(Received for publication, November 20, 1996, and in revised form, January 8, 1997)
From the Department of Pharmaceutical Chemistry,
School of Pharmacy, University of California, San Francisco,
California 94143-0446 and the § Department of Chemistry,
Biochemistry, and Molecular Biology, Oregon Graduate Institute of
Science and Technology, Portland, Oregon 97291-1000
Conversion of heme to verdoheme by heme
oxygenase-1 (HO-1) is thought to involve
-meso-hydroxylation and elimination of the meso-carbon as CO, a reaction supported by both
H2O2 and NADPH-cytochrome P450
reductase/O2. Anaerobic reaction of the heme-HO-1 complex with 1 eq of H2O2 produces an enzyme-bound
intermediate identified by spectroscopic methods as
-meso-hydroxyheme. This is the first direct evidence for
HO-1-catalyzed formation of
-meso-hydroxyheme.
-meso-Hydroxyheme exists as a mixture of Fe(III)
phenolate, Fe(III) keto anion, and Fe(II) keto
neutral radical
resonance structures. EPR shows that complexation with CO enhances the
Fe(II)
neutral radical component. Reaction of the
-meso-hydroxyheme-HO-1 complex with O2
generates Fe(III) verdoheme, which can be reduced in the presence of CO
to the Fe(II) verdoheme-CO complex. Thus, conversion of
-meso-hydroxyheme to Fe(III) verdoheme, in contrast to a
previous report (Matera, K. M., Takahashi, S., Fujii, H., Zhou, H.,
Ishikawa, K., Yoshimura, T., Rousseau, D. L., Yoshida, T., and
Ikeda-Saito, M. (1996) J. Biol. Chem. 271, 6618-6624), does not require a reducing equivalent. An electron is
only required to reduce ferric to ferrous verdoheme in the first step
of its conversion to biliverdin.
Heme oxygenase catalyzes the NADPH- and cytochrome P450 reductase-dependent oxidation of heme (iron protoporphyrin IX regardless of oxidation and ligation state) to biliverdin and CO (1). This enzyme is of physiological interest because of the biological properties of its reaction products: biliverdin and CO. Biliverdin is normally reduced to bilirubin by biliverdin reductase and is excreted after conjugation with glucuronic acid (2). Excretion of bilirubin is frequently impaired in newborn children and in individuals with a genetic glucuronyltransferase deficiency (3). Unconjugated bilirubin is neurotoxic, and approaches to the prevention of its accumulation, including the inhibition of heme oxygenase, are of potential clinical utility (4-6). More recently, a potentially important but controversial role as a neurotransmitter akin to nitric oxide has been invoked for the CO produced by heme oxygenase (7-10).
Two forms of the enzyme, denoted as HO-1 1 and HO-2, have been identified (11-13). HO-1 is induced by both a variety of chemical agents and a range of stress conditions and is found in the highest concentrations in the spleen and liver. HO-2 is not inducible and is found in the highest concentrations in the brain and testes. HO-1 and HO-2 are membrane-bound proteins with a C-terminal lipophilic domain that anchors the protein to the endoplasmic reticulum (14, 15). We have reported that a 30-kDa truncated version of HO-1 lacking the 23-amino acid C-terminal membrane anchor can be expressed in Escherichia coli in high yields (16, 17). The truncated protein is soluble and retains full catalytic activity. Site-directed mutagenesis studies, in combination with catalytic and spectroscopic analyses, have led to unambiguous identification of His-25 as the proximal heme iron ligand in the HO-1 enzyme-substrate complex (18-23). Recent studies have established that His-132, the conserved residue in the distal side of the heme pocket, plays a role in stabilizing the distal water iron ligand and facilitates, but is not absolutely required for, coupled turnover of the enzyme (24).
Heme oxygenase employs heme as both the prosthetic group and substrate.
The oxidation of heme by heme oxygenase is thought to involve
sequential -meso-hydroxylation,
oxygen-dependent fragmentation of the resulting
-meso-hydroxyheme product to verdoheme, and oxidative
cleavage of verdoheme to biliverdin (Scheme 1).
-meso-Hydroxylation, the first step, requires
NADPH-cytochrome P450 reductase-dependent reduction of the
iron to the ferrous state, oxygen binding to the reduced iron, and a
second one-electron reduction of the oxy-ferrous complex (25). The
resulting ferric peroxide (Fe(III)-OOH) complex reacts as an
electrophile with the porphyrin ring (16, 26) and incorporates an
oxygen atom from molecular O2 into the
-meso-hydroxyheme product (27). Although synthetic
-meso-hydroxyheme has been shown to be converted to
biliverdin IX
both by HO-1 (28, 29) and by model systems (30-33),
the formation of
-meso-hydroxyheme has not been directly
demonstrated in the enzymatic reaction, possibly because of its high
reactivity with oxygen. Intervention of
-meso-hydroxyheme
as a true intermediate in the normal catalytic process therefore
remains unproven. This shortcoming is important in view of the
demonstration that
-meso-methyl-substituted heme groups
are oxidized to the corresponding
-biliverdins without the formation
of CO, a reaction that does not involve
-meso-hydroxylation of the heme (34, 35).
