(Received for publication, March 14, 1995; and in revised form, April 21, 1995)
From the
A gene coding for rat neuronal nitric oxide synthase (nNOS) has
been cloned into pCWori and the vector has been expressed in Escherichia coli. The expressed enzyme has been purified with
a final yield of purified protein of approximately 1 mg/g of wet cells.
The recombinant protein reconstituted with calmodulin and
Ca Nitric oxide (NO) functions physiologically as a
neurotransmitter and vasodilator and pathologically as a cytotoxic
agent. Nitric oxide is produced by nitric oxide synthases (EC
1.14.13.39, NOS)
Figure 1:
Steps in the NOS-catalyzed oxidation of
arginine to nitric oxide.
Neuronal nitric oxide
synthase (nNOS) is one of the constitutive, calmodulin-regulated forms
of the enzyme. In the absence of calmodulin, limited proteolysis
cleaves the protein into heme and flavin domains that retain partial
structural and functional integrity(6) . The flavoprotein
domain contains one FAD and one FMN as prosthetic groups and has
extensive sequence identity with cytochrome P450 reductase(7) .
The absorption and resonance Raman spectra of the heme
domain(8, 9, 10) indicate that the heme
group is coordinated, as in cytochrome P450, to a cysteine
thiolate(8, 9, 10, 11) .
Site-specific mutagenesis of the cysteine residues proposed to be the
iron ligands in endothelial (Cys Despite the differences in their
sequences, the presence of a thiolate iron ligand and the analogy
between the catalytic chemistries of cytochrome P450 and NOS suggest
that approaches used to characterize the P450 monooxygenases may be
useful in defining the structure and function of NOS. Recent work from
this laboratory has established that myoglobin, catalase, and
cytochrome P450 react with phenyldiazene (PhN=NH), or its
precursor phenylhydrazine (PhNHNH
Figure 2:
Scheme illustrating the reaction of
phenyldiazene with the prosthetic group of a hemoprotein to give a
phenyl-iron complex from which the phenyl migrates to the porphyrin
nitrogen atoms. The reagent for stepa is
PhN=NH and for step b is
K
We report here the first successful expression of catalytically
active nNOS in Escherichia coli. The recombinant enzyme has
been used to demonstrate that nNOS is inactivated by reaction with
phenyldiazene in a reaction that produces a phenyl-iron complex. We
have also established that ferricyanide promotes migration of the
phenyl group within the intact active site and have used this reaction
to explore the active site topology of nNOS.
Absorption
spectra were recorded on an Aminco DW2000 spectrophotometer and were
transferred to a Macintosh computer for analysis and display. High
pressure liquid chromatography was performed on a Hewlett Packard 1090
HPLC system.
Cloning of an nNOS gene into pCWori,
transformation of E. coli DH5
Figure 3:
SDS-PAGE analysis of the purified nNOS
isolated from E. coli: lane A, molecular weight
standards; lane B, crude cell lysate; lane C, sample
after calmodulin-Sepharose column; lane D, final sample after
the 2`,5`-ADP- Sepharose column. The gel has been stained with
Coomassie Blue. The full-length nNOS and hydrolytic fragments are
labeled as 1 and 2, respectively. Western blots indicate both bands
react with polyclonal antibodies to nNOS.
