From the Department of Pharmaceutical Chemistry,
University of California, San Francisco, California 94143-0446 and the § Department of Biochemistry and Molecular Biology,
Oregon Graduate Institute School of Science and Engineering at
Oregon Health Sciences University,
Beaverton, Oregon 97006-8921
Received for publication, October 30, 2002, and in revised form, November 13, 2002
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ABSTRACT |
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NO and CO may complement each other as signaling
molecules in some physiological situations. We have examined the
binding of NO to human heme oxygenase-1 (hHO-1), an enzyme that
oxidizes heme to biliverdin, CO, and free iron, to determine whether
inhibition of hHO-1 by NO can contribute to the signaling interplay of
NO and CO. An Fe3+-NO hHO-1-heme complex is formed
with NO or the NO donors NOC9 or
2-(N,N-diethylamino)-diazenolate-2-oxide·sodium salt.
Resonance Raman spectroscopy shows that ferric hHO-1-heme forms
a 6-coordinated, low spin complex with NO. The Nitric oxide (NO)1
functions as a signaling molecule in a diversity of physiological
responses, including vasodilation and regulation of normal vascular
tone, neuronal signal transmission, cytotoxicity against pathogens and
tumors, and regulation of cellular respiration (1-3). Most of these
responses result from interaction of NO with the heme group of the
receptor guanylyl cyclase (1). A role akin to that of NO in signaling
pathways has also been postulated for CO (4). CO is produced in mammals
from heme by two heme oxygenases, HO-1 and HO-2 (5-8). The involvement of CO has been invoked as a factor in atherosclerosis (9), psoriasis
(10), vascular constriction (11), chronic renal inflammation (12),
cellular protection (13), hyperoxia-induced lung injury (14), and other
physiological situations. The role of CO as an NO-like signaling
molecule has received strong support from studies of heme oxygenase and
nitric-oxide synthase knockouts (15), but much of the evidence,
particularly that which depends heavily on inhibition of heme oxygenase
by metalloporphyrins such as tin protoporphyrin IX, is tainted by
ambiguities concerning the specificity of the inhibitors (16).
Nevertheless, the collective evidence makes a persuasive case for at
least a limited role for CO in mammalian signaling systems.
Evidence has accumulated that interactions of CO and NO may influence
the physiological responses to each of these agents through
interactions at the level of the biosynthetic enzymes. Thus, NO has
been shown to elevate the levels of heme oxygenase-1 mRNA and
protein (17-25), and this response appears to be mediated by a
guanylate cyclase-independent mechanism that may subserve a more
generalized antioxidant response (18). Conversely, CO has been reported
to elevate the steady state level of NO (26), but increased levels of
heme oxygenase have been shown to decrease NO concentrations, possibly
by consuming the heme required for assembly of the nitric-oxide
synthases (27-29).
As both NO and CO are small molecules that can coordinate to the iron
in heme proteins, it is possible that at physiological concentrations
NO may directly inhibit the heme oxygenases, and conversely, CO may
inhibit the nitric-oxide synthases. In studies of the identity of the
proximal ligand in HO-1, NO has been shown by resonance Raman, and EPR
to bind to the ferrous heme iron atom (30, 31). In HO-2, a heme
regulatory motif binds a secondary non-catalytic heme that binds NO
and, through an undetermined mechanism, inactivates the protein (32).
Indirect evidence also exists for the inhibition of heme oxygenase in
tissue homogenates by endogenously formed NO (33), but no focused study
has been carried out of the inhibition of HO-1 by NO.
Materials--
NADPH, ampicillin, heme, bovine serum album,
glucose, horse myoglobin, MAHMA NONOate (NOC9), and NO gas (98.5%)
were purchased from Sigma. High purity argon (99.98%) was from
Matheson (Newark, CA).