The second step of the catalytic process is elimination of the
-meso-carbon and attached hydroxyl as CO (36) concomitant with replacement of the carbon in the ring structure by an oxygen atom
(37, 38). However, the nature of the intermediates and the reaction
mechanism for this transformation remain unclear. Several studies of
the conversion of
-meso-hydroxyheme to verdoheme have
been carried out with chemical model systems (31, 32, 39-41) as well
as systems in which
-meso-hydroxyheme has been reconstituted into apomyoglobin (31) or HO-1 (29, 42). These studies
indicate that a radical species forms when
-meso-hydroxyheme reacts with O2 prior to the
formation of verdoheme, but the reduction equivalent requirements for
the subsequent reaction are a matter of debate. Matera et
al. (42) concluded that 1 reducing eq is required for the
HO-1-catalyzed reaction, whereas Sano et al. (31) did not
find such a requirement in the myoglobin reaction. Our previous
demonstration that NADPH-cytochrome P450 reductase and O2
can be replaced by H2O2 in supporting the
HO-1-catalyzed conversion of heme to verdoheme is in accord with the
conclusion that exogenous electrons are not required for this reaction
(16).
We report here that -meso-hydroxyheme is formed as a
stable intermediate when the heme-hHO-1 complex reacts anaerobically with H2O2 and characterize this intermediate by
UV-visible, resonance Raman, and EPR spectroscopy. In the presence of
CO,
-meso-hydroxyheme exists predominantly as a
CO-complexed Fe(II)
neutral radical. On exposure to O2,
this porphyrin radical reacts with O2 to yield Fe(III)
verdoheme with no requirement for exogenous reducing
equivalents. An electron is only required to reduce Fe(III) to Fe(II)
verdoheme.
hHO-1 without the 23-amino acid membrane anchor was expressed in E. coli and was purified as previously reported (16, 17). The truncated protein has the same catalytic activity as the full-length form. The catalytic activity was assayed by monitoring the formation of bilirubin at 37 °C in the presence of biliverdin reductase, and the biliverdin products were analyzed by high pressure liquid chromatography as described previously (16).
ChemicalsH2O2 (30%), NADPH, protoporphyrin IX, methyl viologen, sodium dithionite, and pyridine were obtained from either Aldrich or Sigma. High purity argon (99.998%) and CO (99.95%) were obtained from Matheson and Aldrich, respectively. All other chemicals were the highest grade available and were used without further purification.
Anaerobic Reaction of the Heme-hHO-1 Complex with H2O2A custom-made anaerobic cuvette
(glass shop, University of California, Berkeley, CA) was assembled from
a fluorescence cuvette with four optical windows (1- and 0.4- or 0.2-cm
path length, respectively) by attaching it to a spherical chamber (for
gas exchange and mixing) bearing a standard ground-glass joint, a septum-sealed adapter, and a side arm for the addition of reagents after gas exchange is complete. The reconstituted heme-hHO-1 complex (35 µM in 1.2 ml of 100 mM potassium
phosphate buffer, pH 7.4) was placed in the UV cuvette, and 1 eq of
H2O2 (50 µl) was placed in the side arm. The
concentration of H2O2 was quantitated by titration with iodide (43) or by measuring the absorbance at 240 nm
(240 = 43.6 M
1
cm
1). The protein solution and
H2O2 were made anaerobic by flushing the
cuvette with oxygen-free argon for at least 40 min. The argon was
passed through an oxygen scrubber to remove the trace O2
contamination from the gas, and a water-filled bubbler was used to
humidify the argon and to prevent drying of the solutions in the
cuvette. During the gas exchange, the protein solution was placed in
the round bowl of the cuvette to maximize the surface area and the argon exchange rate. After the solutions were fully anaerobic, the
reaction was initiated by mixing the protein solution with the
H2O2 in the side arm at 23 °C and recording
the UV-visible spectrum until no further changes were observed. For
subsequent reaction with O2, the sealed septum was removed;
the protein solution was flushed with 100% O2; and the
UV-visible spectrum was recorded.
For the experiments performed under an atmosphere of CO, the protein and H2O2 solutions were flushed with O2-free CO. The CO (99.95%) gas was bubbled through a sodium dithionite solution containing reduced methyl viologen (blue) as an indicator to remove oxygen impurities prior to entry into the cuvette. The procedure was otherwise the same as that for the anaerobic experiments.
