(Received for publication, December 26, 1995; and in revised form, January 24, 1996)
From the
Inducible nitric oxide (NO) synthase (iNOS) is comprised of an oxygenase domain containing heme, tetrahydrobiopterin, the substrate binding site, and a reductase domain containing FAD, FMN, calmodulin, and the NADPH binding site. Enzyme activity requires a dimeric interaction between two oxygenase domains with the reductase domains attached as monomeric extensions. To understand how dimerization activates iNOS, we synthesized an iNOS heterodimer comprised of one full-length subunit and one histidine-tagged subunit that was missing its reductase domain. The heterodimer was purified using nickel-Sepharose and 2`,5`-ADP affinity chromatography. The heterodimer catalyzed NADPH-dependent NO synthesis from L-arginine at a rate of 52 ± 6 nmol of NO/min/nmol of heme, which is half the rate of purified iNOS homodimer. Heterodimer NO synthesis was associated with reduction of only half of its heme iron by NADPH, in contrast with near complete heme iron reduction in an iNOS homodimer. Full-length iNOS monomer preparations could not synthesize NO nor catalyze NADPH-dependent heme iron reduction. Thus, dimerization activates NO synthesis by enabling electrons to transfer between the reductase and oxygenase domains. Although a single reductase domain can reduce only one of two hemes in a dimer, this supports NO synthesis from L-arginine.
Nitric oxide (NO) ()has important roles as a signal
and cytotoxic molecule in biology (1, 2, 3) and is synthesized from L-arginine by enzymes termed NO synthases (NOSs). The NOSs
exhibit a bi-domain structure in which a N-terminal oxygenase domain
that contains binding sites for iron protoporphyrin IX (heme),
tetrahydrobiopterin (H
biopterin), and L-arginine
is fused to a C-terminal reductase domain that contains binding sites
for calmodulin, FMN, FAD, and
NADPH(4, 5, 6, 7) . L-Arginine binds near enough to the heme to influence its
reduction and its interactions with small ligands such as CO or NO (8, 9, 10) . L-Arginine or substrate
analog binding to the oxygenase domain is also influenced by
H
biopterin, which may bind near the
heme(11, 12, 13) .
Conversion of L-arginine to NO occurs in two steps with N-hydroxy-L-arginine forming as an
enzyme-bound intermediate (for review see Refs. 1, 14, and 15). Both
steps require that NADPH-derived electrons be transferred from the
reductase domain flavins to the heme group in the oxygenase domain,
which is bound to the protein via a cysteine thiolate as in the
cytochromes P-450(16, 17, 18, 19) .
The flavin to heme electron transfer is critical for catalysis because
it is thought to enable the heme iron to bind and activate oxygen at
both steps in the mechanism, resulting in oxygen insertion into L-arginine and into N
-hydroxy-L-arginine.
In spite of
each subunit containing the required flavin and heme groups, the NOSs
appear to be active only in dimeric form(20, 21) .
Dimeric structure does not seem to influence the electron transfer
properties of the NOS reductase domain, because it reduces electron
acceptors like cytochrome c with equal activity when present
in a NOS dimer or a monomer(20, 22) . Indeed, recent
studies with the cytokine-inducible NOS (iNOS) suggest that the subunit
dimeric interaction only involves the oxygenase domains of each
subunit, with the reductase domains existing as independent monomeric
extensions (7, 24) (Fig. 1). The reductase
domains can be removed with trypsin to generate a dimer comprised
exclusively of two oxygenase domains. This oxygenase domain dimer
maintains all of its native properties, including the ability to bind
substrate and catalyze NO synthesis from N-hydroxy-L-arginine when supplied
with electrons from an external reductase
protein(7, 23, 24) . Although the proposed
subunit alignment for iNOS raises intriguing possibilities regarding
electron transfer and cooperative catalysis between the oxygenase
domains during NO synthesis(7) , exactly how dimerization
activates iNOS catalysis remains unclear.
