(Received for publication, November 28, 1995; and in revised form, February 6, 1996)
From the
Bovine endothelial nitric-oxide synthase (eNOS) expressed in Escherichia coli does not have the post-translational
modifications found in the native enzyme and is free of
tetrahydrobiopterin (BH). In the presence of
BH
, eNOS has an absorption maximum at 400 nm that shifts to
395 nm when the substrate L-arginine is added. The low-spin
component of the spectrum of the BH
-free protein is
decreased by the addition of BH
without a corresponding
increase in the high-spin component. Addition of BH
decreases the low-spin population of eNOS even in the presence of
excess L-arginine. These results indicate that BH
directly modulates the heme environment. BH
-free eNOS
is completely inactive, but catalytic activity is recovered when
BH
(EC
200 nM) is added. The
spectroscopically determined binding constants for L-arginine
are
1.9 µM in the presence and
4.0 µM in the absence of BH
. The BH
-supplemented
enzyme has an activity of 90-120 nmol of
citrulline
min
mg
and K
values of 3 and 14 µM for L-arginine and N-hydroxy-L-arginine,
respectively. Of particular interest is the finding by
SDS-polyacrylamide gel electrophoresis that BH
-free eNOS
exists in a monomer-dimer equilibrium very similar to that observed
with the BH
-reconstituted protein. Addition of
BH
increases the percent of the dimer by only
5%. The
results establish that BH
influences the heme environment
and stabilizes the protein with respect to heme loss but is not
required for dimer formation.
Nitric oxide (NO), a major regulatory factor in
the immune, nervous, and cardiovascular systems, mediates vasodilation,
causes inhibition of platelet aggregation, has a role as an important
effector molecule of the host defense system, and functions in neuronal
transmission. Nitric-oxide synthases (NOS, (
)EC 1.14.13.39)
oxidize L-arginine in a process that consumes NADPH and
produces stoichiometric amounts of L-citrulline and
NO(1, 2, 3, 4, 5) .
Two general categories of NOS are known, (a) constitutive
enzymes that are regulated by Ca
and CaM and (b) inducible enzymes with a tightly bound CaM that are not
regulated by Ca
. Both types of NOS are modular
proteins in which a P-450-like heme domain is connected to a two-flavin
domain by a linker peptide that displays a consensus calmodulin-binding
sequence.
Studies carried out with bovine aortic endothelial cells
first demonstrated the presence of nitric-oxide synthase activity that
is principally associated with the particulate fraction and is both
Ca- and CaM-dependent(6) . Affinity
chromatography and gel filtration yield a purified protein that, after
denaturation, migrates as a single band on SDS-PAGE with a molecular
mass of 135 kDa(7) . As reported for the neuronal (8) and inducible (9, 10) isoforms, the
endothelium-derived enzyme also requires BH
for full
activity(7) . This eNOS normally accounts for both basal and
stimulated
NO synthesis throughout the vascular
system.
Cloning of the human (11, 12) and bovine (13, 14, 15) eNOS shows that these proteins have approximately 60% sequence identity with nNOS and 50% with iNOS. The presence of a consensus sequence for N-terminal myristoylation, absent in the other two isoforms, accounts for the observation that more than 90% of the enzymatic activity remains associated with the particulate fraction(6) . Replacement of Gly-2, the myristic acid acceptor site, by an alanine converts eNOS into a cytosolic enzyme without detectably altering its enzymatic properties (16, 17) . eNOS can also be palmitoylated at cysteines 15 and 26 (18) in a process that may be dynamically regulated as part of a response to enzyme agonists such as bradykinin(19) . More recently, a specific interaction between recombinant eNOS and acidic phospholipids has been reported(20) . Surprisingly, deletion of the entire CaM-binding region of eNOS produces a cytosolic myristoylated protein no longer able to interact with lipids(20) .
