Disruption of an Active Site Hydrogen Bond Converts Human
Heme Oxygenase-1 into a Peroxidase*
Luke Koenigs
Lightning
,
Hong-wei
Huang§,
Pierre
Moënne-Loccoz§,
Thomas M.
Loehr§,
David J.
Schuller¶,
Thomas L.
Poulos¶, and
Paul R. Ortiz
de Montellano
From the
Department of Pharmaceutical Chemistry,
University of California, San Francisco, California 94143-0446, the
§ Department of Biochemistry and Molecular Biology, Oregon
Graduate Institute of Science and Technology,
Beaverton, Oregon 97006-8921, and the ¶ Department of Molecular
Biology and Biochemistry and Program in Macromolecular Structure,
University of California, Irvine, California 92697-3900
Received for publication, November 15, 2000, and in revised form, December 15, 2000
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ABSTRACT |
The crystal structure of heme oxygenase-1
suggests that Asp-140 may participate in a hydrogen bonding network
involving ligands coordinated to the heme iron atom. To examine this
possibility, Asp-140 was mutated to an alanine, phenylalanine,
histidine, leucine, or asparagine, and the properties of the purified
proteins were investigated. UV-visible and resonance Raman spectroscopy
indicate that the distal water ligand is lost from the iron in all the mutants except, to some extent, the D140N mutant. In the D140H mutant,
the distal water ligand is replaced by the new His-140 as the sixth
iron ligand, giving a bis-histidine complex. The D140A, D140H, and
D140N mutants retain a trace (<3%) of biliverdin forming activity,
but the D140F and D140L mutants are inactive in this respect. However,
the two latter mutants retain a low ability to form verdoheme, an
intermediate in the reaction sequence. All the Asp-140 mutants exhibit
a new peroxidase activity. The results indicate that disruption of the
distal hydrogen bonding environment by mutation of Asp-140 destabilizes
the ferrous dioxygen complex and promotes conversion of the ferrous
hydroperoxy intermediate obtained by reduction of the ferrous dioxygen
complex to a ferryl species at the expense of its normal reaction with
the porphyrin ring.
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INTRODUCTION |
Heme oxygenase (HO)1
catalyzes the regiospecific oxidation of heme to
-biliverdin, CO,
and free iron (1). All of the oxidative steps in the heme catabolic
pathway have been extensively studied over the past 30 years, but
significant gaps still exist in our understanding of this enzyme
system. During its catalytic cycle, HO consumes 3 eq of O2
and 7 reducing eq supplied by NADPH-cytochrome P450 reductase (P450
reductase) (2). The enzyme catalyzes a sequence of reactions that
includes the conversion of heme to
-meso-hydroxyheme,
-meso-hydroxyheme to verdoheme, and verdoheme to
-biliverdin (Fig. 1). The
intermediates remain bound to the enzyme throughout the catalytic cycle
until
-biliverdin is produced and released. It is remarkable that HO
can catalyze such a diverse set of reactions, because they involve the
oxidation of compounds that possess different electronic and
coordination properties and that have different reactivities with
O2. This enzyme is also distinguished from all other
hemoproteins in that the heme serves as the prosthetic group and
substrate, and the first oxidizing species appears to be a ferric
hydroperoxide (Fe(III)-OOH) rather than ferryl oxene (Fe(V)=O)
intermediate (3). These characteristics suggest that unique
interactions exist between the heme, the iron-bound O2, and
the amino acid residues within the active site of the enzyme.
In humans, HO exists in two well established forms, HO-1 and HO-2, that
share moderate (~45%) amino acid sequence identity but vary in their
inducibility and localization (4). HO-1, also known as heat shock
protein 32, is highly inducible and is the major form present in the
spleen, whereas HO-2 is a constitutive enzyme that is found in highest
concentration in the brain and testes (5). Under normal conditions,
HO-2 predominates by ~2-fold over HO-1 in the liver (4). However,
HO-1 can be up-regulated about 100-fold in response to various
oxidative stress agents and thus can be the dominant form in that
tissue. Due to their respective physiological locations, HO-1 is
considered to be important for heme homeostasis, and HO-2 is thought to
be primarily responsible for the effects of CO as a neurotransmitter
(4). Despite these differences, HO-1 and HO-2 exhibit similar heme
coordination and electronic properties and catalyze the same set of
reactions. By virtue of their ability to catabolize the oxidant heme
and to produce the antioxidant biliverdin, both enzymes are intricately involved in providing protection against the formation of reactive oxygen species and heme-mediated lipid peroxidation.