Figure 4:
Absorbance spectra of recombinant nNOS in
the presence (-) and absence(- - - -) of L-arginine. The spectrum of the reduced, CO-bound
protein minus the reduced protein is shown in the inset. The
samples are in 50 mM HEPES (pH 7.5) containing 10 µM BH
The
recombinant protein is active with a V
Figure 5:
Typical Michaelis-Menton plot of the
activity of nNOS with increasing concentrations of L-arginine. The inset is a Hanes plot of the
data with the axes [L-arginine]/
Figure 6:
Time dependence of the formation of the
iron-phenyl complex and the inhibition of NOS by phenyldiazene. The
formation of the phenyl-iron complex (
Figure 7:
Effect of phenyldiazene and calmodulin on
the rates of L-citrulline formation and cytochrome c reduction by nNOS. The first three bars are for citrulline
formation at 25 °C (in nmol/min/mg protein): in the absence of CaM (A), in the presence of 10 µg/ml CaM (B), and in
the presence of 10 µg/ml CaM after preincubation with phenyldiazene (C). The absolute value for the bar in lane B is 125 nmol
Figure 8:
A typical HPLC trace of the separation of
the four N-phenylprotoporphyrin IX regioisomers obtained from
phenyldiazene-treated nNOS. The ratio of the
N
Expression in E. coli of catalytically competent
nNOS makes the enzyme more readily available in large amounts and more
accessible to site-specific mutagenesis than it currently is. This
should facilitate elucidation of the detailed structure and mechanism
of NOS. The recombinant nNOS obtained from E. coli has the
same ferric and ferrous CO-complexed spectra as the enzyme from rat
brain (Fig. 4). Furthermore, although the enzyme expressed in E. coli is free of calmodulin, reconstitution of the enzyme
with exogenous calmodulin yields a protein with the same dependence on
Ca Several classes of NOS inhibitors, in addition to NO
itself (12, 33) , are currently known. Arginine
analogues such as N As found
previously for cytochrome P450, the phenyl group of the nNOS
phenyl-iron complex shifts to the porphyrin nitrogens when the complex
is oxidized in situ with ferricyanide. This provides
independent support for the contention that a cysteine thiolate is
ligated to the iron because thiolate ligation is required for phenyl
group migration within the intact active
site(15, 18, 19, 20, 21, 23) . The ratio of the four N-phenylprotoporphyrin IX isomers
derived from the N-phenyl heme group extracted from nNOS is
N The
formation of all four N-phenylprotoporphyrin IX isomers with
nNOS differs from the pattern observed with most P450 enzymes, although
there is limited similarity to the pattern
(N In sum, the structural and
mechanistic links between nitric oxide synthases and cytochromes P450
are strengthened by the present demonstration that nNOS, like
cytochrome P450, is (a) inactivated by phenyldiazene via
formation of a phenyl-iron complex, (b) undergoes
ferricyanide-mediated migration of the phenyl from the phenyl-iron
complex to the porphyrin nitrogens within the intact active site, and (c) has a P450-like active site topology.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
exhibits spectroscopic and catalytic properties
identical to those reported in the literature for nNOS. Reaction of
recombinant nNOS with phenyldiazene produces a phenyl-iron (Fe
Ph)
complex with a maximum at 470 nm. Formation of this complex is
paralleled by inactivation of the enzyme and is inhibited by arginine,
the natural substrate of the enzyme. Phenyl-iron complex formation does
not alter the rate of electron transfer from the flavin domain to
cytochrome c. Addition of ferricyanide triggers migration of
the phenyl residue from the iron to the porphyrin nitrogens. The N-phenylprotoporphyrin isomers with the phenyl on the
nitrogens of pyrrole rings B, A, C, and D are formed in, respectively,
approximately a 14:20:21:45 ratio. The regioisomer pattern indicates
that the active site of NOS is open to some extent above all four
pyrrole rings but more so above pyrrole ring D. Arylhydrazines are thus
not only a new class of inhibitors of nNOS but provide useful
information on the active site topology of the enzyme.
(
)that catalyze the conversion
of L-arginine to nitric oxide and citrulline (Fig. 1)(1, 2, 3, 4, 5) .
Two general categories of NOS are known: (a) constitutive
enzymes that are regulated by Ca
and calmodulin, and (b) inducible enzymes with a tightly bound calmodulin unit
that are not regulated by Ca
. Both types of NOS
consist of a heme and a two-flavin domain linked by a polypeptide that
serves as the binding site for calmodulin.
