From the Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970
From the Department of Biochemistry and Cell Biology and W. M. Keck Center for Computational Biology, Rice University, Houston, Texas 77005-1892
From the Institute for Life Support Technology, Yamagata Technopolis Foundation, Yamagata 990, Japan
From the Department of Biochemistry, Yamagata University School of Medicine, Yamagata 990-23, Japan
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ABSTRACT |
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The O2 and CO
reactions with the heme, -hydroxyheme, and verdoheme complexes of
heme oxygenase have been studied. The heme complexes of heme oxygenase
isoforms-1 and -2 have similar O2 and CO binding
properties. The O2 affinities are very high,
KO2 = 30-80
µM
1, which is 30-90-fold greater than
those of mammalian myoglobins. The O2 association rate
constants are similar to those for myoglobins (kO2
= 7-20
µM
1 s
1), whereas the
O2 dissociation rates are remarkably slow
(kO2 = 0.25 s
1),
implying the presence of very favorable interactions between bound
O2 and protein residues in the heme pocket. The CO
affinities estimated for both isoforms are only 1-6-fold higher than
the corresponding O2 affinities. Thus, heme oxygenase
discriminates much more strongly against CO binding than either
myoglobin or hemoglobin. The CO binding reactions with the ferrous
-hydroxyheme complex are similar to those of the protoheme complex,
and hydroxylation at the
-meso position does not appear to affect
the reactivity of the iron atom. In contrast, the CO affinities of the
verdoheme complexes are >10,000 times weaker than those of the heme
complexes because of a 100-fold slower association rate constant
(kCO
0.004 µM
1 s
1) and a 300-fold
greater dissociation rate constant (kCO
3 s
1) compared with the corresponding rate constants of the
protoheme and
-hydroxyheme complexes. The positive charge on the
verdoporphyrin ring causes a large decrease in reactivity of the
iron.
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INTRODUCTION |
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Heme oxygenase (HO)1 is
an amphipathic microsomal protein that catalyzes the regiospecific
oxidative degradation of iron protoporphyrin IX (heme hereafter) to
biliverdin, CO, and iron in the presence of NADPH-cytochrome P-450
reductase as an electron donor (1-3). In the catalytic cycle the
enzyme first binds 1 equivalent of hemin. This binding results in the
formation of the heme-enzyme complex, which exhibits spectral
properties similar to those of ferric myoglobins and hemoglobins (4,
5). The first electron donated from the reductase reduces the hemin
iron to the ferrous state, and then O2 binds rapidly to
form a metastable oxy complex (6). Electron donation to the oxy form
initiates the three-step conversion of oxyheme to the ferric
iron-biliverdin complex through -hydroxyheme and verdoheme
intermediates (Scheme 1). The final step
involves electron donation from the reductase to convert the ferric
iron-biliverdin complex to ferrous iron and biliverdin (7). Thus, heme
participates both as a prosthetic group and as a substrate, a property
unique to heme oxygenase (5, 7).
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HO has two isoforms, referred to as HO-1 and HO-2 (3). HO-1, an inducible form with a molecular mass of 33 kDa, is distributed mainly in reticuloendothelial cell-rich tissues such as spleen and liver. HO-2, with a molecular mass of 36 kDa, is expressed constitutively and is distributed mainly in the brain and testis. The amino acid sequence similarity between HO-1 and HO-2 is about 40%. However, both isoforms display the same enzymatic activity, active site structure, and heme iron coordination and electronic structures, hence the molecular mechanism of enzyme action is considered to be analogous between the two isoforms (8).
We and others have demonstrated that a neutral form of the imidazole
group of histidine (His25 in the HO isoform-1 sequence) is
the axial heme ligand in the heme-enzyme complex of the water-soluble
forms of recombinant HO (9-12). Although the distal residues have yet
to be identified (13), an ionizable group with a pKa
value of 7.6 is linked to the water molecule, which is coordinated to
the ferric heme-HO-1 complex (9). The heme-HO complex appears to have an axial ligand coordination structure that is similar to that in
myoglobins and hemoglobins. Resonance Raman studies on the oxy form of
the heme-HO complex show that the bound oxygen molecule assumes a
highly bent configuration because of strong interactions with the
residues in the distal pocket (14). The CO-bound verdoheme-HO complex
exhibits a Fe-CO stretching frequency at 470 cm1 which is
much lower than the Fe-CO ~505 cm
1 stretching frequency
of the heme- and
-hydroxyheme-HO complexes (15). The Fe-CO bond in
the verdoheme complex appears to be quite different and weaker than
that in either the heme or
-hydroxyheme complex.