There is considerable interest in elucidating those factors that
control the reactivity of HO, because of its unique and intrinsically interesting catalytic mechanism and its role in several important physiological processes. HO is a membrane-bound protein, but truncated, water-soluble, fully active forms of human (hHO-1) and rat HO-1 have
been expressed in Escherichia coli (3, 7, 8). Furthermore, the x-ray crystal structures of truncated versions of human and rat
HO-1 have recently been determined (9, 10). The rat and human HO-1
isoforms share a high degree (~80%) of sequence identity (4).
Analysis of the hHO-1 crystal structure indicates that Asp-140, a
residue within the distal heme pocket, could facilitate catalytic
activity by hydrogen bonding to a water molecule that could, in turn,
hydrogen bond with the putative Fe(III)-OOH species (Fig.
2). In the present study, Asp-140 was
replaced by amino acids that vary in size, charge, and hydrophobicity,
and the resulting mutant proteins were characterized with regard to
their HO and peroxidase activities. The results clearly establish that
Asp-140 is of critical importance to the outcome of the catalytic
cycle.

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Fig. 2.
The active site of hHO-1 depicting the
Asp-140 residue and its proximity to the heme iron and nearby residues
based on the x-ray crystallographic coordinates (9). The distal
water ligand is depicted as a sphere.
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EXPERIMENTAL PROCEDURES |
Materials--
H2O2 (30%), NADPH,
ampicillin, isopropyl-
-D-thiogalactopyranoside, hemin,
bovine serum albumin, sodium dithionite, and guaiacol were obtained
from Sigma. High purity argon (99.998%), CO (99.95), and
O2 (99.9%) were obtained from Matheson (Newark, CA) and Aldrich.
Enzymes--
Catalase was from Sigma. Rat P450 reductase was
expressed and purified from bacterial cultures according to published
procedures (11). Biliverdin reductase was also expressed and purified
according to a published procedure (12) with the following
modifications: (i) BL-21 cells were used for expression in LB-Amp
media; (ii) cells were grown at 30 °C for 8 h after addition of
isopropyl-
-D-thiogalactopyranoside; (iii) the Ni(II)
column was washed with 20 mM Tris-HCl (pH 8.0) containing 5 mM imidazole; and (iv) biliverdin reductase was eluted from
the Ni(II) column with 20 mM Tris-HCl (pH 8.0) containing 100 mM imidazole.
Expression and Purification of hHO-1 and Asp-140
Mutants--
Wild-type hHO-1 and its Asp-140 mutants were expressed
using E. coli strain DH5
(F'araD(lac-proAB)rpsLf80dlacZDM15hsdR17) and the hHO-1 construct encoding the human liver protein lacking the 23 C-terminal amino acids (7). The general sequence for the mutant primers
that were employed was 5'-ACC CGC TAC CTG GGG XXX CTG TCT
GGG GGC-3' (Life Technologies, Inc., Cell Culture Facility, University
of California, San Francisco), where XXX denotes the
nucleotides encoding the desired mutation. The hHO-1 Asp-140 mutants
were generated using the polymerase chain reaction and QuickChange
site-directed mutagenesis kit from Stratagene (La Jolla, CA). Plasmid
purification, subcloning, and bacterial transformations were carried
out by standard procedures (13). Antibiotic selection using ampicillin
afforded a high frequency of mutants. Transformants were confirmed by
sequence analysis (Biomolecular Resource Center, University of
California, San Francisco). The hHO-1 proteins were expressed,
purified, and reconstituted with hemin according to published
procedures (3, 7) except that, after reconstitution, a hydroxyapatite
chromatographic step using 75 mM potassium phosphate (pH
7.4) as the elution buffer was found to be optimal. The proteins were
obtained with yields ranging from 0.5 to 5 mg of purified protein per
liter. All experiments using the purified proteins were performed in
triplicate in 100 mM potassium phosphate buffer (pH 7.4)
(standard buffer) unless otherwise stated.
Spectral Characterization--
The UV-visible spectra of the
hHO-1 proteins were recorded in standard buffer on a Hewlett-Packard
8452A diode array spectrophotometer. The ferrous carbon monoxide
(Fe(II)-CO) complexes were formed by saturating the solutions with CO
by bubbling with the gas for 1 min followed by reduction of the Fe(III)
complexes with a few grains of sodium dithionite. The ferrous dioxygen
(Fe(II)-O2) complexes were formed by bubbling the samples
containing the Fe(II)-CO complexes with O2 for up to
30 s. The pKa value of the water ligand
coordinated to the iron (if present) of hHO-1 and its Asp-140 mutants
was determined by recording the UV-visible spectrum over the pH range
6-11.