2-(N,N-Diethylamino)-diazenolate-2-oxide·sodium salt
(Dea/NO) was obtained from Alexis Corp. (San Diego, CA). All chemicals were used without further purification. The NO donors have the following half-lives in 0.1 M potassium phosphate buffer at
pH 7.4: NOC9, 3 min at 22 °C and 1-2 min at 37 °C; Dea/NO 16 min at 22 °C and 2-4 min at 37 °C.
Enzymes--
Catalase and glucose oxidase were from Sigma. Rat
biliverdin reductase and rat cytochrome P450 reductase were purified by published procedures (34-36). The hHO-1 construct used encoded human
heme oxygenase-1 lacking the 23 C-terminal amino acids (37). Oligonucleotide synthesis was carried out by Invitrogen, through the
Cell Culture Facility of the University of California, San Francisco.
Mutants of hHO-1 were generated with a Quick-change site-directed
mutagenesis kit (Stratagene). Plasmid purifications and bacterial
transformations were performed by standard procedures (38).
Transformants were screened initially by ampicillin resistance and
confirmed by sequence analysis. Wild type hHO-1 and its E29A, G139A,
S142A, D140A, G143A, G143F, and K179A/R183A mutants were expressed,
purified, and reconstituted with heme as reported previously (36, 39,
40). All experiments using these proteins were carried out in 0.1 M potassium phosphate buffer at pH 7.4 (standard buffer),
unless otherwise stated.
NO Treatments--
The concentrated NO donor solution stocks
were prepared fresh in 0.01 N NaOH. NOC9 and Dea/NO are
stable under alkaline conditions, but they decompose spontaneously when
small amounts of the stock solutions are added to the standard pH 7.4 buffer. Concentrated stock solutions of the NO donors were used to
minimize the changes in pH. NOC9 treatment was carried out at room
temperature (~25 °C) and that with Dea/NO at 37 °C. These
temperatures were chosen so that the two NO donors had similar rates of
NO release. All treatments with NO donors were performed aerobically.
The NO gas solutions used in stopped-flow experiments were made
anaerobic by bubbling NO into argon-equilibrated standard buffer.
The concentration of the NO donors in the enzyme binding assays was
varied at a fixed enzyme concentration of 4 µM. NO
binding was monitored at wavelengths between 250 and 700 nm. Formation of the hHO-1-heme Fe3+-NO complex was determined from the
ratio of the 416 to 404 nm absorbance. Formation of the corresponding
met-Mb Fe3+-NO complex was estimated from the ratio of the
absorbance at 417 to 408 nm.
Spectroscopic Characterization--
The UV-visible spectra of
the hHO-1 proteins were recorded in standard buffer on a Cary Varian
model 1E spectrophotometer.
The enzyme concentration for RR experiments was ~125 µM
in 0.1 M potassium phosphate buffer at pH 7.4. 14NO and 15NO gas purchased from Aldrich was
bubbled through a 0.1 M KOH solution to remove higher
nitrogen oxides. Formation of the NO adduct was achieved by addition of
an NO-saturated buffer solution to an argon-purged hHO-1 solution in
the Raman capillary cell to reach a final concentration of ~1
mM NO. Before freezing the samples, the completion of the
reaction was confirmed by UV-visible spectroscopy in the same Raman
capillary cell using a Cary 50 spectrophotometer. Once the RR
experiments were completed, the sample was thawed to obtain its
UV-visible spectrum and confirm the stability of the complex during the
laser illumination at 90 K.
RR spectra were obtained on a custom McPherson 2061/207 spectrograph
(set at 0.67 m with variable gratings) equipped with a Princeton
Instruments liquid N2-cooled CCD detector (LN-1100PB). Kaiser Optical supernotch filters were used to attenuate Rayleigh scattering. Excitation sources consisted of an Innova 302 krypton laser
(413 nm). Spectra were collected on frozen samples kept at ~90 K with
N2 cold finger in a backscattering geometry (41). Frequencies were calibrated relative to indene and CCl4
standards and are accurate to ±1 cm
In the FTIR experiments, the enzyme concentration was increased to ~1
mM using a Microcon 10 ultrafiltration device (Amicon). The
concentrated hHO-1 solution was made anaerobic in a vial before exchanging the head space with pure NO gas to reach a final
concentration of ~2 mM NO in solution. The protein sample
was then injected into an IR cell consisting of CaF2
windows separated by a 50-µm Teflon spacer. The formation of the NO
adduct was confirmed by UV-visible spectroscopy in the IR cell using a
Cary 50 spectrophotometer.