During catalysis, CO is formed when -hydroxyheme is converted to
verdoheme (16). CO binds to ferrous forms of the heme-,
-hydroxyheme-, and verdoheme-HO complexes (4, 10, 15, 17).
Generally, CO binds to ferrous heme iron with a higher affinity than
O2 does. Hence, CO generated during the verdoheme formation
can coordinate to either heme or verdoheme resulting in product
inhibition, and Noguchi et al. (18) have shown that CO can
inhibit the HO enzyme activity by binding to either the ferrous heme-
and verdoheme-HO complexes. Yoshida and his co-workers (19) have
demonstrated that when a single turnover enzyme reaction is carried out
using an 80% air and 20% CO gas mixture, the CO complex of the
verdoheme-HO is generated. This shows that in the presence of 80% air,
20% CO does not completely inhibit the first two mono-oxygenase cycles
of HO catalysis, but the conversion of verdoheme to biliverdin may be
inhibited. However, a quantitative assessment of CO inhibition requires
accurate determinations of the O2 and CO affinities of the
ferrous heme-HO complex. A low CO affinity was reported for
verdoheme-myoglobin (20), but the affinity of verdoheme-HO for CO has
not been determined.
To this end, we have examined the reactions of O2 and CO
with the heme, -hydroxyheme, and verdoheme complexes of heme
oxygenase. The heme-HO complex has an extremely high O2
affinity because of a very slow O2 dissociation reaction.
However, the CO affinities of the heme and
-hydroxyheme complexes
are very similar to those of mammalian myoglobins and hemoglobins. In
contrast, the verdoheme-HO complexes have extraordinary low affinities
for CO. We attribute the low CO affinity of the verdoheme-HO complex to
the partial ferric character of the verdoheme iron atom. The high
O2 affinity of the heme-HO complex and the low CO affinity
of the verdoheme-HO effectively prevent product inhibition by CO that
is generated during catalysis of hemin degradation.
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EXPERIMENTAL PROCEDURES |
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Truncated water-soluble forms of recombinant rat HO-1 (30 kDa)
and human HO-2 (28 kDa) and the corresponding heme, -hydroxyheme, and verdoheme complexes were prepared as described previously (8, 11,
14, 15, 17).
The CO forms of the heme-, -hydroxyheme-, and verdoheme-HO complexes
were prepared by diluting the ferrous forms of the complexes into the
anaerobic buffer solutions containing known CO concentrations. The oxy
form of the heme-HO complexes was made as follows. First, the ferric
heme-HO complex was reduced by sodium dithionite in deoxygenated
buffer. Then the reduced form of the complex was passed through a
column of Sephadex G-25 equilibrated with buffer containing the known
O2 concentrations. Formation of the complex was confirmed
by optical absorption measurements.
O2 and CO binding reactions were measured using stopped-flow and flash photolysis techniques as described previously (21). The sample integrity was confirmed by the optical absorption spectra measured before and after the flash photolysis measurements. All the measurements were carried out in 0.1 M phosphate buffer, pH 7, at 20 °C.
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RESULTS |
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The association and dissociation rate constants for O2
and CO binding to ferrous forms of the heme-HO-1 and HO-2 complexes are
listed in Table I. The kinetic traces for
bimolecular rebinding of O2 to the heme-HO complex after
flash photolysis are monophasic, and the observed rates depend linearly
on O2 concentration (Fig. 1).
The association rate constants,
kO2, are similar to those of
mammalian myoglobins. However, the O2 dissociation rate
constants, kO2, of the heme-HO
complexes are ~0.25 s
1, about 100 times slower than the
dissociation rat constants of myoglobins (15 s
1) (22).
The O2 equilibrium constants,
KO2, are 28 and 77 µM
1 for the HO-1 and HO-2 complexes,
respectively, 30-90 times greater than the equilibrium constant of
myoglobin. The O2 affinity of the heme-HO complexes is
equal to or higher than that of leghemoglobin, which has one of the
highest known O2 affinities.
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The CO association reactions of both the HO-1 and HO-2 heme complexes
are biphasic with the faster phase showing a bimolecular rate that is
about 4~5 times larger than that of the slower phase. The amplitudes
of the two phases are independent of CO concentrations (between 0.1 and
1 mM). The association rate constants,
kCO, for the slower phases are between
0.3 and 0.6 µM
1 s
1, which are
similar to those of myoglobin (0.5-0.8 µM
1
s
1) (22). The kinetic traces for CO dissociation from the
heme-HO complexes are monophasic. The dissociation rate constants,
kCO, (0.007-0.009 s
1) are
slightly smaller than those of myoglobin (~0.02 s
1)
(22). Because of the biphasic nature of the CO association reactions,
the overall CO equilibrium association constants,
KCO, can only be estimated as between 34 and 150 µM
1 and 89 and 420 µM
1 for the HO-1 and HO-2 complexes,
respectively. It should be noted that the ratios of CO and
O2 affinities,
KCO/KO2, are
between 1.2 and 5.6 for the heme-HO complex. These ratios are much
smaller than those for myoglobin (~40) and hemoglobin (~200) (23).