Although numerous reports have been published describing the spectral, catalytic, and structural properties of purified nNOS and iNOS, the corresponding information on the endothelial isoform is limited. Both nNOS (21, 22, 23) and iNOS (24, 25) are homodimers, the dimer being the catalytically active form(23, 25) . These two isoforms contain an average of 1 FAD, 1 FMN(24, 25, 26, 27) , and 1 iron-protoporphyrin IX(28, 29, 30, 31) per subunit and have the spectroscopic properties of a cytochrome P-450 (31, 32) . This fact, together with sequence homologies with P-450 reductase (32) and to a very small extent with the heme-binding sequence of P-450 3A4(33) , suggests that NOS is a self-sufficient relative of the P-450 superfamily.
eNOS has been purified from endothelial cells (6, 7, 34) and heterologous expression has
been achieved in COS
cells(13, 15, 16, 17) , NIH3T3
cells(12) , and in baculovirus
systems(35, 36, 37, 38) . However,
only small amounts of protein have been obtained from these sources. We
report here the first successful expression of catalytically active
bovine eNOS in Escherichia coli. The recombinant enzyme
displays spectral properties comparable with those reported for the
neuronal and macrophage isoforms and is present in a monomer-dimer
equilibrium with the dimer surviving in 0.1% SDS. Of particular
interest is the fact that BH-free eNOS, which has an
absorbance spectrum that differs from that of the BH
-bound
enzyme, retains the ability to dimerize even though it is catalytically
inactive.
To facilitate
complete purification of the protein, a polyhistidine tag (6
histidines) was placed at the N terminus. Due to the presence of two BamHI sites in pCWeNOS, a BamHI and NdeI
double-digestion was performed on a pCW-putidaredoxin plasmid, and a small fragment of approximately 25 bp was excised. Two
synthetic oligonucleotides of 43 and 45 bases were designed so that
upon annealing and ligation between the BamHI and NdeI sites, the Shine-Delgarno region was reinserted with the
additional 18 bp corresponding to the polyhistidine tag. A new BsmI site was also introduced between the BamHI and NdeI sites for screening purposes. The construct was
designated as poly-His pCWPdX. An NdeI-XbaI double
digestion was then performed, and the putidaredoxin gene was replaced
by the bovine eNOS gene. This new construct was designated poly-His
pCWeNOS.
In the case of the poly-His
tagged protein, an additional Ni-NTA column was used.
The protein bound to the ADP resin was washed with Buffer C (Buffer B
without BH
or BME), and after elution was loaded onto a
5-ml Ni
-NTA-agarose column. The column was washed
with 25 ml of Buffer C and eluted with Buffer C plus 200 mM imidazole. After elution of the purified protein, BH
was added to a final concentration of up to 10 µM before storage. When the activity of the protein eluted with
imidazole was measured, a small calmodulin-agarose column was used to
eliminate imidazole and to concentrate the sample prior to the assay.
The protein purification was performed at 4 °C, and no L-arginine was included in the buffers during the purification
steps. When protein without BH was desired, this cofactor
was omitted from all the buffers, and the columns were thoroughly
washed with buffers lacking the cofactor before loading the protein to
remove any trace of pterin from previous purifications. The enzyme was
stored in aliquots at -70 °C, and multiple freeze-thaw cycles
were avoided. The desired quantities of eNOS for the various assays
were routinely obtained by scraping the frozen protein.
Figure 1:
SDS-PAGE analysis of the purified eNOS
isolated from E. coli. Lane A, molecular mass
standards (bovine serum albumin, 66 kDa; phosphorylase b, 97.4
kDa; -galactosidase, 116 kDa; myosin, 205 kDa); lane B,
crude cell lysate; lane C, sample after calmodulin-Sepharose
column; lane D, sample after the 2`,5`-ADP-Sepharose column; lane E, final sample after the
Ni
-NTA-agarose column. Approximately 1.5 µg of
protein was loaded per lane.
Figure 2:
Absorbance spectra of eNOS under different
conditions: A, absorbance spectrum of purified ferric eNOS in
the absence (trace 1) and presence (trace 2) of 50
µM L-arginine, and absorbance spectrum of the
dithionite-reduced eNOS (trace 3); B, difference
spectrum obtained by subtracting trace 1 from trace 2 in panel
A; C, ferrous CO difference spectrum. The samples were in
50 mM Hepes, pH 7.5, containing 10 µM BH.
Difference spectrophotometry was used to calculate the spectral
binding constant K for the protein purified in the
presence and absence of BH
(Fig. 2B). The
BH
-bound protein has a spectral binding constant of
1.9 µM for L-arginine that increases to 4.0
µM in the absence of the cofactor. Hence, the pterin
moiety facilitates the binding of the substrate
2-fold.