FTIR spectra were obtained at room temperature on a PerkinElmer Life
Sciences system 2000 equipped with a liquid N2-cooled MCT
detector. Sets of 20-min accumulations were acquired at a 2-cm Stopped-flow Kinetic Analyses--
Kinetic studies were carried
out with an SF-61 DX2 double mixing stopped-flow system (Hi-Tech
Scientific). The stopped-flow instrument was made anaerobic by first
rinsing with argon equilibrated standard buffer, followed by an
overnight incubation with anaerobically prepared
catalase/glucose/glucose oxidase solution. Kinetic traces were taken at
room temperature at wavelengths between 380 and 700 nm. The data were
analyzed with KinetAsyst2 software and were fit to a first-exponential
expression. In this experiment, 3 µM ferric hHO-1-heme
and 1.5 µM horse met-Mb were used. The solubility of NO
gas under 1 atmosphere at 20 °C is ~2 mM. Dilution of
the NO-saturated solution was made with argon-equilibrated standard buffer in gas-tight syringes (Hamilton).
Bilirubin Activity Assay--
The reaction mixture contained
ferric hHO-1-heme (1 µM), hemin (30 µM),
bilirubin reductase (4 µM), and cytochrome P450 reductase (0.4 µM) in the standard buffer. The reaction was
initiated by the addition of NADPH (400 µM). The
production of bilirubin at room temperature was monitored at 468 nm for
100 s. The initial rate of the reaction was calculated using the
value Absorption Spectrum of the hHO-1-Heme Fe3+-NO
Complex--
Ferric hHO-1-heme has a Soret maximum at 405 ± 1 nm
in 0.1 M phosphate buffer at pH 7.4. Upon addition of a 1 mM concentration of the NO donor NOC9 or Dea/NO, the ferric
hHO-1-heme Soret maximum immediately shifted to 416 nm (Fig.
1). This spectroscopic shift was
reversible, suggesting that the NO species generated by the NO donors
interacted with the heme in hHO-1. Ferric heme alone has a very broad
Soret absorption at about 380 nm, and its incubation with the NO donors
caused a sharpening of the Soret band with a decrease in its absorption
intensity (not shown). In contrast to the reaction with ferric
hHO-1-heme, the spectral change observed with free hemin was not
reversible.
RR and FTIR Spectroscopy--
The ferric hHO-1-heme NO complex
prepared by bubbling NO through a solution of the ferric hHO-1-heme
complex was observed to be very photolabile in the resonance Raman
experiments, but at 90 K the efficiency of this process was
sufficiently diminished to allow the experiments to be carried out. As
shown previously (31), the spectrum of ferric hHO-1 reveals a
hexacoordinated high spin/hexacoordinated low spin equilibrium with
In heme ferric nitrosyl complexes, the
In the low frequency region of the RR spectra (Fig.
4), the identification of the Fe-N-O
vibrational modes was facilitated by the use of isotopic labeling and
the close similarity of these frequencies with those observed in other
hemoproteins (43). In the met-Mb ferric-nitrosyl complex, the
Kinetic Analyses of NO Binding to Horse Met-Mb and Ferric
hHO-1-Heme--
To study further the formation of the hHO-1-heme
(Fe3+-NO) complex, stopped-flow experiments were employed
to determine the kon,
koff, and Kd values for the
binding of NO. The experiments in this instance were performed
anaerobically with NO gas instead of NO donors. Our experimental
conditions in the case of met-Mb gave kon,
koff, and Kd values similar
to those in the literature (49), validating the methodology that was
employed (Table I). The
kon for formation of the hHO-1
Fe3+-NO complex was found to be ~50-fold faster, and
koff 10-fold slower, than for horse met-Mb,
resulting in an ~500-fold tighter binding of NO. The resulting
calculated Kd value for the binding of NO to ferric
hHO-1-heme is 1.4 µM.