RR Spectroscopy--
Typical enzyme concentrations for RR
experiments were 100-2500 µM in standard buffer.
Microcon 10 ultrafiltration devices (Amicon) were used for buffer and
water exchange. A final enrichment of 80% 18O- or
2H-labeled water (95% 18O, Cambridge Isotope
Laboratory, 99.9% 2H, Aldrich) was achieved in the
experiments using labeled water. Reduction to the Fe(II) state was
achieved by adding microliter aliquots of a sodium dithionite solution
(10 mM) to an argon-purged sample in the Raman capillary
cell and was monitored by UV-visible spectroscopy in the same cell.
12CO (CP grade, Air Products) and 13CO (99%
13C, Cambridge Isotope Laboratory) adducts were obtained by
injecting CO through a septum into a closed capillary containing ~20
µl of argon-purged, reduced enzyme.
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) and a Liconix 4240NB He/Cd laser (442 nm). Spectra were
collected in a 90° scattering geometry on samples at room temperature
with a collection time of a few minutes. Frequencies were calibrated
relative to indene and CCl4 standards and are accurate to
±1 cm
1. CCl4 was also used to
check the polarization conditions. Optical absorption spectra of the
samples were obtained on a PerkinElmer Life Sciences Lambda 9 spectrophotometer to monitor the samples (fully oxidized, fully
reduced, CO complex) before and after laser illumination.
HO-1 Bilirubin Activity Assay--
A solution of hHO-1 (1 µM) or an Asp-140 mutant (10 µM), P450
reductase (1 µM), biliverdin reductase (4 µM), catalase (200 units), hemin (30 µM),
and bovine serum albumin (1 µM) in standard buffer was
preincubated for 3 min at 37 °C. The reaction was initiated by the
addition of NADPH (500 µM), and the production of
Fe(III)-verdoheme, Fe(II)-verdoheme, and bilirubin was monitored at
680-700, 636-640, and 468 nm, respectively, for 0-100 s. The initial
rate of the reaction was calculated using the value
468 = 43.5 mM
1
cm
1 for the bilirubin product.
HO-1 Biliverdin Activity Assay--
A solution of hHO-1 (10 µM) or an Asp-140 mutant (100 µM) and P450
reductase (1 µM) in standard buffer was preincubated at 37 °C for 3 min. The reaction was initiated by addition of NADPH (1 mM) (final incubation volume, 500 µl) and was allowed to
proceed for 30 min. In other experiments, guaiacol (1 mM)
was included in the incubation. The sample was then extracted, and the
biliverdin regioisomer formed was determined by HPLC on an ODS-AQ
column (3 × 250 mm, spherical, 120 Å, YMC, Wilmington, NC)
according to a published procedure (14).
HO-1 Verdoheme Activity Assays--
A solution of hHO-1 or an
Asp-140 mutant (10 µM) in standard buffer was
preincubated at 23 °C in a cuvette. The reaction was initiated by
the addition of H2O2 (100 µM to
10 mM) (final incubation volume, 500 µl), and the
production of Fe(III)-verdoheme was monitored at 680-690 nm for 0-300
s. Alternatively, the P450 reductase-dependent reaction was
initiated by the addition of NADPH (1-2 eq) in standard buffer or in
standard buffer that had been presaturated with CO to determine
Fe(III)-verdoheme or Fe(II)-CO-verdoheme formation, respectively, for
0-100 s. Fe(II)-CO-verdoheme formation was monitored spectrophotometrically at 636-640 nm.
HO-1 Peroxidase Activity Assays--
A solution of hHO-1 or an
Asp-140 mutant (125 nM-10 µM) and guaiacol
(90 mM) in standard buffer was preincubated at 23 °C in
a cuvette. The reaction was initiated by the addition of
H2O2 (100 mM) (final incubation
volume, 500 µl), and the peroxidase activity was monitored at 470 nm
for 0-100 s. The initial rate of peroxidase activity was calculated
using
470 = 3.80 mM
1 cm
1
for the guaiacol oligomer products. The Km and
Vmax values for H2O2
were calculated using guaiacol (90 mM) and
H2O2 (10 µM to 100 mM).