Resonance Raman SpectroscopyResonance Raman experiments
were performed using the same anaerobic cuvette used for the absorption
spectroscopy. The procedures used to prepare samples for absorption
measurements were repeated for resonance Raman experiments with higher
protein concentrations (~150 µM). Optical absorption
data could be concomitantly obtained using the cuvette with a 0.2-cm
path length. Resonance Raman spectra were recorded on a custom
McPherson 2061/207 spectrograph (0.67-m focal length, 1800 grooves/mm
of grating, and 6 cm1 spectral resolution) using a Kaiser
Optical holographic super-notch filter and a Princeton Instruments
liquid N2-cooled CCD detector (LN-1100PB). The excitation
source was provided by an Innova 302 krypton laser (413 nm, ~5 mW).
Spectra were collected in a 90°-scattering geometry at room
temperature with a collecting time of a few minutes. Peak frequencies
were calibrated relative to an indene standard and are accurate to ±1
cm
1.
Anaerobic sample handling was as described
above except that higher concentrations of the reconstituted heme-hHO-1
complex were used for EPR experiments. The protein solution (300 µM, 250 µl) in a Teflon-sealed constantly stirred 1-ml
reaction vial was made anaerobic by flushing, as required, with either
O2-free argon or CO. The reaction, initiated by anaerobic
addition of 1 eq of H2O2, was allowed to
proceed for 5 min at 23 °C before the protein solution was
transferred to an anaerobic EPR tube, in which it was frozen by
immersion in liquid nitrogen. For the reaction with O2,
100% O2 was used to flush the head space over the protein solution with constant stirring. Sodium dithionite was prepared in
anaerobic buffered solutions and was quantitated by titration with
potassium ferricyanide solution (420 = 1.03 mM
1 cm
1). X-band EPR spectra
were recorded using a Varian E-109 spectrometer equipped with an Oxford
Instruments ESR-910 liquid helium cryostat, a Hewlett-Packard 436A
power meter, and a Hewlett-Packard 5350B microwave frequency counter.
Temperature, g value calibrations, data acquisition,
subtraction, and integration procedures were as described previously
(44-46).
When the
heme-hHO-1 complex (Fig. 1, ) reacts anaerobically
(argon atmosphere) with 1 eq of H2O2 at pH 7.4, the Soret band (- · -) becomes broader, decreases in intensity,
and crosses at 425 nm that of the original heme-hHO-1 complex. In the
visible region, the
and
bands at 574 and 536 nm are greatly
attenuated; the absorbance at 635 nm increases; and a new band appears
around 820 nm. These spectroscopic changes differ dramatically from
those observed when verdoheme is formed by aerobic addition of 1 eq of
H2O2 to the heme-hHO-1 complex (16). The new
species that is formed anaerobically is stable at 23 °C for at least
30 min if anaerobicity is maintained. The broad Soret band at 405 nm and the relatively featureless visible region of the anaerobic intermediate are similar to those of the reconstituted
-meso-hydroxyheme-HO-1 complex reported by Matera
et al. (42) and the
-meso-hydroxyheme-myoglobin complex reported by Sano
et al. (31). When the anaerobically generated intermediate
is exposed to O2, the Soret intensity undergoes a further
slight decrease; the broadening of the Soret band and the band at 820 nm disappear; and the absorption at 660-690 nm increases markedly
(Fig. 1, - - -). This spectrum, which is the same as that obtained
upon aerobic addition of H2O2 (16), suggests that verdoheme is formed by reaction of the anaerobically generated intermediate with O2. The formation of verdoheme was
confirmed by the addition of 20% pyridine to the protein solution,
which produced the typical spectrum of the verdoheme-pyridine complex (Fig. 1, - ·· -) (16, 37, 38).
Additional evidence for the formation of
-meso-hydroxyheme in the anaerobic reaction of
H2O2 with the heme-hHO-1 complex is provided by
comparison of the resonance Raman spectrum of the product with that
reported for HO-1 reconstituted with synthetic
-meso-hydroxyheme (42). The resonance Raman spectrum of
the ferric heme-hHO-1 complex (Fig. 2, trace
A), as previously reported (18, 22), is characteristic of a
mixture of high and low spin hexacoordinate heme dominated by a
4 porphyrin skeletal mode at 1375 cm
1.
