(Received for publication, October 6, 1994; and in revised form, November 18, 1994)
From the
The nitric oxide synthase-catalyzed conversion of L-arginine to L-citrulline and nitric oxide is known
to be the sum of two partial reactions: oxygenation of arginine to N-hydroxyarginine, followed by oxygenation of N-hydroxyarginine to citrulline and nitric oxide. Whereas the
conversion of N-hydroxyarginine to citrulline and nitric oxide
has been the subject of a number of studies, the oxygenation of
arginine to N-hydroxyarginine has received little attention.
Here we show that substrate amounts of rat cerebellar nitric oxide
synthase, in the absence of added NADPH, catalyze the conversion of
arginine to N-hydroxyarginine as the dominant product. The
product appears not to be tightly bound to the enzyme. A maximum of
0.16 mol of N-hydroxyarginine/mol of nitric oxide synthase
subunit was formed. The reaction requires oxygen and the addition of
Ca/calmodulin and is stimulated 3-fold by
tetrahydrobiopterin. Upon addition of NADPH, citrulline is formed
exclusively. Conversion of N-hydroxyarginine to citrulline,
like the first partial reaction, requires
Ca
/calmodulin and is stimulated by
tetrahydrobiopterin but differs from the first partial reaction in
being completely dependent upon addition of NADPH. These results
indicate that brain nitric oxide synthase contains an endogenous
reductant that can support oxygenation of arginine but not of N-hydroxyarginine. The reductant is not NADPH, since the
amount of nitric oxide synthase-bound NADPH is appreciably less than
the amount required for N-hydroxyarginine synthesis. Possible
candidates for this role are discussed in relation to proposed
mechanisms of action of nitric oxide synthase.
Nitric oxide synthase (NOS) ()catalyzes the
oxygenation of arginine in the presence of NADPH to form nitric oxide,
citrulline, and NADP
. The enzyme is of great interest
because nitric oxide participates in a variety of physiological
processes including vasodilation, macrophage toxicity, and
neurotransmission(1) . There is now convincing evidence that
the overall reaction catalyzed by NOS proceeds via two partial
reactions,
The intermediary role of L-N-hydroxyarginine (NHA) was first
suggested by Marletta et al. (2) and detected as a
minor product during the conversion of arginine to citrulline by
mammalian cells induced for NO synthesis as well as by crude extracts
of these cells (3, 4) and by purified macrophage
NOS(5, 6) . In the last study,
[
H]NHA formation from
[
H[arginine was detected only under special
conditions, namely in the presence of a high concentration of a pool of
unlabeled NHA, indicating that this intermediate may not normally be
released from the enzyme. The amount of [
H]NHA
accumulating was only 20% of the amount of
[
H]citrulline formed(4, 6) .
Recently, Klatt et al. (7) reported the formation of
trace amounts of [
H]NHA from
[
H]arginine catalyzed by purified porcine brain
NOS. Here again, the amounts of NHA accumulating were small relative to
the amounts of citrulline formed. Under normal reaction conditions, NHA
accounted for a maximum of 2% of citrulline formed; in the presence of
the redox-active inhibitors of citrulline formation, nitroblue
tetrazolium or methylene blue, this value increased up to 20%. The
small amounts of NHA relative to citrulline formed under these
conditions have hampered attempts to elucidate the characteristics of
the first partial reaction.
Here we show that stoichiometric amounts of purified rat brain NOS convert arginine predominantly to NHA in the absence of added electron donor. This experimental system permits examination, for the first time, of the characteristics of the first partial oxygenation reaction. Our findings indicate that the rat brain enzyme contains an endogenous reductant that can support a single cycle oxygenation of arginine to NHA but not of NHA to citrulline. Possible candidates for this role are discussed in relation to the proposed mechanisms of action of nitric oxide synthase.
HPLC-HPLC was
performed by cation exchange chromatography on a Zorbax 300-SCX column
(DuPont) (dimensions, 4.6 mm internal diameter 25 cm) using the
solvent system described by Chenais et al. (3) or
Klatt et al.(7) . Reverse phase ion pair
chromatography was performed on a Waters µ Bondapak C18 column (3.9
mm internal diameter
30 cm) with the use of a gradient of
0-75% 1-propanol with a stationary phase of 17 mM sodium
citrate, pH 2.7, containing 0.5% sodium dodecyl sulfate(11) .