Thus, O2 binding to the ferrous heme iron in HO is
inhibited to a much lesser extent by CO than it is in myoglobin and
hemoglobin.
The reaction of CO with the ferrous -hydroxyheme-HO-1 complex is
also biphasic (Table II), and the
association rate constants for the fast and slow phases are similar to
those of the heme-HO-1 complex. Again, CO dissociation is monophasic,
and the dissociation rate constant is essentially the same as that of
the heme-HO complex. Thus, the hydroxyl group at the
-meso position
does not affect the reactivity of the ferrous iron toward CO. The oxy
form of the
-hydroxyheme-HO complex is unstable (17) and cannot be prepared for O2 binding and dissociation rate
measurements.
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The reaction of CO with verdoheme-HO complexes is different from that
of the heme- and -hydroxyheme-HO complexes. The rebinding reaction
is monophasic after flash photolysis. Plots of the dependence of the
observed pseudo-first order rate constant, kobs,
on CO concentration are shown in Fig.
2A for the verdoheme-HO-1
complex. The slope and the y axis intercept of this plot
yield association and dissociation rate constants equal to 0.0054 µM
1 s
1 and 2.9 s
1, respectively, and a CO equilibrium association
constant equal to 0.0018 µM
1. Thus, the CO
affinity of the verdoheme-HO-1 is more than 10,000 times weaker than
that of the protoheme complex (Table II). Low CO affinity is confirmed
by the CO equilibrium curve generated by plotting the absorption
changes induced by the flash photolysis as a function of the CO
concentration (Fig. 2B). Because the quantum yield of the CO
form of the verdoheme-HO complex is almost 1 (15), the absorption
changes after photolysis are proportional to the equilibrium fraction
degree of saturation with this ligand. The experimental points are
compared with a CO equilibrium curve calculated from the association
and dissociation rate constants. The agreement between the calculated
and observed results is excellent. The verdoheme HO-2 complex has the
CO binding parameters similar to those of the corresponding HO-1
complex. As in the case of
-hydroxyheme-HO, an oxy form of the
verdoheme-HO complex cannot be obtained for kinetic measurements.
Verdoheme-myoglobin also shows a large CO dissociation rate, but the
association rate and equilibrium constants of the myoglobin complex are
about 10 times greater than those of the HO complex (20) (Table
II).
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DISCUSSION |
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The oxygen equilibrium constants, 30-80
µM1, of the heme-HO complex are similar to
those of leghemoglobin (23 µM
1) (24).
However, the mechanism responsible for the extraordinarily high
O2 affinity for the heme-HO complex is different from that for leghemoglobin. The high O2 affinity of leghemoglobin is
caused by a very large association rate constant that is determined by enhanced reactivity of the heme iron toward O2 (24). In the heme-HO complex, high O2 affinity is caused by a very slow
O2 dissociation rate constant. Decreases in O2
dissociation rate constants for myoglobins and related heme proteins
are associated with increases in hydrogen bonding or other favorable
electrostatic interactions between bound oxygen and surrounding amino
acids. For example, replacing the distal valine [E11] with an
asparagine residue in sperm whale myoglobin decreases
kO2 from 15 s
1 to 0.5 s
1 presumably by creating a second hydrogen bond with the
bound ligand (22). The presence of strong interactions between distal residues in heme oxygenase and bound O2 has already been
proposed based on resonance Raman studies (14) and on cobalt EPR work (25).
The CO association reactions with the heme- and -hydroxyheme-HO
complexes are biphasic. One of the possible origins of the biphasic
reaction is heme orientational disorder around the porphyrin
-
axis, which has been reported for the heme-HO complex (26). However, if
this were the case, the O2 association reaction would also
be expected to be biphasic, but this is not the case. In addition, the
ligand binding properties of myoglobin are independent of the heme
orientation disorder (27). Thus, the structural origin of the biphasic
CO association reactions with the heme- and
-hydroxyheme-HO complex
is not clear.
There are subtle differences in the kinetic parameters between the HO-1 and HO-2 complexes. The neutral imidazole form of histidine is the proximal ligand in both isoforms (8-10). In the hemin-enzyme complex, the pKa value of the acid-base transition of the HO-1 complex (pKa 7.6) is 0.9 unit lower than that in the HO-2 complex (pKa 8.5) (8, 9). Thus, the polarities of the distal pockets appear to be different between the isoforms, which might explain the small difference in kinetic parameters.