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RESULTS |
Expression, Purification, and Spectral Characterization of the
Asp-140 Mutants--
The D140A, D140F, D140H, D140L, and D140N hHO-1
mutants were expressed and purified as described previously for
wild-type hHO-1 (3, 7). Each protein was judged to be >95% pure by SDS-polyacrylamide gel electrophoresis. The Soret maxima of the Fe(III)
state of the proteins ranged from a low
of 402 nm for the D140A mutant to a high of 412 nm for the D140H mutant
(Table I and Fig. 3). In contrast,
the Asp-140 mutants exhibit the same Soret maxima at 410 and 420 nm for
the Fe(II)-O2 and Fe(II)-CO complexes, respectively, as
wild-type hHO-1 (Table I). These results suggest that the iron
coordination in wild-type hHO-1 and its Asp-140 mutants is similar in
the Fe(II)-O2 and Fe(II)-CO states but differs in their
Fe(III) resting states.
The water coordinated to the iron as the sixth ligand in wild-type
hHO-1 has a pKa of ~8 as determined by the
observation of a red shift in the Soret maximum and changes from a high
spin (HS) to a low spin (LS) species in the RR spectra between pH 7 and
9 (15, 16). Similarly, the Soret maximum of the D140N mutant exhibited
a small, but detectable, shift between pH 7 and 9. However, the Soret
maximum of the other Asp-140 mutants did not shift at pH values up to
11 (Table I). These results indicate that the distal water ligand is at
least partially retained in the D140N mutant but is completely absent
from all the other Asp-140 mutants.
RR of the Asp-140 Mutants--
RR spectra of the Fe(III) state of
wild-type hHO-1 and the Asp-140 mutant heme complexes were obtained
with Soret excitation (Fig. 4). The
3 modes at 1483 and 1503 and
2 modes at
1565 and 1582 cm
1 observed in wild-type hHO-1
are characteristic of a hexacoordinate high spin/low spin mixture
(6cHS/6cLS) with a predominance of the HS state (15, 16). In D140N, the
6cHS species is still clearly observed, but a pentacoordinate high spin
(5cHS) species emerges with
3 at 1491 cm
1. This 5cHS configuration dominates the RR
spectra of the Fe(III) state of the D140A, D140L, and D140F mutants
(Fig. 4). When Asp-140 is replaced by a His, the Fe(III) state is
mostly 6cLS as indicated by the intense and dominant
3 and
2 at
1503 and 1582 cm
1, respectively, with only a
minor 5cHS contribution (Fig. 4 and Table I). These results indicate
that replacement of Asp-140 by other amino acids destabilizes the heme
distal water ligand.

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Fig. 4.
High frequency region of the RR spectra of
the Fe(III) state of wild-type, D140N, D140A, D140L, D140F, and D140H
hHO-1 heme complexes. Spectra were obtained at room temperature
with 413 nm excitation.
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Of all the Asp-140 mutants examined by RR spectroscopy at alkaline
rather than neutral pH, only the D140N mutant presents significant
changes in the Fe(III) state (Fig. 5). A
subpopulation of the D140N mutant heme complex shows the
pH-dependent 6cHS to 6cLS conversion observed for wild-type
hHO-1 (Fig. 5). In the low frequency RR spectrum of the D140N mutant
complex, a band at 549 cm
1 downshifts by 32 cm
1 in 18O-labeled water and is
assigned to the
(Fe-OH) of a 6cLS hydroxo adduct (Fig. 5). In
wild-type hHO-1, the Fe(III)-OH vibration is observed at 546 cm
1 (17). These results demonstrate that the
D140N mutant partially retains the distal aqua/hydroxo ligand.
Moreover, the similarity in the
(Fe-OH) frequencies for both
wild-type hHO-1 and the D140N mutant shows that the hydroxyl group is
engaged in comparable H bond interactions in both distal pockets.

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Fig. 5.
High frequency region of the RR spectra of
the Fe(III) state of wild-type hHO-1 and the D140N mutant heme
complexes at pH 7.4 (solid line) and pH 10.0 (dashed line). The inset spectra show
the 18O-sensitive (Fe-OH) band in the D140N mutant.
Spectra were obtained at room temperature with 413 nm excitation.
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The Fe(III) state of the D140H mutant exhibits a red-shifted Soret
maximum at 412 nm, and its RR spectrum confirms that the heme adopts a
6cLS configuration (Fig. 4). Unlike wild-type hHO-1 and the D140N
mutant, the D140H mutant shows no 18O-sensitive bands in
the low frequency region (data not shown). After reduction with
dithionite, the high frequency RR spectrum of the D140H mutant is
indicative of an LS configuration, and its electronic absorption maxima
at 425, 531, and 558 nm are reminiscent of the Fe(II) state of
cytochrome b558 (18). These results support formation of a bis-histidine heme iron complex in the D140H mutant, although in the Fe(II) state the bis-histidine complex is labile, and
the engineered histidyl ligand can be displaced by exogenous ligands
such as CO and O2. The formation of a similar complex was
recently reported in the high pH form of manganese peroxidase, where
the distal histidine also acts as a labile iron ligand (19).