The
recombinant protein gave similar activity values with both of the
assays used in this study, with a V at 37 °C
of 95-120 nmol
NO
min
mg
eNOS and K
values of 3 and 14 µM for L-arginine and N-hydroxy-L-arginine, respectively. No significant
activity changes were observed when superoxide dismutase, FAD, or FMN
were added to the reaction mixture. Unlike previous results with
nNOS(23, 41) , addition of high concentrations of L-arginine (up to 100 µM) in the absence of high
concentrations of BH
does not result in enzyme inhibition
by
NO. The rate of reduction of cytochrome c at 37 °C by eNOS was 3-5
µmol
min
mg
,
with approximately a 7-fold decrease in the reduction rate in the
absence of CaM. The presence of L-arginine in the cytochrome c reduction assay did not detectably alter these rates.
In order to calculate an
EC value for the cofactor, increasing concentrations of
BH
were added, and the conversion of L-[
H]arginine to L-[
H]citrulline was measured after
incubation for 1, 3, and 5 min. BH
is able to rescue enzyme
activity with an EC
of 200 nM using the data from
the 1-min reaction time point. The assay was not linear at longer time
points, particularly in incubations with low BH
concentrations.
BH appears to directly modulate
the heme environment of eNOS. The absorbance spectrum of the
BH
-free protein is depicted in Fig. 3. In the
absence of the cofactor, there is an elevated low-spin population of
the heme (Fig. 3, trace 1) that immediately decreases
on addition of BH
without a clear increase in the high-spin
population (trace 2), unlike the addition of L-arginine (trace 3) which produces a clear shift
from low to high spin. As shown in Fig. 4, these effects are
additive, since the sequential addition of L-arginine and
BH
produces a more profound decrease in the low-spin
population than that caused by L-arginine alone. Conversely,
sequential addition of BH
and L-arginine produces
a decrease in the low-spin population when BH
alone is
added followed by a low- to high-spin conversion when the substrate is
also added.
Figure 3:
Effect of BH on the
spectroscopic properties of recombinant eNOS. Absorbance spectra of
eNOS purified in a complete absence of BH
(spectrum
1) plus 40 µM BH
(spectrum 2). Spectrum 3 shows the effect of 100 µML-arginine on spectrum 2. The samples were in 50 mM Hepes, pH 7.5, 50 mM NaCl, 10%
glycerol.
Figure 4:
Heme modulation induced by BH
and by L-arginine. Panel A, difference spectra
obtained on a 200 µg/ml eNOS solution upon the sequential addition
of 100 µML-arginine (spectrum 1) and 40
µM BH
(spectrum 2). Panel B,
difference spectra obtained on a 200 µg/ml eNOS solution upon the
sequential addition of 40 µM BH
(spectrum
1) and 100 µML-arginine (spectrum
2). The samples were in 50 mM Hepes, pH 7.5, 50 mM NaCl, 10% glycerol.
Figure 5:
SDS-PAGE analysis of the monomer-dimer
equilibrium. A 7.5% acrylamide, 0.1% SDS gel was used to determine the
quaternary associations of eNOS. Lane A, SDS-boiled molecular
mass standards (bovine serum albumin, 66 kDa; phosphorylase b,
97.4 kDa; -galactosidase, 116 kDa); lane B, unboiled
-amylase standard (
200 kDa); lane C, SDS-boiled eNOS
purified in the presence of BH
; lane D, unboiled
eNOS purified in the presence of BH
; lane E,
unboiled eNOS purified in the absence of BH
. Approximately
3 µg of protein were loaded per lane. The gel was stained with
Coomassie Blue.
Successful expression of eNOS in E. coli and
purification of the recombinant enzyme (Table 1) for the first
time provide ready access to the large amounts of pure protein required
for physical characterization. Although eNOS has recently been
expressed in baculovirus
systems(35, 36, 37, 38) , expression
in E. coli is simpler and provides higher amounts of
functional protein. Heterologous expression of eNOS is particularly
useful because eNOS is currently the least readily available of the
three types of NOS. Furthermore, the protein obtained by bacterial
expression is devoid of BH because bacteria do not produce
BH
(44) . Roman et al.(46) have
reported that nNOS expressed in E. coli contains 10% of the
theoretical content of pterin. The reason for this discrepancy in the
BH
content of enzymes expressed in E. coli is
unclear but might reflect contamination if the enzyme is purified on
columns also used to purify the BH
-supplemented protein.
The BH
requirements of the protein can be satisfied by
adding the cofactor (see below), but the protein purified in the
absence of added BH
provides an excellent source of enzyme
for studies of the role of BH
in catalysis.