As shown in Scheme 2, -hydroxyheme
undergoes a redox-linked transition between ferric oxophlorin and
ferrous
-hydroxyheme (17). In the ferrous form, the prosthetic group
assumes a porphyrin macrocycle structure with a hydroxyl group at the
-meso position (structure 2 of Scheme 2). The
-hydroxyl group should be electron-donating, and electron withdrawal
or electron donation from heme side chains has been shown to affect the
reactivity of the heme iron (23, 28). However, the CO binding
properties of the
-hydroxyheme-HO complex are similar to those of
the heme complex, indicating that the
-meso hydroxyl group does not
affect either heme iron reactivity or iron-CO bond strength. The
porphyrin
-electron spin density at the
-meso carbon could be so
low that the electron donation at this position does not affect the
iron reactivity appreciably. The Raman results show that the Fe-CO and
C-O stretching frequencies of the CO-bound
-hydroxyheme-HO complex
are similar to those of the heme-HO complex (15). This implies that the
electronic states of the Fe-CO unit and the Fe-C bond strengths are
similar in these complexes, which is consistent with our kinetic
results.
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In the verdoheme-HO complex the iron atom is less reactive, and the
iron-CO bond is much weaker than that in the heme and -hydroxyheme-HO complexes. Two resonance structures have been suggested for verdoheme and are shown in Scheme 2 (29). Structure 4 possesses a positive charge on the ring oxygen atom, whereas the charge is on the iron atom in structure 3. The resonance Raman studies show that a significant contribution from
structure 3 is present in ferrous verdoheme-HO (15). Because CO binds only to ferrous iron, the presence of a significant amount of a ferric character will reduce the reactivity of verdoheme-HO toward CO significantly.
The Fe-CO stretching frequency of the CO verdoheme-HO complex is 474 cm1, whereas those for the heme- and
-hydroxyheme-HO
complexes are 503 and 508 cm
1, respectively (10, 15).
Clearly, the Fe-CO bond order in verdoheme-HO is lower than that in the
heme- and
-hydroxyheme complexes. Because the rate of CO
dissociation is determined by the rate of the thermal Fe-CO bond
breakage (30), the lower Fe-CO bond order is probably the cause of the
large kCO value shown by verdoheme-HO.
The conversion of -hydroxyheme to verdoheme by HO generates CO
stoichiometrically (16). Yoshida and co-workers (16, 18) have shown
that CO can inhibit the mono-oxygenation reactions of the HO catalytic
cycle by binding to the ferrous heme- or verdoheme-HO complex. Hence,
CO could potentially act as a product inhibitor. When catalysis is
carried out in air-saturated buffer, hemin is stoichiometrically
converted to biliverdin, and the product inhibition by CO does not
appear to occur. The results in Tables I and II explain why the CO
produced does not severely inhibit heme oxygenase activity. The first
oxygenation cycle is not inhibited because the ferrous iron atom in
protoheme-HO has an extremely high affinity for O2 which is
roughly equal to that for CO. In air, the concentration of
O2 will always be much greater than that of CO, preventing product inhibition. When
-hydroxyheme is converted to verdoheme in
HO, the CO molecule generated by oxygenation of the heme group could
also bind to the verdoheme product. In this case, CO binding to the
verdoheme-HO complex is extremely weak, and the CO dissociation rate
constant is very large, preventing product inhibition until the CO
concentration is greater than 0.5 atmospheres or 500 µM. Previous experimental work has also shown that CO does not inhibit the
conversion of
-hydroxyheme to verdoheme (17, 18). Thus, the active
site of HO has evolved to discriminate strongly against CO binding and
in favor of the binding of O2, which is the oxidizing substrate for the overall reaction.
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FOOTNOTES |
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* This work was supported in part by United States Public Health Service Grants GM35649 and HL47020 (to J. S. O.) and GM51588 (to M. I.-S.); Robert A. Welch Foundation Grant C-612 (to J. S. O.); Grants-in-aid for Scientific Research 08044240, 08249101, 08680675, and 09235101 (to T. Y.); and Grants 09235236 and 09740504 from the Ministry of Education, Science, Sports, and Culture, Japan (to H. F.).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.
Permanent address: School of Allied Health Sciences, Yamaguchi
University, Ube, Yamaguchi 755, Japan.
§ Present address: Dept. of Chemistry, Baldwin-Wallace College, Berea, OH 44017.
¶ To whom correspondence should be addressed.
1 The abbreviation used is: HO, heme oxygenase.
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REFERENCES |
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