Aside from the D140H mutant, the Fe(II) state of the Asp-140 mutants
studied here presents high frequency RR spectra indicative of a 5cHS
heme configuration and display in the low frequency region
(Fe(II)-His) vibrations within 2 cm
1 of
the 216-cm
1 value observed for wild-type
hHO-1 (data not shown). Such Fe(II)-histidine stretching frequencies
are consistent with a proximal histidine that retains the N-H proton
and is only weakly or not hydrogen bonded (15, 16).
Catalytic Turnover of the Asp-140 Mutants--
Under the standard
conditions of our bilirubin activity assay (1 µM hHO-1
enzyme), all of the Asp-140 mutants were found to be catalytically
inactive. However, by increasing the concentration of the Asp-140
mutants 10-fold, a small amount of activity was observed using the
D140A, D140H, and D140N mutants. The initial rates and total amounts of
bilirubin formation catalyzed by these three mutants were 1.8/1.4%,
0.2/0.003%, and 2.8/3.04%, respectively, of the wild-type hHO-1
activity (Table II). The spectroscopic changes observed using these mutants indicated that, similar to wild-type hHO-1, the Fe(III)-verdoheme (680-700 nm) and
Fe(II)-verdoheme (636-640 nm) complexes were formed as intermediates
prior to bilirubin formation (Fig. 6).
Even at the higher concentration, the D140F and D140L mutants were
incapable of catalyzing bilirubin formation. However, the D140L mutant
and, to a much lesser extent, the D140F mutant were able to form the
verdoheme intermediate. HPLC analysis revealed that the
-isomer of
biliverdin was formed from the D140A, D140H, and D140N mutants (Table
II). This result established that the regiospecificity of the HO-1
catalytic mechanism was not altered by the Asp-140 amino acid
substitutions.
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Table II
Catalytic activities of hHO-1 and its Asp-140 mutants
HO-1 catalytic activity reflects the initial rate and total amount of
bilirubin (BR) formation observed in the presence of NADPH (1 mM) and P450 reductase (1 µM). The
regioisomer of biliverdin formed was determined by HPLC analysis. The
values given are the average of three separate determinations (±S.D.).
ND, none detected; WT, wild type.
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Fig. 6.
Spectroscopic changes observed following the
addition of NADPH to initiate the bilirubin activity assay using
wild-type hHO-1 (WT), the D140A mutant, the D140H
mutant, and the D140F mutant. Bilirubin,
Fe(II)-verdoheme, and Fe(III)-verdoheme formation is observed as an
appearance of a band at 468, 636-640, and 680-700 nm,
respectively.
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The HO-1 reaction can be arrested at the Fe(II)-CO-verdoheme stage by
pre-saturating the buffer with CO prior to the addition of NADPH. By
using wild-type hHO-1, the sequence of events leading up to
Fe(II)-CO-verdoheme formation includes generation of the heme-Fe(II)-CO
and heme-Fe(II)-O2 complexes. Under these conditions, the
HO-1 reaction is dependent upon the ability of the enzyme to readily
exchange the bound CO ligand with O2 for catalysis to
occur. Although the Asp-140 mutants could form the
heme-Fe(II)-O2 complex, the Fe(II)-CO-verdoheme
complex characteristic of normal turnover
was not observed (Table III and Fig. 7).
As previously observed for the Gly-139 and Gly-143 hHO-1 mutants (19),
formation of the heme-Fe(II)-O2 complex from the Asp-140
mutants was accompanied by gradual degradation of the heme to
nonbiliverdin products.
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Table III
Fe(III)-verdoheme and Fe(II)-CO-verdoheme formation by hHO-1 and its
Asp-140 mutants in the presence of NADPH/P450 reductase
The initial rate of Fe(II)-CO-verdoheme and Fe(III)-verdoheme formation
catalyzed by NADPH/P450 reductase under an atmosphere of CO or
O2, respectively. Fe(II)-CO-verdoheme and Fe(III)-verdoheme
formation was monitored at 636-640 and 680-700 nm, respectively. The
values given are the average of three separate determinations. ND, none
detected. WT, wild type.
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Fig. 7.
Spectroscopic changes observed following the
addition of NADPH to initiate the Fe(II)-CO-verdoheme formation using
wild-type hHO-1 (WT), the D140A mutant,
and the D140N mutant. Fe(II)-CO-verdoheme formation is
observed as an appearance of a band at 636-640 nm.