Columns were operated at a flow rate of 1 ml/min.
For determination of the amount of NHA synthesized under carbon monoxide, reaction mixtures were the same as described for the standard reaction mixture, except that the arginine concentration was 11.2 µM. Reaction mixtures (NOS and calmodulin omitted) were pipetted into a Pierce Reactivial (0.3 ml), which was then sealed with a Teflon/silicone disc and flushed for 5 min at room temperature with a mixture of carbon monoxide/oxygen (4/1, v/v). The gas mixture entered the Reactivials through a hypodermic needle and was allowed to impinge on the surface of the reaction mixture, which was shaken frequently to facilitate equilibration. After the initial equilibration, cloned NOS (1.6 µg) was injected with a Hamilton syringe, and equilibration with the gas phase continued for another 5 min. Reactions were started by injection of calmodulin with a Hamilton syringe to a final reaction volume of 14 µl. The final reaction mixture was incubated for 5 or 15 min. For determination of the overall reaction (citrulline synthesis), the above procedure was modified to include 667 µM NADPH and NOS (2.3 ng) in a final volume of 42 µl. Procedures for measurements under air were identical except for substitution of air for the carbon monoxide mixture.
Figure 1:
Products derived from
[H]arginine in the absence and presence of NADPH. Panel A, reaction was incubated for 30 min and contained 2
µg of rat cerebellar NOS and the components specified in the
standard reaction mixture for assay of the first partial reaction. Panel B, addition of 670 µM NADPH. Panel
C, same as panel A except for omission of enzyme.
H is shown as histograms. The truncated values for
H in arginine (panel A) are 3.58 and 4.80
10
(37 and 38 min, respectively), and for citrulline (panel B) the values are 4.07 and 4.80
10
(9 and 10 min, respectively). Positions of carrier citrulline and
NHA determined by fluorescamine assay are shown as solid
squares. These values have been displaced on the y axes
to avoid confusion with the values for
H
dpm.
The dominant radioactive
product formed under the conditions used in Fig. 1, panel A , was characterized as [H]NHA by its
comigration with authentic NHA during HPLC on cation exchange and
reverse phase ion pair columns and thin layer chromatography on
cellulose and silica gel. A summary of the chromatographic properties
of the radioactive product compared with those of related amino acids
is given in Table 1. Further evidence for the identity of the
radioactive product with NHA is provided by its conversion
predominantly to [
H]citrulline when incubated
with rat cerebellar NOS (see below).
Table 2summarizes the
effects of components of the reaction mixture on the synthesis of
radioactive products from [H]arginine. Omission
of Ca
/calmodulin or BH
resulted in marked
decreases in [
H]NHA synthesis. Omission of
catalase increased the proportion of radioactivity in citrulline
without affecting the total radioactivity in NHA plus citrulline. In
the presence of added NADPH, total
H incorporated into
product was increased 15-fold, and radioactivity was detected only in
citrulline (see also Fig. 1, panel B).
Figure 2:
Time
course and effect of BH on products derived from
[
H]arginine. Conditions are those described in Fig. 1(panel A) except for omission of BH
where indicated. Values of pmol of [
H]NHA
and [
H]citrulline were calculated by dividing the
amounts of radioactivity in these compounds by the specific activity of
[
H]arginine.
Figure 3:
Enzymic conversion of
[H]NHA to [
H]citrulline.
The complete system (described under ``Methods'') is shown in panel A. NADPH is omitted in panel B. Panel C is the
same as panel A except for omission of NOS. Symbols are the same as for Fig. 1.
Figure 4: Effect of enzyme and arginine concentration on NHA synthesis. Reactions were as described for the standard reaction mixture except that those of panel A contained 2.6 µM arginine and varying amounts of cloned NOS, whereas those of panel B contained varying concentrations of arginine and 2.8 µg (17 pmol) of rat cerebellar NOS. All reactions were incubated for 30 min.
When assayed in the absence of NADPH, the relative amount of radioactivity accumulating in citrulline relative to that in NHA was much higher for ferricyanide-treated enzyme. When assayed in the presence of NADPH, no significant accumulation of NHA was detected with the ferricyanide-treated enzyme. These observations are consistent with ferricyanide treatment inhibiting the first partial reaction to a greater extent than the second partial reaction.
