(Received for publication, April 7, 1995; and in revised form, June 16, 1995)
From the
Nitric oxide (NO) has recently been recognized as an important
biomolecule playing diverse physiological roles. It is synthesized in
several different tissues from L-Arg and O, using
NADPH as an electron donor, by a family of heme-containing
catalytically self-sufficient monooxygenases known as nitric oxide
synthases (NOS). Recently, the CO complex of reduced NOS has been shown
to exhibit an absorption maximum near 450 nm, a characteristic spectral
feature of cytochrome P-450 (P-450). Yet, the amino acid sequences of
NOS and P-450 have no homology. To further probe the active site heme
coordination structure and the heme environment of NOS, we have
employed magnetic circular dichroism (MCD) and CD spectroscopy in the
present study. MCD spectra of several derivatives of rat brain neuronal
NOS strikingly resemble those of analogous derivatives of bacterial
P-450-CAM and fungal chloroperoxidase, two known thiolate-ligated heme
proteins. Given the proven fingerprinting capability of MCD
spectroscopy, this provides convincing evidence for endogenous thiolate
(cysteinate) ligation to the heme iron of NOS. Furthermore, the
heme-related Soret CD bands of NOS (positive) and P-450s (negative), as
represented by P-450-CAM, are almost mirror images, whereas
chloroperoxidase exhibits totally different CD band shapes. This
suggests that the active sites of NOS and P-450 may share some common
structural features, but significant distinctions exist between their
heme environments in certain aspects such as hydrophobicity or size.
Nitric oxide (NO), ()a moderately reactive
(relatively unstable) and potentially toxic inorganic free radical gas,
has recently been recognized as an important biomolecule playing
diverse physiological roles. It is synthesized in the brain cerebulleum
as a neurotransmitter, in vascular endothelial cells as an
``endothelial-derived relaxing factor'' which helps control
blood pressure, and in macrophages as a cytotoxic
agent(1, 2, 3, 4, 5, 6) .
The biosynthesis of NO in these tissues is carried out from L-Arg and O
by a family of enzymes called nitric
oxide synthases (NOS) (130-160 kDa) which utilize NADPH as an
electron donor. The overall reaction is a two-step oxidative conversion
of L-Arg to NO and L-citrulline via N
-hydroxy-L-Arg as the
intermediate(7) . The brain neuronal and macrophage NOSs are
soluble while endothelial NOS is membrane bound with its N-terminal
myristoylated (i.e. attached with n-tetradecanoic
acid)(1, 5, 6) .
NOS is unusual among
oxygenases in that it employs all of the following five prosthetic
groups, NADPH, FAD, FMN, iron protoporphyrin IX (heme), and
Hbiopterin(1, 3, 4, 5, 6) .
The first four components serve as redox co-factors, but
H
biopterin has been reported to regulate the enzyme
activity as a structural
stabilizer(1, 5, 6) . The binding sites for
the flavins and NADPH co-factors are located in the C-terminal half
(the reductase domain) and the binding site for heme in the N-terminal
half (the oxygenase domain). Thus, NOS is a catalytically
self-sufficient oxygenase and, in that respect, is similar to a
recently discovered fatty acid monooxygenase from Bacillus
megaterium, cytochrome P-450
(8) , except
for the H
biopterin requirement. Unlike
P-450
, however, NOS possesses an additional binding site
for calmodulin, the Ca
complex of which is absolutely
required for the catalytic activity of NOS. It has been reported that
NOS is only catalytically active in its homodimer form (1, 4) . The amino acid sequence of the reductase
domain of NOS has been found to be strikingly similar to that of
mammalian P-450 reductase (6) , which also has a high sequence
homology to the reductase domain of P-450
(8) .
However, the heme-binding domain of NOS does not share significant
amino acid sequence with P-450 monooxygenases (6) .