->Ala) and neuronal
(Cys
->His) NOS produces inactive
proteins(12, 13) . Sequence comparisons with the known
cytochrome P450 enzymes, however, reveal no identifiable sequence
identity with the exception of 4 or 5 residues within a decapeptide
surrounding the proposed cysteine iron ligand
(F/W-X-X-A/G-X-R-X-C-hydrophobic
aliphatic-G)(14) . The flavin domain is thus closely related to
cytochrome P450 reductase, but the heme domain is virtually unrelated
to cytochrome P450. Nevertheless, the catalytic chemistry of NOS,
involving hydroxylation of L-arginine to N-hydroxyl-L-arginine followed by oxidation
of this intermediate to nitric oxide and citrulline, is reminiscent of
P450-catalyzed transformations.
), to give stable
-bonded phenyl-iron complexes (Fig. 2) (15, 16, 17, 18) . Formation of
these complexes results in loss of hemoprotein function. The structures
of the phenyl-iron complexes obtained with myoglobin and cytochrome
P450
have been confirmed by x-ray
crystallography(16, 17) . Oxidation of the P450
phenyl-iron complexes with ferricyanide results in migration of the
phenyl group from the iron to the heme porphyrin nitrogens within the
intact protein active site(19, 20, 21) . The
four nitrogen atoms of the porphyrin are distinguishable due to the
substitution pattern at the heme periphery (Fig. 2). The extent
to which the phenyl group migrates toward each of the pyrrole nitrogens
can therefore be determined by HPLC analysis of the N-phenylporphyrins derived from the N-phenyl
hemes(22) . As the shift occurs within the intact active site,
the N-phenylporphyrin ratio provides information on the
location, with respect to a grid defined by the heme group, of active
site residues that interfere with migration of the phenyl group.
Although phenyl-iron complexes are obtained with various hemoproteins,
migration of the phenyl group within the intact active site has only
been observed with thiolate-ligated hemoproteins (i.e. cytochrome P450 and
chloroperoxidase)(15, 18, 19, 20, 21, 23) .
Fe(CN)
. The structure of the N
regioisomer of N-phenylprotoporphyrin IX obtained by
removal of the iron from the corresponding N-phenylheme is
shown (V = CH=CH
, p = CH
CH
CO
H). The
N
, N
, and N
regioisomers bear the
phenyl group on the corresponding pyrrole nitrogen
atoms.
Materials
L-Arginine and L-citrulline were obtained from Aldrich. 2`,5`-ADP-Sepharose,
calmodulin-Sepharose 4B, and Phast System products were from Pharmacia
LKB Biotechnol. Calmodulin, (6R)-5,6,7,8-tetrahydrobiopterin
and N-hydroxy-L-arginine were from Alexis
Biochemicals (San Diego, CA). Bradford protein assay kits, Chelex-100,
and Dowex 50W-X8 were from Bio-Rad. BioScint scintillation mixture was
from Fisher. Restriction enzymes, Bacto yeast extract, IPTG, and
DH5
E. coli cells were purchased from Life Technologies,
Inc.. L-[2,3,4,5-
H]Arginine was
purchased from Amersham Corp. Methylphenyldiazenecarboxylate azo ester
was purchased from Research Organics, Inc. (Cleveland, OH). All other
reagents and materials were from Sigma. The cDNA clone for rat brain
NOS was a gift from Solomon Snyder (Johns Hopkins University) (6) . The pCWori vector was a gift from Robert Fletterick
(University of California, San Francisco)(24) .
Cloning
The polymerase chain reaction was used to
amplify a region of the nNOS gene between the initiator methionine and
the unique ApaI site. This allowed the removal of 348-base
pairs of non-coding sequence and introduction of a unique NdeI
site for cloning purposes. This product was ligated into the vector
pCRII (Invitrogen, San Diego), then into the vector Bluescript
(SK) containing the nNOS gene using KpnI and ApaI restriction sites. The gene was cloned into pCWori using
the NdeI and XbaI sites to give pCWnNOS that was used
to transform competent E. coli DH5
cells. The subcloned
fragment was sequenced by the method of Sanger et
al.(25) .
Expression
An overnight culture of pCWnNOS was
used to inoculate 2 liters of 2xYT media in a 2.8-liter Fernbach flask.