The system described here permits a determination of the
characteristics of the first step in nitric oxide synthesis, i.e. the oxygenation of arginine to NHA. One of the most interesting
findings is that NHA synthesis proceeds in the absence of added NADPH.
This is in sharp contrast to the stringent requirement for added NADPH
for further conversion of NHA to citrulline. Synthesis of NHA was
stimulated approximately 6-fold by Ca/calmodulin and
approximately 3-fold by BH
It is well established that
BH
participates in the second partial
reaction(5, 6, 7) , and it has been suggested (17) that the site of action of BH
may be
restricted to this reaction. Our results clearly demonstrate a role of
BH
also in the first partial reaction. Omission of catalase
did not affect total radioactivity accumulating in NHA plus citrulline
but increased radioactivity in citrulline approximately 4-fold at the
expense of that in NHA. This effect may result from accumulation, in
the absence of added catalase, of hydrogen peroxide, which can then
react with NHA in the presence of NOS to form citrulline. Hydrogen
peroxide is a major product of NOS activity under conditions of low
concentrations of arginine (less than approximately 10 µM)
or of BH
(18) . Further, hydrogen peroxide in the
absence of NADPH can support the conversion of NHA to citrulline
catalyzed by murine macrophage NOS(19) .
Ferricyanide treatment of the enzyme appears to preferentially inhibit the first partial reaction. As can be seen in Table 3, the ratio of citrulline to NHA formed increases from 0.02 for the native enzyme to 0.34 for the ferricyanide-treated enzyme. This suggests that ferricyanide treatment of the enzyme may preferentially interfere with its ability to catalyze the conversion of arginine to NHA or with the ability of NHA to dissociate from the enzyme. The observed decrease in the formation of the oxygenated amino acid products (i.e. NHA plus citrulline) by ferricyanide treatment can also be explained by a ferricyanide-mediated inhibition of the conversion of arginine to NHA. Although experimental conditions are not comparable, other redox compounds such as nitroblue tetrazolium and methylene blue have been reported to decrease the ratio of citrulline/NHA formed, suggesting that these compounds may interfere with the conversion of NHA to citrulline(7) .
NOS is believed to belong to the
cytochrome P-450 class of enzymes(1) , which show varying
susceptibilities to carbon monoxide. This variation has been ascribed
to differences in relative affinities of oxygen and carbon monoxide for
the ferrous form of heme and differences in the steady-state
concentrations of Fe in heme(20) . Inhibition
by carbon monoxide/oxygen (4/1) in the range of 60-80% has been
reported for the overall conversion of arginine to citrulline (21, 22, 23) and of approximately 30% for
conversion of NHA to citrulline (23) . Whereas these combined
findings are consistent with a role of heme at least in the second
partial reaction, they do not critically address its role in the first
partial reaction. In our studies, comparable inhibitions by the carbon
monoxide/oxygen mixture of 66% for the overall reaction and 28% for NHA
synthesis support a role for heme in the first partial reaction. The
requirement of the first partial reaction for calmodulin, which has
recently been demonstrated to trigger the transfer of electrons from
flavin to heme(24) , provides further support for this
suggestion. The apparent role of heme in the first partial reaction
argues against the suggestion (25) that this reaction may
proceed via a heme-independent mechanism similar to that established
for the aromatic amino acid hydroxylases.
