)O2·
complex are found in cytochrome P450scc, which interacts
with cholesterol, (22R)-hydroxycholesterol and
(20R,22R)-dihydroxycholesterol. These substrates
induce significant changes of the ferric spin state and thus of the
P450 Soret absorbance spectrum. However, the
max values
of the respective oxyferrous complexes range between 423 and 416 nm,
while that of the complex in the absence of substrate lies at 420 nm
(31), so that the substrate-induced shift never exceeds 4 nm. In
contrast, the nNOS oxygen complex in the presence of substrate is
blue-shifted by 12 nm.
Alternatively, the blue-shifted intermediate spectrum in nNOS may
reflect a further intermediate state (reduced by a second electron) in
the course of the activation of oxygen. The observation that the
decomposition of this enzyme state yields NOHLA, the formation of which
requires two electrons, provides strong supporting evidence in favor of
this possibility. Similarly, with cytochrome P450, product formation is
only possible if the oxygen complex is reduced by a second electron.
Possible configurations of such a reduced oxygen complex are shown in
Fig. 6. They comprise a peroxo- (or
hydroperoxo-) Fe(III) (2), an oxyferryl porphyrin
-cation
(3), or a hydroxyferryl complex (4). Forms
2 and 3 have been characterized for different
cytochromes P450 and peroxidases. An oxyferrous complex reduced by one
electron maintaining an intact dioxygen bond was recently reported for
the D251N mutant of P450cam (32, 33), which showed a
red-shifted
max of the Soret band (see Table
II). In contrast, compound I of
horseradish peroxidase (3), is characterized by a
blue-shifted maximum (34). A similar blue-shifted intermediate (405 nm), reflecting form 3, was also reported for cytochrome
P450scc as an intermediate compound in the reaction with
(22R)-hydroperoxycholesterol, and for cytochrome
P450cam as an intermediate in the reaction of the
oxyferrous complex with reduced putidaredoxin (35-37). It thus appears
that, relative to the oxyferrous form 1, form 2 is red-shifted, and form 3 is blue-shifted. Our
interpretation of the blue-shifted NOS intermediate is therefore that
it reflects form 3. Additional support for this hypothesis
comes from the very recently determined crystal structure of inducible
nitric-oxide synthase (38), which suggests a stabilization of form
3 by a stacking of aromatic residues with the heme. But
clearly, the definite assignment of its electronic structure awaits
further investigation.

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Fig. 6.
Proposed mechanism of
BH4-dependent reductive activation of
oxyferrous NOS leading to the formation of NOHLA. The
numbers correspond to the intermediates cited in the text.
1 is observed in the absence of BH4;
3 is the probable structure observed in the presence of both
L-arginine and BH4. The dashed line
shows the uncoupled pathway in the absence of BH4.
P+ indicates porphyrin -cation radical.
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The Role of BH4 in the Reductive Activation of the
Oxygen Complex--
Our results clearly show that the decomposition of
the oxygen complex of nNOS in the presence of both
L-arginine and BH4 results in the formation of
NOHLA. This reaction requires that the
Fe(III)O2·-L-arginine complex be
reduced by one electron to
Fe(III)O2
-L-arginine, which would then
decompose in several steps to Fe(III) and NOHLA. But where does the
second electron, necessary to reduce Fe(III)O2·-L-arginine stem from? For
several reasons, it is highly unlikely that it originates from
dithionite. (i) For the related cytochrome P450 system, the second
electron is exclusively provided by a specific reductase, the analogous
counterpart of the reductase domain in NOS. (ii) Replacement of
dithionite by NADPH did not affect the yield of NOHLA formation. (iii)
Reduction of nNOS by dithionite was much slower than the formation of
NOHLA. In control experiments, we measured the reduction rate between 5 and 30 °C (data not shown) and determined an activation energy of
Ea = 115 kJ/mol. Extrapolation of the linear
Arrhenius plot to
30 °C yielded a prohibitively slow reduction
rate (
= 2 weeks), whereas NOHLA was formed in less than 5 min at
the same temperature.
The fact that we performed our experiments in the absence of
calmodulin, essential for electron transfer from the flavins to the
heme (9) and that NOHLA formation was also observed with the isolated
oxygenase domain rules out the flavins as the electron source.