Casein N-Z plus hydrolyzate (Sigma) was used in place of tryptone in
the media. The cultures were grown to an OD of
0.6-0.8 and induced with 1 mM IPTG. The cultures were
then grown at 22 °C at a rotation rate of 190 revolutions/min for
16-20 h before they were harvested by centrifugation. Expression
of nNOS was checked by SDS-PAGE and Western blotting using an antibody
raised against a section of the heme-domain of nNOS.
(
)
Purification
The purification
sequence was typically carried out with the protein from 4 to 8 liters
of culture. Due to the instability of nNOS, the purification was
carried out in the presence of 10% glycerol within a period of 1 day,
and the resultant protein was quickly frozen in liquid nitrogen and
stored at -70 °C. The protein was purified using a
modification of a published protocol(26, 27) . The
cells were lysed in Buffer A (50 mM Tris-Cl (pH 8.0) buffer
containing 0.5 mML-arginine, 10 µM
BH, 5 mM 2-mercaptoethanol, 10% glycerol, and
protease inhibitors (1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 1 µM leupeptin, 1
µM pepstatin, 1 µg/ml antipain), 2 mg/ml lysozyme, 32
units/ml DNase I, and 3 units/ml RNase A). The cells were sonicated
briefly then centrifuged to sediment the cell debris. The supernatant
was brought to 2 mM CaCl
and the protein loaded
onto a 15-ml calmodulin-Sepharose column (1.8
2.5 cm)
equilibrated with Buffer A containing 2 mM CaCl
.
The column was washed with 100 ml of Buffer B (50 mM HEPES (pH
7.5) containing the same protease inhibitors as Buffer A, 2 mM CaCl
, and 0.3 M NaCl) and eluted with Buffer
B supplemented with 0.3 M NaCl and 5 mM EGTA. The
protein was then loaded onto a 5-ml 2`,5`-ADP-Sepharose column
equilibrated with Buffer B supplemented with 0.3 M NaCl. The
column was first washed with 50 ml of Buffer B containing 0.5 M NaCl and then with 50 ml of Buffer B minus L-arginine.
Neuronal NOS was eluted with 10 ml of Buffer B minus L-arginine plus 10 mM NADPH. The NADPH was removed by
passing the protein over a small calmodulin affinity column. The
protein was concentrated (if necessary) to a concentration of greater
than 1 mg/ml, aliquoted into small vials, and quickly frozen in liquid
nitrogen. The enzyme was stored at -70 °C. Multiple
freeze-thaw cycles were avoided in using the protein.
Enzyme Assays
The activities of the protein
preparations were determined by measuring either the production of NO
using the conversion of HbO to met-Hb or by measuring the
conversion of L-[
H]arginine to L-[
H]citrulline(27) . The
conditions of the NO assay were 50 mM HEPES (pH 7.5), 10
µM BH
, 100 µM DTT, 100 µM EDTA, 2 mM CaCl
, 10 µg/ml calmodulin,
5-10 µM HbO
, 100 µM NADPH,
1-2 µg of nNOS, and 10 µML-arginine.
The initial rates of NO production were determined at 37 °C from
A
(
= 38 mM
) or, if catalase was
present, from
A
(
=
12 mM
)(27) . The conversion of L-arginine to L-citrulline was determined under the
following conditions: 50 mM HEPES (pH 7.5) containing 100
µM BH
, 100 µM DTT, 100 µM EDTA, 2 mM CaCl
, 10 µg/ml calmodulin, 100
µM NADPH, 0.5 µg of nNOS, 16 nML-[2,3,4,5-
H]arginine (20 µCi/ml),
and 0.5-50 µML-arginine in a final volume
of 50 µl. The assay was initiated by the addition of nNOS and
carried out at 25 °C. The reaction was quenched after 5 min by the
addition of 200 µl of ice-cold 0.1 M HEPES (pH 5.5)
containing 5 mM EGTA, and the samples were boiled for 1 min.