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By using 1-2 eq of NADPH in the presence of P450 reductase, but in the
absence of CO, the HO-1 reaction can be arrested at the
Fe(III)-verdoheme stage. In such experiments, the D140A, D140H, D140L,
and D140N mutants produced significant amounts (18-87% with respect
to wild-type HO-1) of the corresponding Fe(III)-verdoheme complexes
(Table III and Fig. 8). In agreement with
the results from the bilirubin activity assay, very little (3%) of the
Fe(III)-verdoheme intermediate was formed with the D140F mutant. These
results suggest that the Asp-140 substitutions in the D140A, D140H,
D140L, and D140N mutants have altered the ability of HO-1 to convert
the heme-Fe(II)-CO complex to the catalytically active
heme-Fe(II)-O2 species and that the D140F mutant is
completely unable to convert heme to verdoheme.

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Fig. 8.
Spectroscopic changes observed following the
addition of NADPH to initiate the Fe(III)-verdoheme formation using
wild-type hHO-1 (WT), the D140A mutant,
and D140N mutant. Fe(III)-verdoheme formation is
observed as an appearance of a band at 680-700 nm.
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Peroxidase Activity of Asp-140 Mutants--
We reported previously
that the Gly-139 hHO-1 mutants exhibit a peroxidase activity with
H2O2 and guaiacol as substrates (20). After
addition of H2O2 to the Asp-140 mutants, a
significant amount of heme loss was observed with a corresponding
increase in the formation of a species at 426 nm. Inclusion of guaiacol
in the incubation prevented the heme loss and led to the formation of a
species at 470 nm indicative of guaiacol oligomerization. Furthermore, the addition of guaiacol during the reaction at least partially regenerated the chromophore of the Fe(III) enzyme. Based on these results, it seemed likely that the 426 nm absorbing species represented a Compound II-like ferryl species. To ensure that the conditions used
in the peroxidase assay were optimal, the Km and Vmax values for H2O2 for
each Asp-140 mutant were determined (Table IV and Fig. 9). The
Asp-140 substitutions led to a
substantial decrease (8-54-fold) in the affinity of the enzyme for
H2O2 relative to the wild-type enzyme. By using
the optimized peroxidase assay, the Asp-140 mutants were found to
possess peroxidase activities significantly greater (8-119-fold) than
wild-type hHO-1 (Table IV), with the D140A mutant exhibiting the
highest peroxidative activity. These results demonstrate that
disruption of the putative Asp-140-dependent active site
hydrogen bonding network converts hHO-1 into a peroxidase.
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Table IV
Km and Vmax parameters for
H2O2 with hHO-1 and its Asp-140 mutants as determined
using the peroxidase activity assay
The Km and Vmax parameters were
determined using the peroxidase activity assay described under
"Experimental Procedures." Peroxidase activity represents guaiacol
oligomer formation activity in the presence of guaiacol (90 mM) and H2O2 (100 mM). The
values given are the average of three separate determinations (±S.D.).
WT, wild type.
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Fig. 9.
Eadie-Hofstee plot used for the determination
of Km and Vmax
parameters of the D140A mutant and H2O2 in the
peroxidase activity assay.
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Reaction of the Asp-140 Mutants with
H2O2--
Under the standard conditions used
for monitoring Fe(III)-verdoheme formation (10 µM enzyme
and 100 µM H2O2), only the D140F, D140H, and D140L mutants were found to produce this intermediate, with
initial rates of 0.7, 1.7, and 0.4%, respectively, of the wild-type
hHO-1 rate. The D140A and D140N mutants did not catalyze Fe(III)-verdoheme formation under these conditions. It seemed likely
that the relatively small amount of Fe(III)-verdoheme produced could be
due to the decreased affinity of the Asp-140 mutants for
H2O2. Therefore, a range of
H2O2 concentrations was used in an attempt to
optimize Fe(III)-verdoheme formation. The initial rates of
Fe(III)-verdoheme formation from the D140F, D140H, and D140L mutants
could be increased 5-10-fold at much higher (10-25-fold) H2O2 concentrations (Table
V). Lower H2O2
concentrations were necessary to observe Fe(III)-verdoheme formation
with the D140A and D140N mutants. The total amount of Fe(III)-verdoheme
formed over the time course of the assay was found to correlate well with the increase in the initial rate of formation (Table V). Furthermore, the initial rate and total amount of Fe(III)-verdoheme formation increased significantly (3-51 and 4-30-fold, respectively) in the presence of guaiacol for a majority of the Asp-140 mutants (Table V and Fig. 10).

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Fig. 10.
