(Received for publication, June 29, 1995; and in revised form, October 31, 1995)
From the
Rat cerebellar nitric oxide synthase (NOS) purified from
transfected human kidney cells catalyzes an NADPHdependent reduction of
quinonoid dihydrobiopterin (qBH) to tetrahydrobiopterin
(BH
). Reduction of qBH
at 25 µM proceeds at a rate that is comparable with that of the overall
reaction (citrulline synthesis) and requires calcium ions and
calmodulin for optimal activity; NADH has only 10% of the activity of
NADPH. The reduction rate with the quinonoid form of
6-methyldihydropterin is approximately twice that with qBH
.
7,8-Dihydrobiopterin had negligible activity. Neither
7,8-dihydrobiopterin nor BH
affected the rate of qBH
reduction. Reduction is inhibited by the flavoprotein inhibitor
diphenyleneiodonium, whereas inhibitors of electron transfer through
heme (7-nitroindazole and N-nitroarginine) stimulated the rate
to a small extent. Methotrexate, which inhibits a variety of enzymes
catalyzing dihydrobiopterin reduction, did not inhibit. These studies
provide the first demonstration of the reduction of qBH
to
BH
by NOS and indicate that the reduction is catalyzed by
the flavoprotein ``diaphorase'' activity of NOS. This
activity is located on the reductase (C-terminal) domain, whereas the
high affinity BH
site involved in NOS activation is located
on the oxygenase (N-terminal) domain. The possible significance of this
reduction of qBH
to the essential role of BH
in
NOS is discussed.
Nitric oxide synthase (NOS) ()catalyzes the
conversion of arginine to nitric oxide and citrulline. The enzyme has
attracted much interest because of the involvement of nitric oxide in a
variety of physiological processes, such as vasodilation, cytotoxic
activity, and intercellular signaling in the
brain(1, 2, 3) . All three isozymes of NOS
have FAD, FMN, heme, and tetrahydrobiopterin (BH
) as
required prosthetic groups and either have calmodulin (CaM) tightly
bound or require binding of this factor for activity. The C-terminal
part of the protein, containing NADPH-, FMN-, and FAD-binding sites, is
homologous to NADPH cytochrome P450 reductase, whereas the N-terminal
part of the protein contains the heme-, BH
-, and
arginine-binding
sites(4, 5, 6, 7, 8) .
Based on direct studies of NOS and its similarity to cytochrome
P-450(9) , it has been proposed that the pathway of electron
flow in NOS is NADPH
FAD
FMN
heme
O
.
NOS is unique as a heme oxygenase in requiring
BH, and elucidation of the role of BH
is
essential in understanding how this enzyme functions. In the aromatic
amino acid hydroxylases, BH
acts as a cofactor, providing
electrons for oxygenation; the oxidized biopterin, quinonoid
dihydrobiopterin (qBH
), is recycled to BH
by
dihydropteridine reductase(10) . Whereas it has been proposed
that BH
functions in NOS in the same manner, neither the
oxidation of BH
nor the reduction of dihydrobiopterin
catalyzed by NOS has been
demonstrated(11, 12, 13, 14) .
Giovanelli et al.(15) were unable to detect recycling
of added BH
and suggested that added BH
was not
acting as a stoichiometric reactant as it does in the aromatic amino
acid hydroxylases. The possibility of recycling of enzyme-bound
BH
was not excluded(16) . On the basis of these
results, it was suggested that BH
may function as an
allosteric effector or to maintain some group(s) on the enzyme in a
reduced state required for activity(15) . This suggestion has
been supported by more recent studies showing that BH
helps
stabilize the active dimeric state of the
enzyme(17, 18) , maintains a stable heme pocket that
is accessible to oxygen(19) , and prevents and reverses the
inhibition of NOS by nitric oxide(20) . Because it is not clear
whether BH
undergoes redox reactions during NOS catalysis,
we have examined directly the reduction by NOS of qBH
to
BH
.
Reduction of qBH was
determined in a standard 20-µl reaction mixture containing 25
mM sodium bicine buffer, pH 7.6, 0.6 mM EDTA, 0.9
mM CaCl
, 55 µg/ml CaM, 1.5 mM glucose-6-phosphate, 0.12 units of glucose-6-phosphate
dehydrogenase, and 1 µM NADPH. Unless stated otherwise,
the reaction was started by adding qBH
to a concentration
of 25 µM immediately after adding NOS (0.5-1.5
µg). The reaction mixture was incubated at 25 °C. Blank
reactions in which NOS was omitted were used to correct for nonenzymic
conversion of qBH
to BH
. Reactions were
terminated after 30, 60, and 90 s by adding 4 µl of 1.5 M HClO
followed by partial neutralization to pH 3 with 4
µl of approximately 1.4 M KHCO
. The low pH is
important for stability of BH
and to permit subsequent
assay of the BH
content of the partially neutralized
solution at a pH of 6.8 (see below). In order to compensate for small
variations in concentration of the solutions of HClO
and
KHCO
, the exact concentration of the latter solution
required to achieve a pH of about 3 should be determined in a separate
titration experiment. The precipitate was removed by centrifugation,
and 25 µl of the supernatant solution was immediately added to the
mix described below in order to determine BH
. Unless stated
otherwise, rates of BH
synthesis were determined from an
average of the rates determined at 30 and 60 s.