It has recently been reported that the CO complex of reduced NOS exhibits an absorption maximum at 443-447 nm(9, 10, 11) , a signature spectral feature of the P-450 enzymes. Recent resonance Raman spectral data of NOS are also consistent with thiolate ligation to the heme iron (12, 13) . These findings have added further interest in understanding the active site (i.e. the heme-binding site) structure of NOS in relation to its mechanism of action. The first step of the NOS-catalyzed conversion of L-Arg to NO and L-citrulline is N-hydroxylation (Scheme 1 in (10) ), which is one of the typical mono-oxygenation reactions carried out by P-450 enzymes(14, 15, 16) . However, the second step appears to proceed by a quite unusual mechanism(1, 4, 5, 17) . Therefore, it is important to further define the active site structure of NOS in close comparison with that of P-450. In view of this point, we have employed magnetic circular dichroism (MCD) as well as CD spectroscopy in this study to probe the active site structure of NOS using purified rat brain enzyme. MCD spectroscopy has been proven to have a powerful fingerprinting capability to characterize structurally undefined heme centers by spectral comparison to data for structurally defined iron porphyrins, both from synthetic model complexes and heme protein derivatives(18) . CD spectroscopy, on the other hand, can provide information about the environment surrounding the heme moiety since the method is sensitive to electronic interactions between the heme prosthetic group and nearby aromatic amino acid residues or peptide backbones(19) . We have found that MCD spectral features of all of the several NOS derivatives examined in this study strikingly resemble those exhibited by the analogous derivatives of two known thiolate-ligated heme proteins, P-450-CAM (used as a representative of P-450 monooxygenases) and chloroperoxidase(20) . This provides convincing support for an endogenous thiolate ligation to the heme iron of NOS. On the other hand, the major Soret CD bands of analogous derivatives of NOS and cytochrome P-450-CAM are almost mirror images, suggesting that their active site heme environments differ in certain aspects such as hydrophobicity or size.
Figure 4:
MCD (top) and optical absorption (UV-Vis) (bottom)
spectra of the ferrous-NO derivative. L-Arg-bound NOS
(-), camphor-bound P-450-CAM(- - -), and chloroperoxidase (
). The MCD and UV-Vis absorption spectra of P-450-CAM and
chloroperoxidase are taken from (38) . The absorption spectrum
of P-450-CAM is essentially identical to that reported by Peterson and
co-workers(35) . The spectra for NOS below 370 nm (UV-Vis) and 330 nm (MCD) are omitted because of
optical interference by a large amount of dithionite used. See the
legend to Fig. 1and ``Experimental Procedures'' for
the details for conditions and sample preparations for
NOS.
Figure 1:
MCD (top) and optical
absorption (UV-Vis) (bottom) spectra of the five-coordinate
ferric high spin derivative. Resting NOS without added L-Arg
(--), L-Arg-bound NOS (-), camphor-bound
P-450-CAM(- - -), and chloroperoxidase (
). The data
for NOS were obtained at 4 °C with an enzyme concentration of 31
µM in 40 mM Bis-Tris buffer (pH 7.4) containing 3
mM dithiothreitol, 10% glycerol, 2 µM H
biopterin, and 150 mM NaCl with and without
added 1 mML-Arg. The MCD spectra of P-450-CAM and
chloroperoxidase are taken from Refs. 27 (P-450-CAM) and 20
(chloroperoxidase), and are essentially identical to those reported by
Vickery et al.(28) and Dawson et al.(29) , respectively.
Effects of addition
of L-Arg (1 mM) to resting NOS on its optical
absorption and MCD spectra were relatively small but clearly detectable
as previously shown by difference absorption
spectroscopy(31, 32) . The Soret absorption peak (396
nm) shifted to a shorter wavelength (393 nm) with a concomitant
increase in its intensity by 10%. In the visible region,
absorbance between 540-600 nm decreased slightly while the peak
at
643 nm increased. As compared with substrate-bound NOS, the MCD
spectrum of the resting enzyme has slightly smaller intensities at
395 (Soret trough),
425, and
560 nm, but increased
intensities at
410,
520, and
580 nm. Based on the
established spectral (both optical absorption and MCD) and spin-state
changes accompanying the conversion of substrate-free ferric P-450-CAM
(six-coordinate, low spin) to its camphor-bound form (five-coordinate,
high spin)(20, 33, 34) , the observed
spectral changes for NOS suggest that upon substrate binding, the
predominantly high spin (mixed spin) state of ferric NOS becomes almost
exclusively high spin.