Anaerobic addition of 1 eq of H2O2 leads to a
decrease of the contributions from the ferric heme and the appearance
of new resonance Raman features that signal the formation of a new
species (Fig. 2, trace B), although the residual band at
1375 cm
1 indicates that some unreacted ferric heme is
still present. Reaction with higher concentrations of
H2O2 further decreases the ferric heme bands,
but no increase is observed in the bands attributed to
-meso-hydroxyheme. This is probably due to side reactions of
-meso-hydroxyheme with
H2O2.2 The
difference spectrum (Fig. 2, trace C) obtained by
subtracting the spectrum of the residual starting material from
trace B exhibits major resonance Raman bands at 889, 1125, 1226, 1334, 1354, 1401, 1581, and 1616 cm
1. Allowing for
an experimental error of 2 cm
1, the difference spectrum
reproduces the features of the previously reported spectrum of the
-meso-hydroxyheme complex (42). Although previous studies
have shown that chemically synthesized
-meso-hydroxyheme can be converted to biliverdin (29, 31, 42), this is the first
demonstration of the heme oxygenase-catalyzed formation of
-meso-hydroxyheme.
Kinetics of the Formation of
Rapid
scanning spectroscopy at 23 °C shows that formation of the
-meso-hydroxyheme intermediate in the anaerobic reaction of the heme-hHO-1 complex with H2O2 is complete
within 4 min (Fig. 3A). As before, completion
of the reaction is associated with a decrease in and broadening of the
Soret peak with an isosbestic point at 425 nm and a slight increase in
the absorbance at 635 nm. Aerobic reaction of the heme-hHO-1 complex
with H2O2 also reaches completion within 4 min
at 23 °C (Fig. 3B). However, the isosbestic point at 425 nm and the broadening of the Soret band are not observed, and the
660-690 nm absorption characteristic of verdoheme increases in
parallel with the decrease in the Soret band. In the aerobic reaction,
the heme appears to be directly transformed into verdoheme without the
accumulation of
-meso-hydroxyheme. When preformed
-meso-hydroxyheme reacts with oxygen, there is a slight
loss of Soret absorption and an increase in the 660-690 nm absorption
(Fig. 3C). These changes are complete within 5 s at
23 °C, indicating that the conversion of
-meso-hydroxyheme to verdoheme is very rapid. The
kinetics of these aerobic and anaerobic reactions of the heme-hHO-1
complex with H2O2 indicate that (a)
a new stable intermediate with a different UV-visible spectrum forms
anaerobically with H2O2; (b) the new
intermediate is a precursor of verdoheme and is converted rapidly to
verdoheme upon exposure to O2; and (c) the rate
of the H2O2-dependent oxidation of
heme to verdoheme appears to be limited by the rate of formation of
-meso-hydroxyheme. This latter result is consistent with
the observation that
-meso-hydroxyheme does not
accumulate in the aerobic reaction of the heme-hHO-1 complex with
H2O2.
Monitoring of the Formation of
The resting state of the heme-hHO-1 complex at pH
7.4, as previously reported, gives predominantly a high spin
(S = 5/2) ferric axial EPR signal with
g = 6 and g
= 2 (Fig. 4, trace A) (18, 21). When the complex
reacts anaerobically with 1 eq of H2O2, a
slight loss of the g = 6 signal and a radical signal at
g = 2.008 are observed (Fig. 4, trace B).
The deprotonated ferric
-meso-hydroxyheme can be
represented as a mixture of ferric phenolate, ferric keto anion, and
ferrous keto
neutral radical resonance forms (32). The EPR data in
Fig. 4 (trace B) are consistent with this interpretation:
the
neutral radical species accounts for the g = 2.008 signal, and the EPR-silent ferrous iron accounts for the decrease
in the g = 6 signal. The g = 6 signal
is broader (Fig. 4, trace B) than that in the parent ferric
state (trace A). The broadening of the signal may be due to
either the generation of a rhombic signal contributed by the ferric
-meso-hydroxyheme or the presence of a more heterogeneous
electronic structure in the keto-enol iron complex. It is difficult to
differentiate the signal type due to interference by the
g = 6 axial signal from unreacted ferric heme, the
presence of which is confirmed by the resonance Raman experiments. When
the EPR signal from unreacted ferric heme is subtracted, the remaining
spectrum shows a ferric
-meso-hydroxyheme rhombic signal
(Fig. 4, trace C) consistent with that reported by Matera
et al. (42). The rhombic signal suggests that the ferric
keto-enol form of
-meso-hydroxyheme has a high spin iron
(S = 5/2). However, the ferrous
neutral radical
species at g = 2.008 was not observed previously in
either the reconstituted
-meso-hydroxyheme-HO-1 (42) or
-meso-hydroxyheme-myoglobin (31) system. Strong evidence
for the ferrous
neutral radical character of
-meso-hydroxyheme is provided by the change in the EPR
spectrum observed upon complexation of the iron with CO (Fig. 4,
trace D). The EPR spectrum of the
-meso-hydroxyheme generated with 1 eq of
H2O2 under an atmosphere of CO shows that the
g = 6 signal is virtually eliminated, whereas the
g = 2.008 signal is greatly enhanced (Fig. 4,
trace D). The dramatic increase in the g = 2.008 radical species in the presence of CO suggests that CO forms a
complex with the ferrous form of the intermediate and thereby
stabilizes the Fe(II) keto
neutral radical structure relative to
the enol and keto anion resonance forms. This inference is consistent
with the UV-visible spectrum in Fig. 5, which shows that
when
-meso-hydroxyheme is formed with 1 eq of
H2O2 under an atmosphere of CO, the Soret
maximum is red-shifted (from 405 to 408 nm) relative to its position in
the absence of CO. The Soret shift argues for the formation of a CO
complex of the Fe(II)
neutral radical species.