NO Binding to Mutant Ferric hHO-1-Heme Complexes--
As shown in
Fig. 5, the binding of NO to ferric
hHO-1-heme can also be seen with the NO donors NOC9 and Dea/NO. The
concentrations of NOC9 and Dea/NO that cause half-maximal binding of NO
to ferric hHO-1-heme are ~80 and 100 µM, respectively,
although the actual concentration of NO in these experiments is much
lower. Under the same conditions the binding of NO to horse met-Mb did
not reach a plateau even at much higher concentrations of the NO
donors, as expected from the higher Kd value for
this protein (Table I and Fig. 5).
The high affinity of the ferric hHO-1-heme complex for NO is unusual
for a ferric hemoprotein, although the binding of NO to ferric catalase
reportedly occurs with a comparably low Kd of ~0.5
µM (45, 46). In our efforts to identify specific residues that contribute to this high binding affinity, we investigated the
binding of NO to hHO-1 in which individual active site amino acids had
been mutated. hHO-1 residues that could stabilize distal iron ligands
by hydrogen bonding or other interactions include Glu-29, Gly-139,
Asp-140, Ser-142, Gly-143, Lys-179, and Arg-183. Glu-29 is close enough
to form a hydrogen bond with His-25, the proximal iron ligand (47).
Gly-139, Asp-140, and Gly-143 are part of a hydrogen bonding network on
the distal side that interacts with distal ligands, and mutation of any
one of them to an alanine results in dissociation of the distal water
ligand (36, 39). Mutation of Ser-142 shift the pKa
value of the distal water ligand toward more basic
values.2 Lys-179 and Arg-183
appear to interact with the propionate carboxyl groups of the heme and
may be important for proper orientation of the heme (47, 48). We have
therefore examined the binding of NO to the E29A, G139A, D140A, S142A,
G143A, G143F, and K179A/R183A mutants, all of which were heterologously
expressed in Escherichia coli, purified, and found to have
appropriate Soret maxima (Table II). The
values for half-saturation of NO binding using NOC9 as the NO donor
show that none of the mutations altered the NO binding affinity by more
than a factor of ~2 (Table II). Thus, the high NO affinity of ferric
hHO-1-heme is not very sensitive to the identities of these active site
residues despite the fact that some of them interact strongly with the
distal water ligand that is replaced by NO. Indeed, the high affinity
for NO appears to be insensitive to the presence or absence of the
distal water ligand, as the water ligand is absent in at least the
G139A, G143A, and D140A mutants (36, 39).