Figure 1:
Diagrams of an iNOS
homodimer and the heterodimer used in this study. The heterodimer is
composed of a full-length subunit and an oxygenase domain subunit that
contains a six-histidine tag at its C terminus. Both subunits contain
heme and incorporate Hbiopterin (H
B) and bind L-arginine (L-Arg) during their dimerization.
We recently showed that
full-length and oxygenase domain dimers can both be dissociated into
heme-containing monomers when treated with
urea(22, 24) . Each monomer can reassemble into its
respective homodimer in the presence of L-arginine and
Hbiopterin. Besides confirming that all of the determinants
for dimerization reside within the oxygenase domain, these results
suggest that it may be possible to construct an iNOS heterodimer that
contains a single reductase domain linked to a dimeric oxygenase core (Fig. 1). This species, in conjunction with a full-length iNOS
monomer, could be used to investigate how dimerization influences
electron transfer and catalytic activity in the oxygenase domain. In
this report, we describe a method to synthesize and purify such an iNOS
heterodimer and report on its catalytic and electron transfer
properties.
To form a heterodimer we utilized a full-length iNOS monomer
preparation that was generated from dimeric iNOS and an oxygenase
domain monomer preparation that was generated from an oxygenase domain
dimer that was expressed in E. coli and contained a
six-histidine tag at its C terminus. Preliminary experiments with the
recombinant oxygenase domain dimer showed that the histidine tag did
not alter the spectral or binding properties of the protein relative to
native oxygenase domain dimer(7, 24) . The histidine
tag also did not prevent dimerization between two oxygenase domain
monomers in solution or when one monomer was bound to nickel affinity
resin (data not shown). As detailed under ``Experimental
Procedures,'' the dimerization reaction that formed the
heterodimer was carried out between oxygenase domain monomers bound to
nickel affinity resin and full-length monomers in solution. The nickel
affinity resin enabled us to separate bound heterodimers from most of
the unreacted full-length monomers and full-length homodimers that had
formed during the dimerization reaction. After eluting the immobilized
heterodimer with imidazole, it was separated from contaminating
oxygenase domain monomers and homodimers by virtue of its containing a
NADPH binding site, using 2`,5`-ADP affinity chromatography. The
heterodimer underwent analytical gel filtration as a final step to
remove residual full-length and oxygenase domain homodimers (Fig. 2). The protein eluted at 11.7 ml, which was between the
elution volumes of authentic full-length iNOS homodimer (10.7 ml) and
the oxygenase domain homodimer (13.2 ml). Analysis of the indicated
protein peak fraction by SDS-polyacrylamide gel electrophoresis (Fig. 2, inset) showed that it was comprised of both
full-length and oxygenase domain subunits whose relative staining
densities were 2.4 to 1. Based on this densitometric data and the
relative molecular mass of each subunit (130 and 55 kDa), a 1:1 molar
ratio of full-length and oxygenase domain subunits was present in the
peak fraction. ()Thus, we conclude that the isolated protein
is an iNOS heterodimer. In the example shown, 0.52 nmol of pure
heterodimer was obtained from reacting 9.2 nmol of immobilized
oxygenase domain with an equivalent amount of full-length monomer
preparation.
Figure 2:
Size exclusion chromatographic
purification of the iNOS heterodimer. Elution profile of the
heterodimer following injection onto a Superdex 200 HR column.
Fractions between the dash marks represent the heterodimer
peak fraction and were pooled and concentrated together. The inset shows analysis of the concentrated peak fraction by
SDS-polyacrylamide gel electrophoresis followed by visualization of
protein by silver staining. The two major bands correspond with the
full-length iNOS subunit (130 kDa) and oxygenase domain subunit
(
55 kDa). The results shown are representative of three
purifications.
Activity measurement by the oxyhemoglobin assay showed
that the heterodimer could synthesize NO from L-arginine or
from N-hydroxy-L-arginine. For three
separate heterodimer preparations, the catalytic turnover number with L-arginine as substrate was 52.0 ± 5.8 nmol of
NO/min/nmol of heme. This is about half of the activity obtained with a
purified full-length homodimer when assayed under identical conditions
(125 nmol of NO/min/nmol of heme). In contrast, full-length monomer
preparations generate either no measurable NO or some small amount of
NO in direct proportion to their residual dimer content(22) .