BH formed in the above reaction was determined from the amount of
[
H]tyrosine formed in the
BH
-dependent hydroxylation of [
H]
phenylalanine catalyzed by rat liver PAH. The reaction mixture (35
µl) contained 0.1 mg ml
catalase, 130 mM potassium phosphate buffer, pH 6.8, 0.1 mM lysolecithin,
20 µM [
H]phenylalanine (7.5
µCi), and 1.1 µg of PAH. The mixture was incubated for 30 min
at 25 °C; the reaction was subsequently stopped by adding 20 µl
of a solution containing 5% (w/v) trichloroacetic acid, 0.1 mM phenylalanine, and 0.1 mM [
C]tyrosine (30 nCi), added to correct for
losses during subsequent purification by high performance liquid
chromatography (HPLC). [
H,
C]Tyrosine
was purified by reverse phase HPLC(15) . For amounts of
[
H]tyrosine less than 5 pmol, the product from
the HPLC step was mixed with additional carrier tyrosine and further
purified by crystallization(15) . The
[
H]tyrosine to [
C]tyrosine
ratio was used to calculate the amount of
[
H]tyrosine formed. The efficiency of BH
determination was assessed with separate BH
standards, and appropriate corrections were made for calculating
amounts of enzymic synthesis of BH
. The efficiency was
usually approximately 70% and somewhat lower for amounts of BH
below 25 pmol. These procedures were also used for determination
of the reduction products of 7,8-BH
and q6MPH
;
the reduction product of q6MPH
, 6MPH
, is also a
substrate for PAH(30) .
Figure 1:
Citrulline and BH formation
by NOS. Formation of BH
(
) and citrulline (
)
were determined in 20-µl samples removed from the same reaction
mixture containing 25 µM qBH
and 250
µM [
H]arginine at the time intervals
shown. Further details are described under ``Experimental
Procedures.''
Figure 2:
Effect of qBH concentration on
BH
synthesis. Reactions were started by adding the
indicated amount of qBH
to the standard reaction mixture
containing 0.48 µg of NOS and 100 µM NNA. Reaction
rates were estimated from duplicate determinations of the amount of
BH
formed in 30 s.
Figure 3:
Stimulation of NOS activity by BH and qBH
. The indicated amounts of pterin (
,
BH
;
, qBH
) were added just prior to the
addition of 0.5 µg of NOS. Stimulation of NOS activity was
expressed as the ratio of citrulline synthesis with added pterin to
that measured in the absence of added pterin. The inset includes data obtained with higher concentrations of pterin and is
plotted with the abscissa on a logarithmic
scale.
The novel method described here allows for the sensitive and
specific determination of BH in the presence of relatively
large amounts of qBH
. BH
formed during
reduction of qBH
is coupled to the PAH-catalyzed conversion
of an equivalent amount of [
H]phenylalanine to
[
H]tyrosine. The procedure proved to be
reproducible and easy to use and avoids HPLC of unstable BH
as required in other methods(36, 37) .
A
proposed scheme for qBH reduction by NOS is presented in Fig. 4. The strong inhibition of qBH
reduction by
DPI demonstrates a role for flavin. Three enzymes, one of which is also
a flavoprotein, have previously been reported to catalyze the
pyridine-nucleotide-linked reduction of dihydrobiopterin. They are
dihydropteridine reductase (38, 39) and the
flavoprotein methylenetetrahydrofolate
reductase(40, 41) , both of which catalyze qBH
reduction, and dihydrofolate reductase, which catalyzes the
reduction of 7,8-BH
(42) . Methotrexate is an
extraordinarily potent inhibitor of dihydrofolate reductase (43) and causes appreciable inhibition of the other enzymes at
the concentration of 100 µM(38, 44, 45) used in our work. The
sensitivity of these enzymes to methotrexate is in sharp contrast to
the observed insensitivity of qBH
reduction by NOS to this
pterin analogue. The absence of an inhibitory effect of methotrexate on
qBH
reduction is especially relevant in the light of the
recent report of a ``dihydrofolate reductase module'' on the
oxidase domain of NOS(5) .
Figure 4:
Proposed scheme for qBH
reduction by NOS. The reductase or C-terminal domain (left)
contains the NADPH-, FAD-, and FMN-binding sites, whereas the oxygenase
or N-terminal domain (right) contains the heme-,
BH
-, and arginine-binding
sites(4, 5, 6, 7, 8) . CaM
binding activates by increasing the rates of electron transfer between
NADPH and flavins and also facilitates electron transfer to
heme(46) . DPI is a flavoprotein
inhibitor(31, 32) . 7NI is believed to bind to heme
and is competitive with BH
binding(14, 35) . NNA also inhibits electron
transfer through heme(33) . The symbol e
denotes electrons. Oxygenation of arginine predominantly to NHA
is catalyzed by substrate amounts of NOS in the absence of added
NADPH(25) . Oxygenation is supported by an endogenous reductant
that was shown not to be NADPH(25) . Added BH
stimulates each of the two partial reactions involving NHA as an
intermediate(25, 50, 51, 52) .