The samples were then passed over a 1-ml Dowex 50W-X8 column
(Na
form), and the eluents were collected in
individual scintillation vials. The column was washed two times with
double-distilled H
O, and the eluent from the first wash was
pooled with the eluent obtained during sample loading. BioScint
scintillation mixture (10 ml) was added to each sample, and the amount
of radioactivity present was determined by liquid scintillation
counting. A control sample containing either no NADPH or no nNOS was
included for background determination. The reduction of cytochrome c by nNOS was performed at 25 °C in a volume of 400 µl
using an extinction coefficient of 21 mM
cm
at 550 nm. The reaction mixture contained
50 mM HEPES (pH 7.5), 100 µM EDTA, 100 µg/ml
bovine serum albumin, 2 mM CaCl
, 4 µM FAD, 4 µM FMN, 10 µM BH
, 100
µM DTT, 500 µM NADPH, 50 µM cytochrome c, 50 units of catalase, and 1 µg of NOS
and was initiated by the addition of cytochrome c. Some assays
contained, as indicated, 10 µML-arginine and/or
10 µg/ml calmodulin.
Protein Determination
Protein concentrations were
determined by the Bradford protein assay (28) or micro-assay
(Bio-Rad) using bovine serum albumin as a standard. Concentrations of
heme-containing NOS were determined from the absorption spectrum of the
protein using the extinction coefficients = 72 mM
cm
for the ferric enzyme and
= 76 mM
cm
for the ferrous
CO complex(29) .
SDS-PAGE
Electrophoresis was carried out using a
Pharmacia Phast Gel system according to the manufacturer's
instructions. Homogenous 7.5% gels were used with SDS buffer strips and
were stained with Coomassie Blue. Western blotting was carried out
using the Phast Transfer system, and nNOS was detected using an
antibody raised against the heme domain of nNOS and an alkaline
phosphatase detection system.Formation of Iron-Aryl Complexes
To form a
phenyl-iron complex of nNOS, 1-2 µl of a stock solution of
phenyldiazene was added to either 0.5 or 1 ml of a 1 mg/ml solution of
nNOS (3-6 nmol) in 50 mM HEPES (pH 7.5) containing 10%
glycerol and 10 µM BH. In some experiments the
incubation also contained 0.5 mML-arginine. The
stock solution was made immediately prior to use by mixing 1 µl of
phenyldiazene carboxylate azo ester in 100 µl of an aqueous 1 N NaOH solution. The formation of a phenyl-iron complex was followed
spectrophotometrically by the appearance of a maximum at 470 nm and a
trough at 416 nm in a difference spectrum. When complex formation
reached a maximum, five 1-µl aliquots of a 63 mM potassium
ferricyanide solution were added to the cuvette over a period of 20 min
to shift the phenyl group of the complex to the pyrrole nitrogens. The
reaction was then added to 10 ml of an ice-cold solution of 5% sulfuric
acid in acetonitrile and left overnight at 4 °C. The N-phenylprotoporphyrin IX regioisomers were isolated by
removing the organic phase, adding 2 ml of an aqueous 5% sulfuric acid
solution, and extracting the porphyrin adducts three times with
CH
Cl
. The combined organic extracts were
concentrated to dryness on a rotary evaporator, and the residue was
taken up in 100 µl of HPLC solvent A (6:4:1 methanol/water/acetic
acid) for analysis. The products were analyzed by HPLC as described
previously(21, 30) .
Inhibition Studies with Phenyldiazene
The
phenyl-iron complex was formed as described above in a cuvette at 25
°C. At each time point a spectrum of the solution was taken, and an
aliquot of the reaction was removed and added to the reaction mixture
described above for the determination of citrulline formation. The
reaction was allowed to proceed for 5 min before quenching with the
EGTA stop mix. The samples were analyzed as described above. To
determine the effect of phenyldiazene on the kinetics of cytochrome c reduction, an aliquot of nNOS (typically 10 µg) was
placed on ice, and 1 µl of phenyldiazene solution was added. The
stock solution of phenyldiazene was prepared as above except
immediately before use it was diluted 1:50 with 50 mM HEPES
(pH 7.5) to buffer the NaOH added in preparing the solution. Aliquots
were taken at various time points and added to a reaction mixture as
described above.