Inhibition of Bilirubin Formation--
Incubation of a system
consisting of ferric hHO-1-heme, biliverdin reductase, cytochrome P450
reductase, and hemin with increasing concentrations of NOC9 for various
times, followed by addition of NADPH to assay bilirubin formation,
clearly demonstrated that the protein is reversibly inhibited by NO
(Fig. 6). Little inhibition was
observed with a 1 µM concentration of the NO donor at any time point, but inhibition was observed when the donor concentration was raised to 10 µM. With a 50 µM
concentration of NOC9, bilirubin formation was completely suppressed
when the assay was carried out without NOC9 preincubation, but activity
was recovered when the assay was carried out after 10 min or longer of
preincubation. Similar results were obtained with 100-600
µM NOC9, except that inhibition was observed after even
longer preincubation periods (Fig. 6). A 100 µM
concentration of NOC9 is required to half-saturate the active site of
ferric hHO-1-heme with NO, and this concentration therefore must
roughly correspond to the Kd of 1.4 µM for NO itself. The fact that complete inhibition is observed with half
of this NOC9 concentration, and some inhibition even at lower concentrations, suggests that inhibition may also reflect some binding
of NO to the ferrous intermediate obtained when the enzyme complex is
reduced by cytochrome P450 reductase. Furthermore, the inhibition, like
formation of the spectroscopically determined Fe3+-NO
complex, is reversible. Inhibition is therefore lost with time as the
NO donor is exhausted and the NO concentration decays. This is most
clearly seen in Fig. 7, in which the
recovery of bilirubin forming activity has been measured as a function
of the time a fixed concentration of the NO donor is preincubated with
the enzyme prior to carrying out the activity assay. In this case, two
different NO donors were employed, NOC9 and Dea/NO. As the figure
shows, the activity of the enzyme is completely inhibited when there is
no preincubation and for NOC9 even after a 20-min preincubation, but
the activity with both NO donors recovers as the preincubation time is
prolonged. However, only ~80% of the activity was recovered in these
experiments. To determine whether stable NO donor decomposition
products inhibit hHO-1 activity, ferric hHO-1 was incubated with
solutions of decomposed NO donors for 40 min, and the bilirubin forming
activity was then assayed. As shown in Fig.
8, the products of decomposition of the
NO donors have some hHO-1 inhibitory activity. Only 80% of the control
activity was observed in the presence of the decomposition products
from a 1.0 mM concentration of the NO donors, readily
explaining the recovery of only 80% of the enzyme activity in the
incubations with the NO donors (Fig. 7). Control experiments in which
NOC9 was added to an incubation only containing biliverdin reductase and P450 reductase, followed by addition of hHO-1, heme, and NADPH resulted in negligible inhibition, confirming that hHO-1-heme is the
site of inhibition. High concentrations of NOC9 added to heme alone
prior to addition of the other components of the catalytic system also
inhibited turnover (results not shown), in accord with the finding that
the spectrum of heme in solution was altered by the NO donors.
NO, unlike O2 and CO, can bind to the iron atom of
hemoproteins in both the ferric and ferrous states, although the
binding affinity is generally much higher for the ferrous state (49, 50). For example, at pH 7.4 and 25 °C, the Kd
values for the binding of NO to ferrous deoxymyoglobin and ferric
met-Mb are 7 × 10 The basis for this difference in binding affinity is unclear. The
overall vibrational characterization of these {Fe(NO)}6
structures in hHO-1 and met-Mb demonstrates that these complexes share
the same bonding geometry and strength. Resonance Raman studies show
that the hHO-1-heme Fe3+-NO complex is 6-coordinated low
spin (Fig. 2), as is the met-Mb complex (30). Furthermore, FTIR shows
that the In view of the similarities in coordination properties, the differences
in the Kd for binding of NO to ferric hNO-1-heme and
met-Mb presumably stem from other differences in the active sites of
the two proteins. The ~50-fold increase in kon
rate for NO in hHO-1 compared with met-Mb may reflect a higher steric
hindrance in the distal pocket of met-Mb. In both these ferric proteins the sixth iron coordination site is occupied by a water molecule that
must be displaced for NO to bind, but displacement of the water
molecule in hHO-1 may require relatively little protein side chain
rearrangement relative to that which occurs in met-Mb (58). In an
attempt to identify residues that might contribute to the unique
binding affinity of NO for ferric hHO-1-heme, we have mutated seven
residues that could contribute to this affinity. However, in no
instance did the mutation cause more than a 2-3-fold change in the
observed Kd value. Mutations of Asp-140 and Gly-143
slightly decreased the affinity; those of Gly-139 slightly increased
it; and those of Glu-29, Ser-142, and Lys-179/Arg-183 did not
significantly alter it (Table II). Even though these residues, particularly Asp-140, Gly-139, Ser-142, and Gly-143, have been shown by
mutagenesis and the crystal structure to interact with the distal water
ligand (36, 39, 47), they do not appear to be key determinants of the
high NO affinity of ferric hHO-1-heme.