NOS binds NO, its catalytic reaction product,
to form a ferric-NO adduct (for its significance in catalysis, see the
ferrous-NO NOS section described below) which exhibits absorption peaks
at 442, 545, and 576 nm and a shoulder at 480 nm (spectrum not
shown). The spectrum we obtained is very similar to that reported by
Wang et al.(13) except for slight (4 nm) blue-shifts
in the visible region peak positions for our sample and the lack of a
shoulder between 600-650 nm. Ferric P-450-CAM (35) and
chloroperoxidase (26) also form a complex with NO which
exhibits sharp Soret and visible (
and
) peaks. MCD spectral
features of the NO adducts of the three heme proteins (not shown),
which consist of three sets of derivative-shaped bands with crossover
points corresponding to the absorption peak positions, are similar in
band shapes, but distinguishable in band positions (red-shifted by
10-20 nm), from those observed for histidine-ligated heme
proteins(36) .
With regard to this point, high performance liquid
chromatography analysis of L-Arg in purified resting NOS after
denaturation indicated the presence of no more than 0.08 mol equivalent
of L-Arg (per mol of heme) in the enzyme. ()Yet,
predominantly high spin resting NOS could be converted to a low
spin-type derivative (Soret peak at 416 nm with a shoulder at
390
nm) by gel-filtration column chromatography(32) , followed by
concentration (
0.5 mM), dilution (by 1000-fold), and
storage overnight at 5 °C. (
)The resulting sample was
too low in concentration to be examined with MCD spectroscopy. Addition
of L-Arg (1 mM) to this NOS sample regenerated a high
spin species (
=
393 nm). These
observations suggest that the predominantly high spin nature of resting
NOS could result from an endogenous source within the protein which can
be replaced with L-Arg or can slowly dissociate from the
heme-binding site upon dilution of the enzyme. Thus, the true spectral
nature of L-Arg-free native NOS and the identity of such an
endogenous residue remain uncertain.
Figure 2:
MCD (top) and optical absorption
(UV-Vis) (bottom) spectra of the deoxy-ferrous
(five-coordinate high spin) derivative. L-Arg-bound NOS
(-), camphor-bound P-450-CAM (- - -), and chloroperoxidase
(
). Note that the MCD spectra below and above 500
nm are plotted in different scales. The spectra below 360 nm for NOS
and P-450-CAM are omitted because of optical interference by large
absorbance (
= 315 nm) of dithionite used as
a reductant. The MCD spectra of P-450-CAM and chloroperoxidase are
taken from Refs. 27 (P-450-CAM) and 20 (chloroperoxidase), and are
essentially identical to those reported by Vickery et al.(28) and Dawson et al.(29) ,
respectively. See the legend to Fig. 1and ``Experimental
Procedures'' for the details for conditions and sample
preparations for NOS.
Optical absorption and MCD spectra of substrate-bound ferrous-CO NOS are displayed in Fig. 3together with the previously reported spectra of P-450-CAM (37) and chloroperoxidase(26) . The three enzymes have absorption peaks in both the Soret and visible regions at nearly the same positions and exhibit MCD spectra similar to each other as well as to a thiolate-ligated ferrous-CO heme model (spectrum not shown)(20) . Their MCD spectra consist of a relatively intense, symmetrical derivative-shaped spectral features in the Soret region. In the visible region, the MCD spectrum of NOS is more similar to that of P-450-CAM than to that of chloroperoxidase which exhibits slightly better band shape resolution.
Figure 3:
MCD (top) and optical absorption
(UV-Vis) (bottom) spectra of the ferrous-CO derivative. L-Arg-bound NOS (-), camphor-bound P-450-CAM(- - -), and
chloroperoxidase (
). Note that the MCD spectra
below and above 480 nm are plotted in different scales. The spectra for
NOS below 380 nm (UV-Vis) and 350 nm (MCD) are
omitted because of optical interference by a large amount of dithionite
used. The MCD spectra of P-450-CAM and chloroperoxidase are taken from
Refs. 37 and 26, respectively. The MCD spectrum of P-450-CAM is similar
to that reported by Vickery et al.(28) . See the
legend to Fig. 1and ``Experimental Procedures'' for
the details for conditions and sample preparations for
NOS.