No significant changes are observed in the high frequency region of the
resonance Raman spectrum of the -meso-hydroxyheme-hHO-1 complex when CO is added (data not shown). Several explanations are
possible for the absence of a change in the resonance Raman spectrum on
CO complexation. Although the EPR changes measured at 6 K are dramatic,
they may be tempered at high temperature. For example, the shift in the
Soret absorption at room temperature is relatively minor. Thus,
temperature may affect the population ratio of the different resonance
states of the
-meso-hydroxyheme-hHO-1 complex. In
addition, the formation of radical states on the porphyrin ring is
known to greatly decrease their resonance Raman intensity (47).
Therefore, the signal for the ferrous
neutral radical species may
be difficult to detect under the present conditions, where several
other species are known to coexist. Moreover, the photolability of
heme-bound CO is well known; hence, CO may be dissociated by the laser
beam during data collection. The same results were observed when a
power of ~1 mW was used, but the light sensitivity of this particular
complex is unknown. Similarly, the extent of the resonance Raman
changes of the ferrous
-meso-hydroxyheme upon CO binding
are not known.
As already discussed, the
660-690 nm absorption maximum indicative of verdoheme formation
increases when O2 is added to the -meso-hydroxyheme generated anaerobically by reaction of
the heme-hHO-1 complex with H2O2 (Fig. 1,
- - -). Identical experiments were carried out to determine the
resonance Raman spectroscopic changes caused by the reaction. The
presence of the unreacted ferric heme (
4 mode at 1375 cm
1) from the anaerobically generated
-meso-hydroxyheme (shown in Fig. 2, trace B)
remains unchanged upon exposure to O2. Once the resonance
Raman contributions from the unreacted heme have been subtracted (Fig.
6, trace A), the spectrum clearly shows that the bands characteristic of
-meso-hydroxyheme
(e.g. 889, 1226, 1354, and 1581 cm
1; Fig. 2,
trace C) are replaced by a new set of vibrations with intense peaks at 1258, 1463, and 1612 cm
1 (Fig. 6,
trace A). We have also recorded the resonance Raman spectrum
of the verdoheme-hHO-1 complex generated by aerobic reaction of the
heme-hHO-1 complex with H2O2 (Fig. 6,
trace B). In this reaction, a higher
H2O2 concentration was used, and the conversion of heme to verdoheme is nearly
quantitative,3 as indicated by the smaller
unreacted heme peak at 1375 cm
1. A perfect match in
frequencies is observed between the two spectra (Fig. 6, traces
A and B), clearly establishing that verdoheme is formed
when the
-meso-hydroxyheme complex reacts with
O2. An identical resonance Raman spectrum is also obtained
when CO-complexed
-meso-hydroxyheme reacts with
O2, showing that the same verdoheme product is formed in
the presence of CO (data not shown). These results demonstrate that
oxygen alone is required for the conversion of ferric
-meso-hydroxyheme to verdoheme and specifically show that
an exogenous electron is not required for this transformation. This
finding contradicts the report by Matera et al. (42) that no
verdoheme is formed from
-meso-hydroxyheme unless an
exogenous electron is provided.
Trace B in Fig. 6 is not identical to that reported by
Matera et al. (42) for the Fe(II) verdoheme complex. Major
peaks are found at higher frequencies, 1612 (+3) and 1258 (+4)
cm1, and the single broad band at 1463 cm
1
is shown as a pair of bands at 1446 and 1486 cm
1. To
determine whether these differences are due to the presence of Fe(III)
rather than Fe(II) verdoheme, the resonance Raman spectrum of Fe(II)
verdoheme generated by anaerobically adding a stoichiometric amount of
dithionite to trace B in Fig. 6 was recorded. A direct comparison of the resonance Raman spectra of the Fe(III) and Fe(II) verdohemes (Fig. 6, traces B and C) reveals
several differences. (a) The major bands at 1612 and 1258 cm
1 (Fig. 6, trace B) shift to 1608 and 1252 cm
1, respectively, in the ferrous verdoheme spectrum
(trace C); (b) the band at 1463 cm
1
(trace B) splits into two bands at 1441 and 1484 cm
1 in ferrous verdoheme (trace C); and
(c) the band at 1353 cm
1 in trace C
characteristic of ferrous heme (18) comes from the reduction of the
unreacted ferric heme (1375 cm
1 in trace B).