The amount of NHA formed
was always less than the amount of enzyme subunits added. With low
concentrations of arginine such as 189 nM used in the
experiments of Fig. 2, a maximum value of 0.02 mol NHA/mol NOS
subunit was obtained. This ratio increased with increasing
concentrations of arginine (Fig. 4, panel B) to a value
of 0.13 at 20 µM arginine. The maximum value of
NHA/subunit observed was 0.16 (Table 5). The possibility was
considered that ratios of less than unity may reflect the presence of L-arginine bound to the enzyme, as suggested by spectral
studies(16) . The presence of nonradioactive enzyme-bound
arginine in equilibrium with a small amount of radioactive arginine
would lower the specific radioactivity of arginine and its products and
lead to the underestimation of the amount of NHA formed. Reference to
the results of Table 4shows this not to be the case. Assuming
equilibrium between [H]arginine (157 pmol) and a
maximum of 0.72 pmol (0.06
12) of nonradioactive enzyme-bound
arginine, the ratio of NHA/NOS calculated in Table 4(0.10) would
be underestimated by less than 1%. Even with the extreme assumption
that all enzyme-bound arginine is converted to nonradioactive NHA (i.e. with no equilibration between enzyme-bound arginine and
added radioactive arginine), the total amount of NHA formed would be
increased only from 1.2 pmol to 1.92 (1.2 plus 0.72) pmol,
corresponding to an increase in NHA/subunit from 0.10 to 0.16. The
reason for the apparent discrepancy between the negligible amounts of
bound arginine determined in this work with the values of greater than
0.8 mol of arginine/mol of NOS subunit suggested by spectral studies (16) is not clear. Kinetic studies by Matsuoke et al. (26) also indicate that the purified native cloned enzyme
contains little, if any, bound arginine. The differing amounts of
enzyme-bound arginine may reflect differences in conditions used to
culture the cells or in the procedure used to isolate the enzyme.
Two reasons can be suggested to explain why values of less than
unity were observed for the ratio of NHA/NOS subunit. First, NOS may be
heterogeneous in the sense that not all species contain the putative
enzyme-bound factor (see below) required for NHA synthesis. Second,
formation of NHA may not proceed to completion because this compound
competes with arginine for binding to the enzyme. This reason seems
less likely based on the observation that the product NHA is recovered
in the non-protein fraction obtained by gel filtration at 4 °C.
However, the possibility cannot be excluded that NHA remains
enzyme-bound during the assay but is dissociated during the gel
filtration step. Indeed, spectral studies of the cloned enzyme (16) indicate that NHA may be able to displace bound arginine,
a notion that is consistent with our observation that a pool of
unlabeled NHA essentially abolished the incorporation of H
from arginine into NHA. Based on the evidence presented, it is proposed
that NOS contains one or more endogenous enzyme-bound factors that
substitute for NADPH in the formation of NHA. It is generally believed
the path of electron flow in NOS resembles that proposed for
NADPH-cytochrome P-450 reductase(15, 27) , with which
it shares close sequence
homology(1) .
In analogy with current ideas about the mechanism of oxygenation catalyzed by cytochrome P-450(28, 29) , it has been suggested(7, 23) that ferrous heme formed in this electron chain complexes with oxygen. Further addition of an electron to this ferrous-oxygen complex, followed by electron rearrangement, converts it to a ferric peroxide complex. This complex or its fission product, perferryl oxygen, is believed to be the oxygenation species.
A priori, the endogenous enzyme-bound factor could be either a reductant that generates an oxygenation species, or an oxygenation species itself. The oxygen dependence for NHA synthesis is consistent with the endogenous factor being a reductant rather than an oxygenation species. It is therefore suggested that synthesis of NHA in the system described here proceeds as follows.
NHA is shown here as dissociated from the enzyme, based the finding that NHA appears in the non-protein fraction following gel filtration.
Possible candidates for this reductant are discussed below.
In summary, of the redox factors believed to be
involved in NOS function, neither NADPH, flavins, nor heme appears to
account for the endogenous reductant supporting NHA synthesis.
Photoreduction was also excluded as a source of electrons. This work
does not exclude a possible role of BH. Using a sensitive
method developed for specific assay of BH
in the presence
of the quinonoid form of dihydrobiopterin, we are currently examining
the possible role of enzyme-BH
in the oxygenation of
arginine. (
)
A question with important mechanistic
implications posed by these findings is why the putative endogenous
reductant is available for oxygenation of arginine but not of NHA. One
explanation proposed is that arginine has a higher affinity for
NOS-reductant than does NHA. Another interesting possibility is that
electrons ultimately donated from NADPH may proceed in different
domains, each one subserving only one of the partial reactions. It is
interesting to note that recent sequence and structural homology
studies ()indicate the presence of a second NADPH binding
site, distinct from the NADPH binding site associated with FAD on NOS.
Further, resonance Raman scattering studies (36) of NOS have
been interpreted to suggest that either there are two different
heme-protein conformations or that the two protein subunits bind heme
differently.