Expression of nNOS in E. coli
Catalytically
active NOS has been heterologously expressed in mammalian cells and,
more recently, in insect cell
systems(9, 12, 13, 31, 32, 33, 34, 35, 36, 37) .
Although expression of the separate heme and flavin domains in E.
coli has been reported(38) , expression of an intact,
catalytically active NOS in E. coli has not been described.
The first task addressed in this study was therefore expression of nNOS
in E. coli. cells with the resulting
vector, and growth of the cells at low temperature (22 °C) resulted
in expression of functional nNOS as determined by Western blotting and
activity assays. Purification of the enzyme in the presence of a
mixture of proteolytic enzyme inhibitors was achieved by two-step
affinity chromatography, first on a calmodulin-Sepharose 4B column and
subsequently on a 2`,5`-ADP-Sepharose column (Table 1). The
procedure typically yields 1 mg of protein/g of wet cells that is
greater than 80% pure by SDS-PAGE and absorption spectroscopy. The
principal contaminants of the protein are shown by SDS-PAGE and Western
blotting to be proteolytic fragments of nNOS (Fig. 3). When
cultures were grown at temperatures above 30 °C, a large amount of
insoluble nNOS was produced, but no soluble protein was obtained. As
the incubation temperature was decreased, the total amount of nNOS
produced also decreased, but the amount of soluble nNOS increased.
Increasing the cell density at the time the cells were harvested by
increasing the rotation rate, aeration, or growth time after induction
by IPTG enhanced the degree of proteolytic digestion. For example, when
growth was allowed to continue longer than 20 h, a 70-kDa SDS-PAGE band
that cross-reacts with the nNOS antibody increased in intensity with
respect to the band for the full-length protein (see Fig. 3).
Under controlled conditions, however, the E. coli system is
capable of delivering large amounts of purified, catalytically active
nNOS. Efforts to further optimize the expression and purification
protocols are continuing.
Characterization of nNOS Expressed in E. coli
The
spectra of the purified, recombinant ferric protein in the presence and
absence of L-arginine, and of the ferrous carbon monoxide
complex of the protein, are shown in Fig. 4. In the absence of
substrate, the enzyme has an absorbance maximum at 400 nm with a
shoulder at 418 nm. Upon addition of either L-arginine or N-L-hydroxyarginine, the absorbance
maximum shifts to 394 nm with a shoulder at 370 nm, in agreement with a
shift in the equilibrium from low to high spin. Both forms have a
charge-transfer band at 634 nm. The dithionite reduced, CO-bound form
has an absorbance maximum at 444 nm. These spectral changes are the
same as those seen with native nNOS(29, 39) .
.
at 25
°C of 150-400 nmol NO min
mg
(25-65 nmol min
nmol
)
nNOS and a K
of 0.8 µM (Fig. 5). The variability in the V
correlates with the degree of proteolysis in the sample. An
apparent loss of activity is seen in incubations with high
concentrations (>10 µM) of L-arginine if the
concentration of BH
is less than 10 µM, as it
is in the oxyhemoglobin activity assay. This affect has been observed
previously and is due to product inhibition by
NO(12, 40) . In agreement with results on the native
enzyme, recombinant nNOS is completely inhibited by >5
µMN
-nitro-L-arginine and is
not inhibited by [D]-arginine. Furthermore,
the enzyme is completely dependent for activity on exogenously provided
calmodulin and CaCl
, and the rate of the reaction is
enhanced by exogenous BH
. The addition of FAD and/or FMN
does not greatly enhance the activity. The enzyme catalyses the
reduction of cytochrome c at a rate similar to that reported
for the native enzyme (6) and the rate is calmodulin-dependent:
44 µmol
min
mg
with calmodulin versus 3.9
µmol
min
mg
without.
versus [L-arginine]. Conditions are as described under
``Experimental Procedures,'' using 0.2 µg of nNOS. The
values calculated from the plot are V
=
425 nmol citrulline/min/mg nNOS and K = 0.8
µM.