This change in the residual heme peak serves to confirm that the iron
atoms in the solution are in the ferrous state. Thus, it is seen that
the ferrous verdoheme spectrum (Fig. 6, trace C) is
essentially identical to that reported by Matera et al.
(42). The only significant difference is that the bands at 1338 and
1366 cm
1 in their spectrum are masked in trace
C by the unreacted ferrous heme band at 1353 cm
1,
but shoulders at higher and lower frequencies are noted. Further support for identification of trace C as that of ferrous
verdoheme is provided by the similarities between it and the resonance
Raman spectrum of a model Fe(II) verdoheme in
pyridine.4 These data prove that the iron
in the verdoheme obtained from reaction of
-meso-hydroxyheme with oxygen in the absence of reducing equivalents is in the ferric state, as expected.
Another piece of evidence for the formation of a ferric product is
provided by the finding that the UV-visible spectrum of the verdoheme
complex generated by aerobic reaction of heme-hHO-1 with
H2O2 (max = 404, 544, and 680 nm) (Fig. 7,
) remains unchanged when CO is added
(····). In contrast, the addition of a stoichiometric amount of
dithionite produces ferrous verdoheme (Fig. 7, - ·· -) with a
distinct UV-visible spectrum (
max = 400, 534, and 690 nm). The addition of CO to the reduced sample yields a UV-visible spectrum with maxima at 412 and 638 nm that clearly identifies the
product as the Fe(II) verdoheme-CO complex (Fig. 7, - - -) (16,
37, 38). Thus, reaction of the heme-hHO-1 complex with oxygen in the
absence of an external electron source readily yields Fe(III)
verdoheme.
EPR spectroscopy provides additional evidence for the aerobic
conversion of -meso-hydroxyheme to Fe(III) verdoheme.
When anaerobically generated
-meso-hydroxyheme (Fig.
8, trace A; obtained from Fig. 4, trace
B) is exposed to O2, the g = 2.008 radical species disappears, and the g = 6 signal
decreases (trace B). The remaining g = 6 axial signal comes from the unreacted ferric heme species also observed
in the resonance Raman experiments. A fairly weak rhombic signal in the
g = 2 region (gz = 2.57, gy = 2.14, and gx = 1.86;
gav = 2.19) is also obtained that corresponds to
a low spin (S = 1/2) ferric signal. Comparison of this
spectrum with that obtained when 1 eq of H2O2
is added aerobically to the heme-hHO-1 complex (Fig. 8, trace
C) shows that, in the aerobic reaction, the g = 6 signal is similarly suppressed, and the new low spin ferric iron signal
is also generated. However, the residual g = 6 signal
due to unreacted heme is smaller (Fig. 8, traces B and
C), which suggests that the conversion of heme to verdoheme is more complete when the reaction with H2O2 is
carried out aerobically. The loss of the g = 6 axial
signal associated with verdoheme formation indicates that Fe(III)
verdoheme has a different iron spin state relative to that of ferric
heme or
-meso-hydroxyheme, possibly a low spin iron
(S = 1/2) that gives rise to the rhombic EPR signal at
gav = 2.19. The same results, disappearance of
the g = 2.008 radical species and appearance of a weak
rhombic EPR signal at gav = 2.19, are observed
(data not shown) when the CO-complexed ferrous
neutral radical
(sample in Fig. 4, trace D) is exposed to O2.
This finding indicates that the ferrous
neutral radical resonance
structure of the
-meso-hydroxyheme most clearly
represents the species that reacts with molecular oxygen. The new
gav = 2.19 rhombic EPR signal generated in these
reactions suggests that it is a signature of Fe(III) verdoheme,
although the reason why it is a weak signal is unclear.
The first step in heme degradation is thought to be the cytochrome
P450 reductase-, NADPH-, and O2-dependent
-meso-hydroxylation of heme by heme oxygenase. This
transformation requires reduction of the ferric to the ferrous
heme-protein complex by an electron from NADPH-cytochrome P450
reductase, oxygen binding to the iron to give the ferrous dioxygen
(Fe(II)-OO·) complex, and a second reduction to give an
undetected species formally equivalent (after protonation) to a ferric
peroxide (Fe(III)-OOH) complex. The ferric peroxide complex then
undergoes direct reaction as an electrophile with the
bond
structure of the heme to give the
-meso-hydroxylated heme
intermediate (16, 26). The
-meso-hydroxyheme is
subsequently oxidized to carbon monoxide and enzyme-bound verdoheme. Key evidence for this mechanism is provided by the finding that the
conversion of heme to verdoheme is supported by
H2O2 in the absence of cytochrome P450
reductase and NADPH (16). Reaction of the heme-hHO-1 complex with
H2O2 is arrested at the verdoheme stage even
under aerobic conditions because H2O2 is not an
appropriate replacement for NADPH-derived electrons and O2
in the conversion of verdoheme to biliverdin.