The average rate of NADPH oxidation by the three heterodimer
preparations during NO synthesis from L-arginine was 77.6
± 4.3 nmol of NADPH/min/nmol of heme. This gives a calculated
stoichiometry of 1.5 NADPH oxidized per NO formed, which is equal to
the minimum stoichiometry for NO synthesis from L-arginine (26, 27) and indicates that NO synthesis by the
heterodimer was tightly coupled to its NADPH oxidation. We conclude
that dimerization enabled the oxygenase domains to catalyze efficient
NO synthesis when supported by only a single reductase domain.
To
examine how dimerization activated NO synthesis in the heterodimer, we
utilized CO binding as a means to quantitate NADPH-dependent heme iron
reduction. In previous work with a full-length iNOS homodimer, we
observed that the addition of NADPH in the presence or the absence of L-arginine results in rapid reduction of 90-95% of the
heme iron, as determined by build-up of a characteristic ferrous-CO
Soret peak at 444 nm(25, 28) . Because NADPH-derived
electrons can only reduce the heme iron by passing through the
reductase domain(6, 23) , we performed an analogous
experiment with the heterodimer to determine what portion of its heme
is accessible to electrons coming from the single reductase domain. The
experiment was carried out under anaerobic conditions in buffer that
contained Hbiopterin and L-arginine and was
saturated with CO. As shown in Fig. 3(solid line), the
light absorbance spectrum of the heterodimer displays a broad Soret
absorbance centered at 398 nm, indicative of predominantly high spin
ferric iNOS, as observed for a full-length iNOS homodimer under similar
conditions(17, 25, 28) . There also is an
absorbance shoulder between 425 and 500 nm attributable to bound
flavins in the heterodimer. The spectrum obtained immediately after
addition of excess NADPH (dashed line) shows that a portion of
the heterodimer's heme iron remained ferric, as indicated by the
absorbance remaining at 380-420 nm, and a portion had become
reduced, as indicated by the new absorbance peak at 444 nm. The
relative proportion of oxidized and reduced heme iron did not change
with time or upon adding more NADPH (data not shown). Dithionite was
then added to completely reduce all of the heme iron present (dotted line), resulting in a further absorbance decrease in
the 380-420 nm region and an increase at 444 nm. Quantitation of
the amount of heme iron reduced with NADPH relative to the amount
reduced with dithionite indicates that 55% of the heme iron was reduced
by NADPH in the heterodimer. The percentage of heme iron reduction
obtained in two replicate experiments using different heterodimers was
calculated to be 56 and 50% (data not shown). Thus, these results
clearly distinguish the heterodimer from a full-length iNOS homodimer,
where 90-95% of the available heme iron is reduced upon NADPH
addition under identical circumstances(25, 28) .
Figure 3:
Heme iron reduction in an iNOS
heterodimer. Purified iNOS heterodimer was diluted to 0.8 µM in 350 µl of 40 mM EPPS buffer, pH 7.6, containing 4
µM Hbiopterin, 1 mM dithiothreitol,
and 10% glycerol. The solution was made anaerobic in a cuvette,
saturated with CO, and kept at 15 °C during measurements. The solid line is the spectrum of the resting enzyme. The dashed line was obtained following the addition of 6 µl of
NADPH solution to give 5 µM in the cuvette. The dotted
line was obtained following the addition of 5 µl of dithionite
solution to give 20 µM. The experiment shown is
representative of replicate experiments done with three different
heterodimer preparations.
We
next investigated heme iron reduction in three iNOS monomer
preparations that were generated by dissociating a full-length iNOS
homodimer with urea(22) . The addition of NADPH in these cases
resulted in very little ()or no heme iron reduction, as
judged by a lack of CO binding. Adding dithionite to the NADPH-treated
monomer preparation reduced all of its heme iron as judged by the total
loss of Soret absorbance in the 400 nm region and an immediate buildup
of the ferrous-CO complex at 444 nm (data not shown). These results
indicate that electron transfer between the reductase domain and heme
iron in a full-length subunit does not occur when the subunit is in
monomeric form.