Spectroscopic changes observed
following the addition of H2O2 to initiate the
Fe(III)-verdoheme formation using the D140A mutant in the absence
(left) or presence (right) of
guaiacol. Fe(III)-verdoheme formation is observed as an appearance
of a band at 680-700 nm.
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DISCUSSION |
The HO catalytic cycle that ultimately produces
-biliverdin
proceeds via initial electrophilic addition of a reactive Fe(III)-OOH species to the
-meso-carbon of the heme group. This
conclusion is based on the following: (i) ability of
H2O2 to substitute for NADPH and P450 reductase
in the production of verdoheme (3); (ii) formation of an
-ethoxyheme
adduct upon exposure of hHO-1 to ethylhydroperoxide (21); (iii)
electronic effects of meso-methyl and meso-formyl
heme substituents on the reaction (22, 23); and (iv) direct
spectroscopic observation of the Fe(III)-OOH complex and hydroxyheme
intermediate following low temperature photoreduction (24). To define
further the mechanism, it was of considerable interest to test the
effect of point mutations introduced into the hHO-1 active site on the
catalytic activity of the enzyme. We previously reported that the
flexibility of the distal helix of hHO-1 is involved in controlling the
reactivity of the enzyme (20). Two residues implicated by the crystal
structure as critical for this flexibility were Gly-139 and Gly-143
(9). Therefore, several mutations were made at these residues in an
effort to perturb the enzymatic reaction by altering the flexibility of the helix (20). Interestingly, the regiospecificity of the reaction was
not perturbed, but the mutations did cause a substantial shift in the
fate of the key Fe(III)-OOH species, favoring its conversion to a
ferryl species at the expense of
-meso-hydroxylation of the heme. This change in the fate of the Fe(III)-OOH species caused a
conversion of hHO-1 from an oxygenase to a peroxidase.
The hHO-1 x-ray crystal structure was critical for identifying other
residues in the active site that might be essential for the highly
regiospecific oxidation of the heme substrate. One such residue,
Asp-140, appears to be well positioned to participate in a hydrogen
bond network with a water molecule and the Fe(III)-OOH intermediate
(Fig. 2). As shown here, wild-type hHO-1 has almost no peroxidase
activity, but mutations at Asp-140 increase the peroxidative activity
of the enzyme from ~10 to more than 100-fold. In contrast, biliverdin
production, as assayed by the subsequent formation of bilirubin, was
greatly decreased with an almost complete (>97%) loss in the
bilirubin formation activity relative to wild-type hHO-1. Despite this
precipitous decrease in the ability to form biliverdin, the
regiospecificity of the enzyme remained intact, and only the
-isomer
of biliverdin was formed.
The individual steps leading up to bilirubin formation were examined to
determine at which stage the normal catalytic cycle was disrupted. The
relative amount of Fe(III)-verdoheme formed using the NADPH/P450
reductase system was significantly higher than that obtained using
H2O2 even after optimization of the
H2O2 concentration (Tables III and V). Addition
of H2O2 to the Asp-140 mutants caused a gradual
destruction of the heme that, compared with wild-type hHO-1, resulted
in the formation of only a small amount of Fe(III)-verdoheme. This
observation may reflect side reactions known to occur between
H2O2 and heme that lead to fragmentation products other than biliverdin (25). In the present study, this abnormal heme destruction could be suppressed by the inclusion of
guaiacol in the incubation, suggesting that the heme loss was catalyzed
by a ferryl rather than Fe(III)-OOH species. This conclusion derives
from the fact that the production of Fe(III)-verdoheme is mediated by
an Fe(III)-OOH intermediate (3), whereas the peroxidase activity is
catalyzed by the ferryl species derived from this intermediate by
cleavage of the O-O bond. Thus, it should be possible to suppress
reactions that depend on the ferryl species by reducing it with
guaiacol without altering reactions supported by the Fe(III)-OOH
species, which is not expected to react with guaiacol. Indeed, when
guaiacol is present, a significant increase in the amount of
Fe(III)-verdoheme formed with H2O2 was observed (Table V).
All of the Asp-140 mutants could be reduced by NADPH and P450 reductase
to form the characteristic Fe(II)-CO and Fe(II)-O2 complexes (Fig. 11). In addition,
although the amount was significantly less than that observed with
wild-type hHO-1, the Asp-140 mutants were able to generate
Fe(III)-verdoheme in the presence of P450 reductase and a few
equivalents of NADPH. However, the Asp-140 mutants were unable to
catalyze the formation of verdoheme in the presence of CO. In these
experiments, the wild-type HO reaction is arrested at the
Fe(II)-CO-verdoheme stage. Formation of this intermediate on the normal
catalytic pathway is dependent on displacement of CO from the
Fe(II)-CO-heme complex to form the Fe(II)-O2-heme species.