Theoretically, the two hemes within one dimer might be able to provide
the electrons to account for the observed NOHLA-to-heme stoichiometry.
However, since most evidence suggests that the hemes operate
independently (30, 39) and that electron transfer between the hemes
does not occur (40), this option is unlikely. This leaves
BH4 as the prime candidate for the donor of the second
electron, in perfect agreement with the observation that NOHLA
formation does not take place in its absence.
The present results are therefore relevant with regard to the role of
BH4 in catalysis. It has long been suspected that the redox
properties of BH4 are crucial to its function, although direct evidence for its participation in the redox process is still
lacking (reviewed by Mayer and Werner (41) and Hemmens and Mayer (42)).
The best evidence to date in favor of a redox-active role is the
observation that redox-inactive BH4 analogues can mimic the
effects of BH4 on NOS dimerization, substrate affinity, and
heme spin state but fail to sustain L-citrulline production (26, 43-45). However, little information is available on the question
of at which stage the putative BH4 oxidation takes place and whether it occurs in the reaction with L-arginine or
with NOHLA or in both steps. Concerning the latter question, it was reported that oxidized BH4-containing NOS can catalyze the
conversion of substoichiometric concentrations of
L-arginine into NOHLA, whereas further conversion to
L-citrulline was strictly NADPH-dependent, suggesting that BH4 may serve as an electron source for the
first but not for the second step (46). However, in the present
experiments, no NOHLA was formed unless the enzyme was previously
reduced. The cause for the divergent results is unclear. A possible
explanation might be that the enzyme preparation of Campos et
al. (46) contained traces of reductant, e.g. reduced
flavins.
The present results demonstrate that BH4 is absolutely
required for L-arginine oxidation. If our assignment of the
blue-shifted intermediate as the oxyferryl complex (3) is
correct, the observation that its formation is only observed in the
presence of BH4 implies that BH4 is required
for oxygen activation. The simplest explanation and the one favored by
us is that BH4 furnishes the electron needed to reduce
Fe(III)O2· (1) to Fe(III)-O-OH
(2), which immediately transforms then to the Fe(IV)=O
porphyrin
-cation radical (3). Species 3 could
then react with bound L-arginine via hydrogen abstraction to form species 4, the decomposition of which would liberate NOHLA and Fe(III). In the absence of BH4, the uncoupled
reaction (47, 48) will occur, and compound 1 will dissociate to Fe(III) and O
2 (Fig. 6). In this way, our model also
explains why the redox activity of BH4 is crucial for coupling NADPH
oxidation to NO formation, although it is not required for
O2 reduction per se. Since only one electron is
transferred in this reaction, we denote the resulting state of the
biopterin formally as BH3. However, we cannot exclude the
possibility that the state that is actually formed is the
quinonoid-BH2 (6,7-dihydro-L-biopterin), which
is the usual product in enzymatic oxidations of BH4 (41, 49) and which can be reduced to BH4 by NOS (50).
The NOHLA-to-heme ratios of
0.5 suggest that in nNOS as isolated,
which contains approximately one equivalent of BH4 per dimer, only the BH4-containing subunit produces NOHLA. This
confirms that electron transfer between the two subunits in a NOS dimer does not take place (40) and agrees with the role postulated by us for
BH4 in catalysis. Similarly, in experiments with the oxygenase domain, where BH4 was added (20 µM), the NOHLA-to-heme ratio did not reach unity,
although it was a little higher (0.52 instead of 0.36 under the same
buffer conditions). This may be explained by an incomplete binding of
BH4, which could result from a relatively high
Kd of the second BH4 binding site and an
increase of Kd due to the presence of ethylene glycol. The latter idea is supported by our observation that with full-length nNOS the ratio is somewhat decreased in the presence of
ethylene glycol.
Conclusion--
The observations presented in this study provide
important clues to the role BH4 plays in NOS catalysis. The
hypothesis that electron transfer from BH4 is required for
oxygen activation is the simplest explanation of our results. It does,
however, need further corroboration, and it remains also to be
established if the same holds true for the second step in NO catalysis,
the oxidation of NOHLA to L-citrulline.