The unusually low Kd value for the binding of NO to
ferric hHO-1-heme suggests that the catalytic turnover of the enzyme
should be inhibited by NO. Furthermore, as the catalytic cycle of hHO-1
traverses the ferrous state, NO could bind not only to the ferric but
also to the ferrous protein, again inhibiting the enzyme. The
Kd value for the binding of NO to the ferrous
protein is expected to be much lower than that for the ferric enzyme,
but the Kd value for binding to the ferric enzyme is
already low enough that significant inhibition of the protein could
occur at physiological or pathological NO concentrations, which range
from 50 nM to 5 µM (59-65). Indeed,
inhibition of heme oxygenase was observed when the enzyme was incubated
with 100 µM concentrations of either NOC9 or Dea/NO (Fig.
7). These concentrations of NO donors provide a sufficient flux of NO
to inhibit completely the enzyme at short periods (10-20 min) of
incubation, but the inhibition is lost at longer incubation times as
the NO donors are exhausted and the NO concentration falls. Inhibition
by NO is reversible (Fig. 7), although a full recovery of the hHO-1 activity is not observed due to a residual inhibition caused by persistent NO donor decomposition products distinct from NO (Fig. 8).
The inhibition of heme oxygenase by NO and NO donors has received
little attention (32, 33). A possible role for NO as an in
vivo inhibitor of heme oxygenase is suggested by the report that
L-arginine, the substrate of the nitric-oxide synthases, inhibits heme oxygenase activity when added to spleen or brain homogenates, whereas analogous addition of L-NAME, an
inhibitor of the nitric-oxide synthases, stimulates the heme oxygenase
activity (33). In the only molecular level study prior to this work, Ding et al. (32) reported that the NO donors
3-morpholinosydnonimine (SIN-1),
S-nitroso-N-acetylpenicillamine (SNAP), and
sodium nitroprusside inhibit HO-2 but not HO-1. The study focused on
the possible role of the non-catalytic heme binding domains that are
only present in HO-2. However, in that study the NO donors were removed
by dialysis prior to measuring the residual catalytic activity, so that
the reversible inhibition by NO reported here would not have been
detected because the NO would be removed at the same time as the NO
donors. Furthermore, Ding et al. (32) reported that enzyme
inactivation was not observed with HO-2 when the cysteine residues
within the two heme-binding motifs had been mutated to alanines. The
irreversible inhibition they observed therefore reflects modifications
related to the presence of the two cysteines, neither of which is
present in HO-1. This is consistent with the present findings that HO-1
is not irreversibly inactivated by NO or NO donors.
In sum, the present results clearly establish that NO inhibits HO-1 by
binding to the catalytic iron atom and show that this inhibition can
involve binding to even the ferric heme complex due to its unusually
high affinity for NO. The inhibition of heme oxygenase by NO occurs at
concentrations of this agent that are pathologically and possibly
physiologically accessible. The cross-talk between the nitric-oxide
synthase and heme oxygenase systems may play some role in the
pleiotropic responses that are associated with these two hemoprotein
signaling systems.
(N-O) vibration of
this complex detected by Fourier transform IR is only 4 cm
1 lower than that of the corresponding metmyoglobin
(met-Mb) complex but is broader, suggesting a greater degree of ligand
conformational freedom. The Fe3+-NO complex of hHO-1 is
much more stable than that of met-Mb. Stopped-flow studies indicate
that kon for formation of the hHO-1-heme Fe3+-NO complex is ~50-times faster, and
koff 10 times slower, than for met-Mb,
resulting in Kd = 1.4 µM for NO. NO
thus binds 500-fold more tightly to ferric hHO-1-heme than to met-Mb. The hHO-1 mutations E29A, G139A, D140A, S142A, G143A, G143F, and K179A/R183A do not significantly diminish the tight binding of NO,
indicating that NO binding is not highly sensitive to mutations of
residues that normally stabilize the distal water ligand. As expected
from the Kd value, the enzyme is reversibly inhibited upon exposure to pathologically, and possibly
physiologically, relevant concentrations of NO. Inhibition of hHO-1 by
NO may contribute to the pleiotropic responses to NO and CO.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1. CCl4
was also used to check the polarization conditions.