-meso-Hydroxyhemes have been synthesized, reconstituted
into myoglobin and heme oxygenase, and shown to be converted to the corresponding verdohemes and biliverdins (29, 31, 42).
-meso-Hydroxyheme, however, has never actually been
identified by spectroscopic or other means as an intermediate in the
enzymatic conversion of heme to verdoheme. This is a significant
shortcoming because the synthetic species could be converted to the
observed product without actually being an intermediate in the normal
process. Evidence that this is possible is provided by the recent
finding that
-meso-methyl-substituted hemes are oxidized
to give normal
-biliverdin, but not CO (34, 35). The kinetic,
optical, resonance Raman, and EPR studies of the intermediate formed
when 1 eq of H2O2 is added anaerobically to the
heme-hHO-1 complex provide the missing evidence that
-meso-hydroxyheme is a true intermediate in the catalytic
reaction.5
The electronic structure of -meso-hydroxyheme can be
presented by three resonance structures: a ferric phenolate ion, a
ferric keto anion, and a ferrous keto
neutral radical (Scheme
2, 1). The rhombic EPR signal of
-meso-hydroxyheme, which represents the contribution of
the ferric phenolate and keto resonance structures, suggests that the
iron is in the high spin state (S = 5/2). This finding
agrees with the results obtained when
-hydroxyheme is reconstituted
into either HO-1 or apomyoglobin (31, 42). Matera and co-workers (48)
and Bogumil et al. (49) also reported that the complex is a
five-coordinate species based on the similarity of its rhombic high
spin EPR signal to that of pentacoordinate myoglobin mutants. Rhombic
high spin signals are often observed for five-coordinate high spin
ferric hemoproteins (48, 50). The observation that CO binds to and
increases the contribution of (Fig. 4, trace D) the ferrous
neutral radical at the expense of the ferric keto-enol resonance
forms provides strong support for the electronic ferrous keto-enol
neutral radical resonance structure of
-meso-hydroxyheme.
After exposure of the
-meso-hydroxyheme complex to
O2, the g = 2.008 radical signal
disappears, and verdoheme is formed, in accord with reaction of the
radical with O2. The ferrous
neutral radical
(g = 2.008 signal) was not detected in earlier EPR
studies of
-meso-hydroxyheme-reconstituted apomyoglobin or HO-1 (31, 42). We have found that the g = 2.008 radical signal is increasingly saturated when the instrument power is increased from 1 to 500 µW at 6 K (data not shown). When the power is
raised above 400 µW at 6 K, the radical signal is completely saturated and disappears. It is therefore very likely that, under the
conditions (10 K, 1000 µW) used in the earlier studies, the radical
signal was saturated and thus was not observed.
The reaction of -meso-hydroxyheme with O2 to
give verdoheme, as shown here by kinetic, optical, EPR, and resonance
Raman studies, is rapid and proceeds without a requirement for
additional reducing equivalents. This finding is consistent with
the reaction mechanism in Scheme 2. The ferrous
neutral radical of
-meso-hydroxyheme (1) binds O2 on
the ring carbon adjacent to the keto group to form a ferrous
hydroperoxy radical (O-O·; 2). Intramolecular
electron transfer from the iron to the peroxy radical produces a ferric
peroxide intermediate (3), the terminal oxygen of which
coordinates to the iron to form a peroxo-bridged intermediate
(4). Heterolytic dioxygen bond cleavage then yields a ferryl
(Fe(IV)=O) species and an alkoxy radical (5) that fragments
with the elimination of CO. Internal electron transfer from the
resulting pyrrole ring A carbon radical to the ferryl oxygen followed
by reaction with the pyrrole ring B carbonyl group produces ferric
verdoheme (6). Formation of ferric rather than ferrous
verdoheme is established by several lines of evidence. (a)
The addition of CO to the verdoheme generated from the heme-hHO-1
complex and 1 eq of H2O2 does not alter the UV-visible spectrum, which suggests that verdoheme is in the ferric state (Fig. 7, ····). (b) The addition of 1 reducing
eq and CO to the verdoheme-hHO-1 complex yields the characteristic
Fe(II) verdoheme-CO UV-visible spectrum (Fig. 7,- - -).