Substrate-bound ferrous-NO NOS exhibits optical absorption and MCD
spectra which also closely resemble those of P-450-CAM and
chloroperoxidase (38) as shown in Fig. 4. The optical
absorption spectrum of the NOS complex is essentially identical to that
reported by Wang et al.(13) . Ferrous-NO NOS has been
shown to be formed as an inhibited species during the catalysis only
under O-limited conditions, although the ferric-NO
derivative has not been detected under such conditions(13) .
The MCD spectra of the three proteins in the Soret region are
asymmetric and have a predominant peak at
430 nm. The overall
features of the visible region MCD spectra of the three heme proteins
are similar. However, the spectrum of ferrous-NO NOS is more similar to
that of P-450-CAM than to that of chloroperoxidase which exhibits a
slightly more resolved band pattern as in the ferrous-CO case.
Figure 5:
Soret
CD spectra of the ferric high spin (five-coordinate) (A),
deoxy-ferrous (high spin, five-coordinate) (B), ferrous-CO (C), and ferrous-NO (D) derivatives. Resting NOS
(--) (A only), L-Arg-bound NOS (-),
camphor-bound P-450-CAM(- - -), and chloroperoxidase (
). The spectra of P-450-CAM are taken from Refs. 39 (A),
27 (B), and 37 (C and D) and are essentially
identical to those (for A-C) reported by
Peterson(33) , except that the intensities in (33) should be multiplied by 10
. The CD unit used
in this paper,
(the molar extinction coefficient, in
mM
cm
) is related to
[
] (the molar ellipticity, in
deg
cm
/decimole) as:
=
[
]/2.303(4500/
)
3.0
10
[
] ((19) ). All of the chloroperoxidase
spectra were obtained in this work in 0.1 M potassium
phosphate, pH 6.0, and 4 °C with enzyme concentrations of
100
µM using a 0.1-cm cuvette. Note that the ordinate
scale for B is expanded by a factor of two as compared
with the scale for the others. The spectra for the ferrous derivatives
for NOS below 320 nm (B-D) and for P-450-CAM below 360 nm (B) are omitted because of optical interference by large
amounts of dithionite used. See the legend to Fig. 1and
``Experimental Procedures'' for the details for conditions
and sample preparations for NOS.
The crystal structure of P-450-CAM revealed that its active site heme environment is composed of mostly nonpolar amino acid residues(43) . Tyr and Thr (a conserved residue for P-450s) are the only polar groups directly surrounding the heme. Significantly, P-450s lack a distal His and another polar group (such as Arg), a pair of which are considered to be important for peroxidase catalysis(43) . Moreover, the known tertiary structures of several peroxidases and of P-450-CAM differ (43) . Chloroperoxidase has a peroxidase-type polar heme environment (26) and an active site topology different from that of P-450-CAM(44) . It is thus not necessarily unexpected that P-450-CAM and chloroperoxidase exhibit totally different Soret CD band patterns as shown in Fig. 5. On the other hand, the Soret CD spectra of NOS and P-450-CAM appear to differ only in sign but are similar in shape.
Certain His-ligated monomeric Hbs such as soybean
leg Hb and lamprey Hb exhibit negative Soret CD bands while mammalian
Mbs have positive bands (19) . Yet, their tertiary structures
are similar, except that leg Hb (as well as lamprey Hb) has a broader
heme distal site than the Mbs (45) . Interestingly, binding of
alkyl isocyanides to deoxy-ferrous lamprey Hb converts its negative
Soret CD band to a positive band in a manner that is dependent on the
length (hydrophobicity) of the alkyl group(19) . Thus, the sign
of the Soret CD bands of heme proteins appears to be sensitive to the
size and/or hydrophobicity of the heme environment. The Soret CD band
sign also changes upon binding of SH (but not alkyl-
or aryl-thiolates) to the heme iron of ferric P-450-CAM (negative to
positive sign) (39) or upon flipping of the heme by 180°
about an inplane axis in ferrous-CO sperm whale Mb (positive to
negative sign)(46) .
Thus the mirror image Soret CD bands for NOS and P-450-CAM may be attributed to likely differences in size or hydrophobicity of their active sites heme environments. Without having tertiary structural information available for NOS, however, it is not possible at present to assess the size of its active site in relation to that of P-450-CAM. Nevertheless, the heme environment of NOS is likely to be more polar than that of P-450-CAM since NOS binds a highly polar substrate, L-Arg.