Inactivation of nNOS by Phenyldiazene
The
formation of a phenyl-iron complex of nNOS can be followed
spectrophotometrically in a difference spectrum by the absorbance
increase at 470 nm and the corresponding decrease at 418 nm. Complex
formation is much faster in the absence of L-arginine: the
rate of complex formation decreases 3-fold, and the maximum degree
of complex formation is diminished, in the presence of 50-500
µML-arginine. These results are consistent with
a sterically restricted active site that is blocked by the presence of L-arginine. Monitoring the rate of formation of L-citrulline at different time points during the reaction of
nNOS with phenyldiazene shows that enzymatic activity decreases in
proportion to the extent of formation of the phenyl-iron complex (Fig. 6). As already noted, the rate of NOS-catalyzed cytochrome c reduction is greatly increased by the binding of calmodulin.
Calmodulin binding promotes reduction of the heme iron, and this
reduction is presumably blocked by the presence of inhibitors that bind
to the iron. Formation of the phenyl-iron complex does not affect
cytochrome c reduction, however (Fig. 7). This lack of
inhibition is seen in the presence or absence of calmodulin, indicating
that the cytochrome c reduction rate is unaffected by the
state of the heme iron. An inability of inhibitors of the heme site to
affect cytochrome c reduction has been reported with L-thiocitrulline, an inhibitor that coordinates to the heme
iron(41) .
) is determined by the
absorbance at 470 nm and the citrulline-forming activity (
) with
respect to the activity of the protein incubated similarly but without
phenyldiazene.
min
mg
. The
second set of four bars are for cytochrome c reduction at 25
°C (in µmol min
ng
): in
the absence of CaM (D), in the absence of CaM after
preincubation with phenyldiazene (E), in the presence of 10
µg/ml CaM (F), and in the presence of 10 µg/ml CaM
after preincubation with phenyldiazene (G). The absolute value
of the bar in lane F is 44 µmol
min
ng
. Preincubations with
phenyldiazene were for 20 min.
Active Site Topology of nNOS
The nNOS phenyl-iron
complex was directly transferred into an acidic acetonitrile solution
or was oxidized with potassium ferricyanide prior to being transferred
to the acetonitrile solution. When the protein complex was directly
added to acidic acetonitrile and the porphyrin products were isolated
and quantitated, the four possible regioisomers of N-phenylprotoporphyrin IX were obtained in equal amounts.
Previous work has shown that there is no inherent regiospecificity of
phenyl group migration when the phenyl-iron heme complex is free in
solution(22) . Isolation of the N-phenylprotoporphyrin
IX products provides clear evidence for phenyl-iron complex formation
in the reaction with phenyldiazene. In contrast to these results, the N-phenylprotoporphyrin IX isomer ratio is not equal when the
phenyl-iron protein complex is treated with ferricyanide prior to
transfer to acidic acetonitrile. Although the precise values obtained
are sensitive to the concentrations of phenyldiazene and ferricyanide
that are employed, the ratio of the N-phenylprotoporphyrin IX
regioisomers (N/N
/N
/N
in order of elution from the HPLC column) is approximately
14:20:21:45 (Fig. 8). The N
-isomer is consistently
the most abundant and the N
-isomer the least. These results
indicate that migration of the phenyl group within the intact active
site favors the nitrogen of pyrrole ring D, although there do not
appear to be gross differences in the accessibilities of the four
pyrrole ring nitrogens.
/N
/N
/N
isomers (listed
in order of elution from the HPLC column) is typically 14:20:21:45.
Isocratic analyses were carried out on a Partisil ODS-3 5 micron column
with a solvent mixture consisting of 20% solvent B (10:1
methanol/acetic acid) in solvent A (6:4:1 methanol/water/acetic acid)
at a flow rate of 1 ml/min. The detector of the HPLC system was set to
monitor the 416 versus 600 nm
difference.
and BH
, a similar calmodulin-dependent
ability to reduce cytochrome c (Fig. 7), and
essentially the same K
and V
for the oxidation of arginine as the native
enzyme (Fig. 5). Furthermore, the recombinant enzyme is
inhibited by high concentrations of arginine (presumably via the
production of excess NO) and N
-nitro-L-arginine in the same manner as
the native enzyme. The recombinant and native enzymes thus appear to be
identical.