A major finding of our study is that dimerization may activate iNOS NO synthesis by enabling the reductase domain to transfer electrons to the heme iron. This is consistent with heme iron reduction being an essential first step that leads to oxygen activation and insertion into the substrate(1, 14, 15) . As evidenced with the iNOS heterodimer, transfer of NADPH-derived electrons to the heme can occur even when only one reductase domain is attached and is sufficient to support NO synthesis. Although the heterodimer synthesized NO at a rate that was half-maximal on a per heme basis when compared with the native iNOS homodimer, only half of the heterodimer heme iron was susceptible to reduction by NADPH. This implies that the portion of heterodimer heme iron that was reducible participates in NO synthesis at a rate that equals the heme iron in a full-length iNOS homodimer.
It is remarkable that only half of the
heme iron in the heterodimer was susceptible to reduction by NADPH. The
partial reduction does not appear to reflect a point in a redox
equilibrium, because adding extra reductant (NADPH) did not cause an
increase in the amount of heme iron reduced. Furthermore, when the same
experiment is carried out using a full-length iNOS homodimer,
90-95% of heme iron is reduced(25, 28) . Thus,
our results suggest that a kinetic barrier exists in the heterodimer
toward reduction of half of its heme iron. Based on the available
evidence, there is little reason to suspect that the kinetic barrier
arises from an inequality between the hemes contained in the oxygenase
and full-length subunits of the heterodimer. For example, a homodimer
formed from two oxygenase domains has the capacity to bind L-arginine and Hbiopterin, accept electrons from
an added reductase domain, and synthesize NO from N
-hydroxy-L-arginine(7, 23, 24) .
In addition, the light absorbance spectrum of the heterodimer indicates
that both of its heme irons are predominantly high spin in the presence
of L-arginine and H
biopterin, as is the case with
both full-length and oxygenase domain
homodimers(7, 17) . Thus, the most likely explanation
for why NADPH reduces only half of the heterodimer heme iron is that
the single reductase domain can provide electrons to only one of the
two heme irons.
If the reductase domain can only transfer electrons
to one of the two hemes in the heterodimer, which heme becomes reduced?
The apparent inability of a full-length iNOS monomer to transfer
NADPH-derived electrons to its heme iron implies that electron transfer
in the heterodimer may occur in trans, i.e. electrons transfer
from the reductase domain in the full-length subunit to the adjacent
oxygenase domain heme iron. However, alternative mechanisms that invoke
electron transfer within the full-length subunit could also explain the
results. For example, dimerization may change the reactivity of the
heme iron in the full-length subunit such that it can accept electrons
from its reductase domain. Indeed, our related work (22, 24) indicates that the heme iron in an iNOS
monomer is exposed to solvent and as a result is predominantly
six-coordinate low spin, which in cytochrome P-450 is associated with a
decrease in heme iron reduction potential (29, 30) .
Dimerization, which shifts the iNOS heme iron spin equilibrium back
toward high spin(22, 24) , could thus enable electron
transfer by increasing the reduction potential of the heme iron in the
full-length subunit. Alternatively, dimerization may cause a protein
conformational change in the full-length subunit that creates a path
for electrons to pass between its reductase and oxygenase domains. In
any case, our current results suggest that dimerization enables
electrons to transfer to a single heme in the heterodimer, and this is
sufficient for activating a normal rate of NO synthesis from L-arginine. Thus, mechanisms that invoke electron transfer
between two adjacent oxygenase domains, participation of both hemes in
oxidizing a single molecule of L-arginine, or concerted
catalysis between the subunits can be ruled out. In addition, the
general method we outline here to create and purify iNOS heterodimers
should now make it possible to study how sequence and structural
alterations in a single subunit will affect dimerization, electron
transfer, Hbiopterin binding, and other aspects of the
reaction mechanism.