(c) The addition of 1 reducing eq to the verdoheme produces
a resonance Raman spectrum (Fig. 6, trace C) that closely
resembles that reported for Fe(II) verdoheme (42). (d) The
generation of a weak low spin signal associated with loss of the
g = 6 signal in the aerobic reaction of the heme-hHO-1
complex with H2O2 suggests that the spin state of the verdoheme intermediate is S = 1/2 Fe(III) (Fig.
8, traces B and C).
Past studies with HO-1 (37, 38, 42) and apomyoglobin (31) suggest that
the iron oxidation state of the verdoheme product is Fe(II). For
example, Yoshida et al. (37, 38) reported for the
NADPH-cytochrome P450 reductase-dependent reaction that a 688 nm intermediate located after hydroxyheme but before
iron-biliverdin has a ferrous iron and binds CO with high affinity to
yield 638 nm species. However, in all these studies, a source of
reducing equivalents, usually NADPH-cytochrome P450 reductase or
ascorbate, was present in the reaction. In the presence of reducing
equivalents, Fe(III) verdoheme is reduced to Fe(II) verdoheme, which in
turn reacts with O2 to give biliverdin unless the high
affinity of ferrous verdoheme for CO is used to arrest the reaction.
The universal finding that ferrous verdoheme is the product of the
reaction of -meso-hydroxyheme with O2 thus
simply reflects the fact that exogenous reducing agents were present in
the experimental systems that were used. In the present studies carried
out without exogenous reducing agents, the reaction product is clearly
identified as ferric verdoheme. A case can be made that the
formation of Fe(III) verdoheme is desirable to prevent the CO that is
released in the reaction from binding to the intermediate and stopping
the reaction process.
Sano et al. (31) reported that reaction of the ferric
-meso-hydroxyheme-myoglobin complex with O2
yields Fe(III)-biliverdin via Fe(II) verdoheme without the
addition of reducing equivalents. Matera et al. (42)
reported that both O2 and 1 reducing eq
are required for formation of Fe(II) verdoheme from the
-meso-hydroxyheme-HO-1 complex. We find that oxygen
alone is required for the conversion of
-meso-hydroxyheme to Fe(III) verdoheme, although an
electron is required to reduce the Fe(III) verdoheme complex to the
ferrous state. It is not clear why Matera et al. (42) failed
to observe the formation of verdoheme in the reaction of the
-meso-hydroxyheme-HO-1 complex with O2. They
apparently monitored verdoheme formation under CO by observing the
Fe(II) verdoheme-CO complex at
max = 402 and 638 nm (37,
38). One possible explanation is that CO binds to the ferrous
neutral radical species (see Scheme 2, 7), causing the
reaction to stop at the ferrous- CO peroxy radical adduct
(8) due to inhibition of electron transfer from the iron to
the peroxy radical by the CO ligand. An external electron might then be
required to reduce the peroxy radical to the hydroperoxide
(9), leading to formation of Fe(II) verdoheme
(10) and the conclusion that an exogenous electron is
required for the reaction to occur. This scheme would also explain
their observation of an organic radical (8) when they
exposed the
-meso-hydroxyheme-hHO-1 complex to
O2 in the presence of CO. However, we have been unable to
reproduce their observations by reacting the
-meso-hydroxyheme-hHO-1 complex with O2 for 5 min under a 50% CO atmosphere. Although these conditions may not
closely reproduce theirs, our observation that the reaction does not
stop at the ferrous CO-peroxy radical adduct (8), but
continues to Fe(III) verdoheme (6) under these conditions, suggests that the affinity of the peroxy radical species for CO is not
high.
Spectroscopically identical verdoheme species are formed when 1 eq of
H2O2 is added aerobically to the heme-hHO-1
complex (one step) or by exposure to O2 of the
-meso-hydroxyheme complex preformed by anaerobic reaction
with 1 eq of H2O2 (two steps). The verdoheme
yields obtained by the two approaches differ, however, with a ratio of
~1:0.6 for the one- and two-step processes, respectively. The higher
intensity of the g = 6 signal in the EPR spectrum of the two-step reaction suggests that the lower yield of verdoheme is
matched by a higher fraction of unreacted heme complex. This finding
suggests that the
-meso-hydroxyheme intermediate may react with H2O2 to give alternative reaction
products, a possibility that is currently under investigation.
In summary, the formation of -meso-hydroxyheme in the
HO-1-catalyzed oxidation of heme has been directly demonstrated, and its subsequent conversion to ferric verdoheme has been shown to require
O2, but not an exogenous electron. The exogenous electron is only required to reduce ferric verdoheme to the ferrous state.
We thank Professor John Lipscomb (University of Minnesota) for providing us with access to the EPR spectrometer.