What is the site of qBH reduction? Does reduction proceed directly at the flavoprotein
NADPH ``diaphorase'' site of NOS, or are electrons
transferred from flavin to the high affinity BH
-binding
site involved in the activation of NOS(14, 15) ? Two
main findings indicate that the flavoprotein diaphorase is the site of
qBH
reduction. First, BH
and
7,8-BH
, two compounds with a high affinity for the
BH
-binding site(14, 15) , have no effect
on qBH
reduction. Second, 7NI, which interferes with
BH
binding to the high affinity
site(14, 35) , stimulates qBH
reduction. This stimulation, as well as that by NNA, can be
tentatively ascribed to the inhibition of reactions proceeding through
heme(14, 33, 35) , thereby increasing the
availability of reducing equivalents for qBH
reduction. The
high EC
(Fig. 2) for qBH
reduction
compared with the relatively low K
values for
binding and activation with BH
(Fig. 3,(13, 14, 15) ) is
consistent with qBH
reduction proceeding at the diaphorase
site, rather than the high affinity BH
-binding site.
The
NADPH diaphorase component of NOS is known to catalyze the oxidation of
NADPH with a range of electron acceptors including cytochrome c, ferricyanide, and various
dyes(11, 46, 47, 48, 49) .
Reduction of q6MPH at a rate of approximately twice that of
qBH
is consistent with this reported low specificity; the
product of q6MPH
reduction, 6MPH
, shows little
or no activation of NOS at concentrations optimal for activation by
BH
(13, 15) .
There are conflicting data
on whether superoxide is an intermediate in the NADPH-cytochrome c reductase activity of
NOS(35, 46, 48, 49) . It was
therefore of interest to examine the effects of superoxide dismutase on
this reaction and also on the NOS-catalyzed reduction of
qBH. Our finding that superoxide dismutase had no effect on
the NADPH-cytochrome c reductase activity of NOS is in
agreement with other reports(35, 46, 48) .
Furthermore, the small inhibition of qBH
reduction by
superoxide dismutase further suggests that superoxide is not involved
in this reaction.
Our studies show that the reduction of both
qBH and cytochrome c by NOS is strongly stimulated
by Ca
/CaM. Other workers have also reported a strong
stimulation of the reduction of the latter substrate by
Ca
/CaM; by contrast, for unexplained reasons, the
NADPH-dependent reduction of ferricyanide and
2,6-dichlorophenolindophenol by NOS is stimulated to only a small
extent or not at all by
Ca
/CaM(46, 48, 49) . With
respect to dependence on Ca
/CaM, qBH
reduction therefore more closely resembles cytochrome c reduction than ferricyanide or 2,6-dichlorophenolindophenol
reduction.
Under the conditions used in our earlier
experiment(15) , where the PAH reaction would have been
expected to generate a steady-state concentration of about 0.05
µM qBH from the added 0.05 µM BH
and the limits of detection were 2.5 pmol, there
was no detectable regeneration of BH
in the presence of
added NOS and NADPH. Because this result appears to contrast with our
present data, it is important to understand why the earlier experiment
did not detect any reduction of qBH
. The explanation, in
all probability, is that in the earlier experiment, the amount of
qBH
converted to BH
was below the limits of
detection. Thus, from the data in Fig. 2, it can be estimated
that 2 pmol of BH
would have been synthesized during the
30-min incubation period used in the earlier experiment; this amount
would be considerably less if the instability of qBH
and
the lack of linearity of BH
synthesis with time (Fig. 1) are taken into account.
The significance of the
qBH reduction described here to the essential role of
BH
in NOS remains to be clarified. Our results indicate
that at the relatively high concentration of 25 µM,
qBH
reduction can proceed at a rate comparable with that of
the overall reaction (citrulline synthesis) and that the efficiency of
added qBH
in stimulating NOS activity approaches that of
added BH
. QBH
seems to stimulate by first being
converted to BH
and subsequent binding to the
BH
-binding site. This postulate is supported by the
observation that added qBH
causes little or no stimulation
of NOS-catalyzed oxygenation of arginine to NHA (Fig. 4), a
reaction that proceeds in the absence of added NADPH and consequently
precludes qBH
reduction.
A central feature of the model
proposed in Fig. 4is that qBH reduction proceeds on
the C-terminal (reductase) domain, remote from the high affinity
BH
-binding site of the N-terminal (oxidase) domain.
Implicit in this model is the notion that if BH
recycling
does occur, it may proceed by a pathway in which qBH
reduction occurs predominantly at the flavin site, followed by
transfer of the BH
so formed to the high affinity
BH
-binding site. A redox cycle would be completed by
transfer of qBH
back to the flavin reduction site. Transfer
of pterins between the flavin and BH
-binding sites may
proceed either by dissociation from the enzyme or by a direct
interdomain transfer. Our present studies also do not eliminate the
possibility that cycling of pterin occurs exclusively at the
BH
-binding site on the oxygenase domain. These
possibilities are currently under investigation.