1 resolution on the samples and the identical cell
filled with buffer for background subtraction.
468 = 43.5 mM
1
cm
1 for the bilirubin product. To study the effects of NO
on hHO-1 activity, two sets of experiments were carried out. In one
set, biliverdin reductase, cytochrome P450 reductase, hemin, and hHO-1 were incubated with various concentrations of NOC9 for different times
before NADPH was added to measure the bilirubin forming activity. In
the other set, hHO-1 was incubated with 1 mM NO donor and
then the rest of the assay mixture was added for the activity assay.
Separately, hemin (30 µM) and bilirubin reductase (4 µM)-cytochrome P450 reductase (0.4 µM) were
separately incubated with the NO donors before the other components of
the reaction system were added, and bilirubin formation was quantitated.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Spectral change of ferric hHO-1-heme
(4.6 µM) in the presence of either
1.0 mM NOC9 or Dea/NO. The spectra were recorded in
the standard buffer. Arrows indicate the direction of
spectral change over time. The Soret band at time 0 was taken
immediately after adding the NO donor and is already that of the NO
complex.
3,
2, and
10 modes at
1482/1508, 1566/1585, and 1608/1640 cm
1, respectively
(Fig. 2A). After exposure to
NO, the porphyrin skeletal modes are observed at higher frequencies
with
4,
3,
2, and
10 at 1378, 1511, 1588, and 1645 cm
1,
respectively (Fig. 2B). These frequencies identify the NO
adduct as a hexacoordinated low spin complex, although a minor
hexacoordinated high spin population revealed by the weak
3 at 1483 cm
1 is assigned to some
photodissociation.
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Fig. 2.
High frequency region of the resonance Raman
spectra of the ferric (A) and ferric-nitrosyl
(B) hHO-1-heme complex. Spectra were obtained at
90 K using 413-nm excitation.
(N-O) is not
resonance-enhanced with Soret excitation, but it is easily observed in the FTIR spectrum. This vibration is detected at 1918 cm
1
in ferric hHO-1-heme, only 4 cm
1 lower than that in
met-Mb (Fig. 3). Such
(N-O)
frequencies are characteristic of linear six-coordinated
{Fe(NO)}6 complexes (42). A significant difference
between these two signals resides in the ~20-cm
1
half-width of this stretching mode in hHO-1 compared with the 9-cm
1 half-width observed in met-Mb. In met-Mb the
configuration of the nitrosyl group is clearly defined by the presence
of the imidazole ring from the distal histidine, but the absence of
distal polar side chains above the heme iron of hHO-1 and a greater
solvent accessibility may permit greater fluctuation of the NO ligand, resulting in substantial inhomogeneous broadening of the
(N-O).
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Fig. 3.
High frequency region of the FTIR spectra of
the ferric nitrosyl complex in met-Mb and hHO-1-heme.
(Fe-NO) and
(Fe-N-O) are observed at 595 (
6) and 573 (
11)
cm
1, respectively. In the ferric nitrosyl complex of
hHO-1, two bands at 596 and 588 cm
1 that shift to 590 and
573 cm
1 with 15NO are assigned to the
(Fe-NO) and the
(Fe-N-O), respectively.
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Fig. 4.
Low frequency RR spectra of the ferric
hHO-1-heme nitrosyl complex.
Kon, Koff, and Kd for formation of the
ferric hHO-1-heme and horse met-Mb Fe3+-NO complexes
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Fig. 5.
Binding of NO to 4 µM ferric hHO-1-heme and 4 µM horse met-Mb. The binding assay
was performed in the standard buffer. The data points are
the averages of two independent determinations, and the bars
indicate the positions of the two averaged values.