The terminal amino acid in the mature eNOS expressed in mammalian cells is a glycine due to post-translational processing of the terminal methionine. Myristoylation of this N-terminal glycine leads to association of eNOS with the membrane. Myristoylation does not occur, however, and the protein does not associate with the membrane when the glycine is replaced by an alanine(16, 17) . The glycine in the protein studied here has been replaced by an alanine due to the observation that the expression of P-450 enzymes in E. coli is improved when the amino acid adjacent to the terminal methionine is an alanine (40) . The eNOS expressed in E. coli is thus a cytosolic protein not only because E. coli lacks N-myristoyltransferase activity (45) but also because the glycine that would be myristoylated in mammalian cells has been replaced by an alanine.
In order to facilitate the preparation of highly purified eNOS for crystallographic studies, a second form of the protein has been expressed with six histidine residues attached to the N terminus. The protein with the poly-His domain is indistinguishable from the recombinant protein in terms of both spectroscopic and catalytic properties.
Due to the
unavailability of eNOS there is little published spectroscopic data on
this form of the enzyme. Spectra of the ferric protein have recently
been reported(36, 37) , but no spectroscopic data are
available on the other oxidation states of the protein or its
interactions with cofactors or ligands. As shown in Fig. 2, the
BH-reconstituted ferric protein has a Soret maximum at 400
nm, the ferrous protein a maximum at 415 nm, and the ferrous-CO complex
a P-450-like maximum at 445 nm. The absorbance maximum of the ferric
protein shifts from 400 to 395 nm on addition of arginine, indicating
that substrate binding triggers a low- to high-spin transition of the
prosthetic heme iron atom. Binding of BH
to the
BH
-deficient enzyme in the absence of arginine decreases
the amount of the low-spin state without detectably increasing the
high-spin component (Fig. 3). These results suggest that
BH
directly modulates the heme environment of eNOS. A
different conclusion is suggested for nNOS by the report that nNOS has
the same spectrum whether BH
is bound or not(47) .
However, we find that nNOS expressed in E. coli and purified
in the complete absence of BH
exhibits a substantial
low-spin component that is even more pronounced than that observed with
eNOS. (
)
The specific activity of the
BH-supplemented recombinant protein at 37 °C is
95-120 nmol min
mg
, as
measured by both
NO and citrulline production, and the K
values for arginine and N-OH-arginine
are 3 and 14 µM, respectively. These values compare well
with those reported in the literature for the protein isolated from
mammalian cells, a K
of 1-5 µM and a specific activity of
150 nmol
min
mg
(20) . Older reports
describe activities in the 1-5 nmol
min
mg
range, and a specific
activity of 900 nmol min
mg
has
also been reported (34) , but this latter value is abnormally
large and is suspect. No catalytic activity was observed in the absence
of added BH
, in accordance with the well established
requirement for BH
for catalytic turnover of nitric-oxide
synthases. The EC
for rescue of the catalytic activity of
eNOS by BH
is 200 nM, a value consistent with that
for the enzyme isolated from mammalian cells(34) . In contrast
to the findings with nNOS(23, 41) , high
concentrations of arginine in the absence of high concentrations of
BH
do not result in enzyme inhibition.
As found
previously for nNOS, reduction of cytochrome c by the
flavoprotein domain of eNOS is stimulated 7-fold by the addition of CaM
and Ca, which indicates that electron flow from the
flavins to even an exogenous electron acceptor is modulated by CaM. The
reduction of cytochrome c was not detectably influenced by the
presence or absence of BH
, essentially the same rates and
the same dependence on CaM were obtained with the BH
-free
and BH
-supplemented enzymes.
Studies carried out with
iNOS suggest that BH is required for formation of the
functional dimer of this protein(48, 49) . In the
absence of BH
, iNOS reportedly does not dimerize. As shown
here, BH
is not required for dimerization of eNOS (Fig. 5, Table 3). Nondenaturing electrophoretic analysis
indicates that the eNOS monomer-dimer equilibrium is similar for the
protein purified in the absence and presence of BH
despite
the fact that the protein purified in the absence of BH
is
catalytically inactive and is less stable. Indeed, it is of some
interest that the dimeric enzyme, whether formed in the presence or
absence of BH
, is stable to electrophoresis in the presence
of 0.1% SDS (Fig. 5). Studies of porcine nNOS indicate that the
formation of a 2% SDS-resistant dimer is synergistically promoted in
that system by BH
and arginine(22) . Our results
with eNOS differ from those with porcine nNOS in that the extent of
dimer formation in the absence of BH
is only slightly
increased by the addition of BH
(Table 3) or L-arginine. The dimerization requirements for dimerization of
eNOS therefore appear to differ from those for dimerization of iNOS and
nNOS.