-nitro-L-arginine and N
-methyl-L-arginine (42) , and
imidazole derivatives that coordinate to the heme
iron(43, 44) , are the two classes of agents that have
received the most attention. Limited information is available on other
classes of inhibitors, including other heterocycles(45) ,
tetrahydrobiopterin analogues(46) , and electron acceptors that
uncouple electron transfer between the flavin and heme
domains(47) . Some of the arginine analogues (e.g.N
-methyl-L-arginine) appear to be
mechanism-based irreversible inhibitors(48, 49) . As
shown here, phenyldiazene is an irreversible inhibitor of nNOS due to
reaction with the heme to form a
-bonded phenyl-iron complex (Fig. 2). Phenylhydrazine autooxidizes to phenyldiazene and
should therefore also be an irreversible inhibitor of
nNOS(50) , but the present studies were carried out with
phenyldiazene to avoid ambiguities caused by the possible sensitivity
of nNOS to H
O
formed by autooxidation of
phenylhydrazine. Irreversible inhibition of nNOS by phenyldiazene, as
suggested by the correspondence of phenyl-iron complex formation with
loss of activity (Fig. 6), is due to interference with the
catalytic action of the iron atom rather than to interference with the
electron transfer capacity of the flavin groups. This is clearly shown
by the fact that phenyl-iron complex formation does not result in loss
of the ability of the flavin groups to accept electrons from NADPH of
to transfer them to cytochrome c (Fig. 7).
/N
/N
/N
=
14:20:21:45 (Fig. 8). These results suggest an active site in
which the region directly above the heme group is more open above
pyrrole ring D than above pyrrole rings A, B, or C. The active site may
be sterically congested because arginine slows down the rate of
formation of the phenyl-iron complex, a finding that suggests that
arginine and phenyldiazene compete for common space above the heme iron
atom or within an access channel leading to the iron atom.
/N
/N
/N
=
13:67:13:07) observed with P450
(CYP102) in that all
four isomers are formed in significant amounts, including the N
isomer that is rarely observed(30) . Among P450 enzymes
examined to date, only in the case of P450
does the
phenyl group migrate significantly toward the nitrogen of pyrrole ring
B(30) . Comparison of the crystal structure of P450
with those of P450
and P450
shows
that in all three the heme group is held in the active site by the
pincer action of two helices, one located above and the other below the
heme group(51, 52) . The distal helix in all three
enzymes sits directly above pyrrole ring B of the heme, although in
P450
the helix is displaced away from the iron relative
to its position in the other two enzymes. This makes the nitrogen of
pyrrole ring B more accessible to the phenyl group than it is in other
two P450 enzymes. The observation that N
is formed as the
minor isomer in the reaction of phenyldiazene with nNOS suggests that
the heme in nNOS may be held in the active site crevice by a similar
two-helix motif. This possibility is supported by a small extent of
sequence identity between the residues surrounding the cysteine iron
ligand in NOS and P450 3A(14) .
, (6R)-5,6,7,8-tetrahydrobiopterin; heme, iron
protoporphyrin IX regardless of the oxidation or ligation state; CaM,
Ca
-dependent calmodulin; Hb, methemoglobin;
HbO
, oxyhemoglobin; PMSF, phenylmethylsulfonyl fluoride;
IPTG, isopropyl
-D-thiagalactopyranoside; DTT,
dithiothreitol; PAGE, polyacrylamide gel electrophoresis; NaPi, a
mixture of mono- and di-basic sodium phosphate buffer; HPLC, high
performance liquid chromatography; N
, N
,
N
, and N
are the isomers of N-phenylprotoporphyrin IX with the phenyl group on the
nitrogens of, respectively, pyrrole rings A, B, C, and D.
We thank Solomon Snyder (Johns Hopkins University) for
kindly providing the nNOS clone, Robert Fletterick (University of
California, San Francisco) for the pCWori vector, and Dr. Stephen Black
for initial work on subcloning of nNOS into Bluescript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.