Clearly, the interactions favoring formation of the
Fe(II)-O2 intermediate from the Fe(II)-CO complex are
disrupted by substitution of an Ala, Phe, His, Leu, and Asn for
Asp-140. It appears that Asp-140 facilitates the formation of the
Fe(III)-OOH intermediate by enhancing the binding affinity of
O2 for the Fe(II) form of the heme iron or, less likely, by
decreasing the binding affinity of CO. Apparently, Asp-140 also
enhances the formation of the Fe(III)-OOH species using
H2O2 as well as its subsequent conversion to
verdoheme. The altered binding of O2 to the Fe(II) state of the protein, and of H2O2 to the Fe(III) state
of the enzyme, is mirrored in the Fe(III) state by loss of the water
molecule that is normally coordinated as a sixth ligand to the iron
atom. These findings support the inference from the x-ray crystal
structure that Asp-140 participates in a critical hydrogen bonding
network. Introduction of other amino acids at this position disrupts
the favorable hydrogen bonding interactions, redirecting the HO
catalytic cycle toward ferryl formation. As a consequence, the ferryl
species promotes abnormal heme fragmentation or, in the presence of
guaiacol, catalyzes the observed peroxidation reaction.
It is clear that the oxidation of heme to verdoheme and verdoheme to
bilirubin is sensitive to the nature of the amino acid that replaces
Asp-140 in the active site. For example, the oxidative steps leading
from heme to verdoheme are significantly inhibited (65, 82, and 97%)
in the D140F, D140H, and D140L mutants, respectively, and, as a result,
no bilirubin formation is detected. On the contrary, mutation of
Asp-140 to either Asn-140 or Ala-140 has less of an impact on the
conversion of heme to verdoheme (13 and 44%, respectively), but
produces a precipitous drop in the relative amounts of bilirubin formation (97 and 99%, respectively). It seems likely that the larger
amino acids interfere with the binding and subsequent activation of
O2 in the hHO-1 active site, whereas the smaller amino acid substitutions simply reduce the efficiency of the reaction. This conclusion is most clearly illustrated by the RR data for the D140H
mutant, which indicate that the distal histidine coordinates to the
iron atom, giving a bis(histidine)-coordinated heme. As might be
expected, bis coordination significantly inhibits the binding of
O2 to the heme iron, thus accounting for the decrease in
the amount of verdoheme formed. It has been reported that the redox
potential of bis(histidine)-coordinated heme proteins is significantly
lower than that of heme proteins in which only one of the iron ligands
is a histidine (6, 26-28). This finding may explain the inability of
the verdoheme that is formed from the D140H mutant to be reduced by
P450 reductase and therefore to catalyze the oxidation of heme to biliverdin.
In summary, replacement of Asp-140 of wild-type hHO-1 by an Ala, Phe,
His, Leu, or Asn significantly decreases bilirubin formation but
increases peroxidative activity. These effects appear to be due largely
to an alteration of the normal catalytic cycle that involved disruption
of the O2 binding facilitated by Asp-140. As a result, a
much smaller proportion of the Fe(III)-OOH intermediate is directed
toward electrophilic addition to the
-meso-carbon edge
and, instead, is channeled by heterolytic O-O bond cleavage into the
formation of a ferryl species. This drastic shift in the partitioning
of the reactive intermediate formed by hHO-1 as a result of the
mutations establishes the importance of Asp-140 as a catalytic residue
and converts heme oxygenase into a peroxidase.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants DK30297 (to P. R. O. M.) and GM34468 (to T. M. L.) and a
Ford Foundation postdoctoral fellowship (to L. K. 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: School of
Pharmacy, Rm. S-926, University of California, San Francisco, CA
94143-0446. Tel.: 415-476-2903; Fax: 415-502-4728; E-mail:
ortiz@cgl.ucsf.edu.
Published, JBC Papers in Press, December 19, 2000, DOI 10.1074/jbc.M010349200
 |
ABBREVIATIONS |
The abbreviations used are:
HO, heme oxygenase;
heme, iron protoporphyrin IX regardless of oxidation and ligation
state;
HO-1, heme oxygenase isoform 1;
hHO-1, truncated human HO-1;
HPLC, high performance liquid chromatography;
RR, resonance Raman
spectroscopy;
HS, high spin;
LS, low spin;
P450 reductase, NADPH-cytochrome P450 reductase.
 |
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