IC50 for NO binding to wild-type and mutant ferric
hHO-1-heme with NOC9 as the NO donor
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Fig. 6.
Effects of NO donors on the hHO-1 bilirubin
forming activity. The reaction system consisting of hHO-1, ferric
heme, biliverdin reductase, and cytochrome P450 reductase was
preincubated with increasing concentrations of NOC9 for the indicated
times, after which NADPH was added, and the formation of bilirubin was
measured. The preincubation and activity assays were both carried out
at room temperature. ND indicates no bilirubin formation was
detected. The values shown are the mean of duplicate determinations.
The bars indicate the range of the two values.
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Fig. 7.
Recovery of hHO-1 bilirubin forming activity
over time. A ferric hHO-1-heme (1 µM) solution was
preincubated with 1.0 mM NOC9 or Dea/NO after which the
bilirubin forming activity of the enzyme was determined by adding the
required additional reaction components.
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Fig. 8.
Effects of decomposed NO donors on the
bilirubin activity of hHO-1. NO donors (1 mM) were
allowed to decomposed at 37 °C for ~4 h, after which 1 µM ferric hHO-1-heme was incubated with the decomposed NO
donor solutions for 40 min prior to adding the necessary additional
components and assaying bilirubin formation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
6 and 905 µM,
respectively, a difference of roughly 108 (49, 51). The
binding of NO to ferric hemoproteins, however, occurs over a
considerable range, the tightest binding reported being
Kd = 0.5 µM for catalase (45, 46). The
binding of NO to the ferric hHO-1-heme complex with
Kd = 1.4 µM thus approaches the
tightest binding so far observed for any ferric hemoprotein. Detailed
analysis shows that this high affinity, when compared with the
~500-fold lower affinity for met-Mb, is due to a 50 times faster
kon and a 10 times slower
koff for NO (Table I).
(N-O) band of the hHO-1 Fe3+-NO complex is at
1918 cm
1, a value only 4 cm
1 lower than for
the met-Mb complex (Fig. 3) (52). This suggests that coordination of
the NO to the heme iron is similar in both hemoproteins, although the
broader bandwidth observed with the hHO-1 complex suggests that the NO
has greater mobility in that protein. Generally, as is the case for the
binding of NO to ferric hemoproteins, these results strongly argue that
the NO is bound perpendicular to the heme face rather than at an angle
(53-56), in contrast to its orientation when bound to ferrous
hemoproteins. Furthermore, in both met-Mb and ferric hHO-1-heme, the
iron in the absence of NO is coordinated to a water molecule. The
pKa values for deprotonation of these
iron-coordinated water molecules are similar in both proteins (30, 57),
again indicating similar coordination states and environment and
suggesting that the energy cost of displacing the water ligand
should be similar in both proteins.
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ACKNOWLEDGEMENTS |
---|
We thank Yi Liu for preparing the E29A, G139A, and S142A hHO-1 constructs and Luke Lightning for construction of the G143A, G143F, and K179A/K183A mutant plasmids.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants DK30297 (to P. R. O. M.) and GM18865 (to P. M. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: University of California School of Pharmacy, San Francisco, CA 94143-0446. Tel.: 415-476-2903; Fax: 415-502-4728; E-mail: ortiz@cgl.ucsf.edu.
Published, JBC Papers in Press, November 13, 2002, DOI 10.1074/jbc.M211131200
2 Y. Liu and P. R. Ortiz de Montellano, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: NO, nitric oxide; heme, iron protoporphyrin IX regardless of oxidation and ligation state; HO-1, heme oxygenase-1; hHO-1, truncated human HO-1; met-Mb, metmyoglobin; FTIR, Fourier transform infrared spectroscopy; RR, resonance Raman spectroscopy; NOC9, (Z)-1-[N-methyl-N-[6-(N-methylammoniohexyl)amino]]diazen-1-ium-1,2-diolate; Dea/NO, 2-(N,N-diethylamino)-diazenolate-2-oxide·sodium salt.
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