From the a Marine/Freshwater Biomedical Center, Duke University Marine Laboratory, Beaufort, North Carolina 28516, the c Department of Chemistry, the University of Alabama, Tuscaloosa, Alabama 35487-0336, the e Department of Chemistry, Duke University, Durham, North Carolina 27708, the f Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, and the h Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213
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ABSTRACT |
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Previous studies showed that
CO/H2O oxidation provides electrons to drive the
reduction of oxidized hemoglobin (metHb). We report here that Cu(II)
addition accelerates the rate of metHb Hemoglobin (Hb)1
functions as an oxygen carrier only when reduced and is so maintained
in vivo through enzymatic NADPH-driven reduction reactions
(1-4). It has been suggested that low levels of copper, normally
present in vivo, promote Hb oxidation and that
copper-induced oxidation contributes to loss of reducing potential in
the red cell and eventual red cell deterioration. Copper is toxic at
elevated levels, and the mechanism of toxicity involves increased rates
of Hb oxidation with adverse side reactions such as increased cell
membrane oxidation (1, 2, 5, 6).
We owe much of our understanding of copper-induced oxidation of Hb to
the work of Winterbourn and Rifkind and co-workers (1, 5), who showed
that copper-induced oxidation of oxyHb occurs through an internal
electron transfer pathway between the iron atoms of the Previous studies carried out in our laboratories have shown that
Fe3+ in isolated heme (hemin), in Hb, in cytochrome
c oxidase and in several other oxygen-binding proteins can
become reduced when maintained under an atmosphere of CO, with the
half-cell reaction given in Eq. 1 driving the overall process (9).
chain reduction by CO by a
factor of about 1000. A mechanism whereby electron transfer occurs via
an internal pathway coupling CO/H2O oxidation to Fe(III)
and Cu(II) reduction is suggested by the observation that the
copper-induced rate enhancement is inhibited by blocking Cys-
93 with
N-ethylmaleimide. Furthermore, this internal
electron-transfer pathway is more readily established at low Cu(II)
concentrations in Hb Deer Lodge (
2His
Arg) and other species
lacking His-
2 than in Hb A0. This difference is consistent with preferential binding of Cu(II) in Hb A0 to
a high affinity site involving His-
2, which is ineffective in
promoting electron exchange between Cu(II) and the
heme iron.
Effective electron transfer is thus affected by Hb type but is not
governed by the R
T conformational equilibrium. The
hemes in
Cu(II)-metHb are reduced under CO at rates close to those observed for
cytochrome c oxidase, where heme and copper are present
together in the oxygen-binding site and where internal electron
transfer also occurs.
INTRODUCTION
Top
Abstract
Introduction
References
chain hemes
and copper bound at or near the
93 sulfhydryls. They found that
Cu(II) binding to Hb facilitates the oxidation process through this
internal pathway only when the amount of Cu(II) exceeds that bound at
unreactive high affinity sites, which in Hb A0 includes the
2 histidine residues. As a consequence, the oxidative reaction
proceeds less readily at low copper concentrations in Hb A0
than in the human Hb variant Hb Deer Lodge (
2His
Arg) and in Hbs
of species that lack His-
2 (1). Hb Deer Lodge has higher oxygen
affinity and increased anion sensitivity and is more readily oxidized
than Hb A0 (7, 8). We have used Hb Deer Lodge and Hb
A0 in the present studies in which we show that
CO/H2O-driven electron transfer can bring about a reduction
of the
chain hemes that utilizes this internal electron transfer pathway.
In the following we refer to metal reduction coupled to this
half-reaction as CO/H2O-driven reduction. The overall
free-energy change is clearly dependent upon the identity and
environment of the metals reduced. The process of reduction under CO
was called "autoreduction" by previous workers (10-14) who noted
that cytochrome c oxidase, with two constituent
electron-accepting metal centers in the oxygen-binding site, becomes
reduced under CO. Heme a3 in the oxygen-binding
site of the oxidase is typically reduced in less than an hour, with a
much slower (overnight) incubation required to bring about reduction of
heme a. The details of the reduction of cytochrome
c oxidase are not well understood, and there are clearly
competing processes that have hindered elucidation of the mechanism
(15).
(Eq. 1)
We now report that Cu(II) addition to metHb dramatically increases the
rate of heme reduction under CO. We show that Cu(II) is reduced to
Cu(I) as the reaction proceeds and that the enhanced rate of reduction
is inhibited by sulfhydryl reagents such as N-ethylmaleimide. The data indicate the existence of an
internal pathway that is used for electron exchange between the heme
iron and the bound Cu(II).
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EXPERIMENTAL PROCEDURES |
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Samples of native adult human hemoglobin (Hb A0) and
Hb Deer Lodge were prepared by the ammonium sulfate method,
chromatographically purified, and stripped of organic phosphate
cofactors as described previously (7). Horse Hb was prepared similarly
but without chromatographic purification. The samples were typically
dialyzed against 0.05 M Tris at pH 8.3 with selected anions
as desired. Concentrations and compositions of samples were determined
spectrophotometrically, using Hewlett-Packard 8451 and 8453 Diode Array
spectrophotometers for routine measurements and a Perkin-Elmer 6
dual beam-scanning spectrophotometer for higher resolution work.
Samples were usually measured in 1-cm path length cuvettes, but cells
with 2-mm path lengths were used to obtain spectral data on samples
whose optical density would have gone beyond the linear response range
of these instruments. Oxidized (met)Hb samples were made by treatment
with potassium ferricyanide at 1.2-fold excess over heme. Sequential Sephadex G-25 chromatographies (high salt then no salt) were used to
remove free and bound ferricyanide and ferrocyanide from the metHb
samples. The amounts of oxidized Hb (metHb), deoxyHb, oxygenated Hb
(oxyHb), carbon monoxyHb (CO-Hb), and hemichrome were determined by
spectral analysis. Samples that contained any detectable hemichrome were discarded. The stock Hb solutions, typically 1-2 mM
in heme units (iron-porphyrin units), were stored in liquid nitrogen
prior to use. Sodium dithionite at 2 mg/ml was used as a reductant to obtain spectra of the fully reduced proteins. Hb with only the
chain hemes oxidized was prepared by addition of a 5-fold excess of
CuSO4 over oxyHb. It has been shown (2, 16, 17) that under
these conditions the
chain hemes are selectively oxidized, whereas
the
chain hemes remain in the reduced, ferrous state.
Isolation of and
chains of Hb A0 was accomplished
by use of a modified version of established methods in which
para-hydroxymercuribenzoate binds to SH groups and
encourages dissociation of the Hb tetramer (18). Our modifications
consisted of carrying out the separations at 4 °C, using
chromatographies with CMC cellulose (for
chains) and DEAE cellulose
(for
chains) that effectively separate the chains. Regeneration of
the SH groups of the
chains was done by treatment with
-mercaptoethanol to reduce SH groups, followed by chromatography
through DEAE-cellulose to remove
para-hydroxymercuribenzoate. Tetramers of
chains form
spontaneously after SH groups are regenerated.
The free SH groups of Hb (at position 93) were blocked by treating
Hb A0 with N-ethylmaleimide (NEM) at a
protein-to-reagent ratio of 1:3. The reaction was carried out at
37 °C for 1 h with the reactants in 0.05 M
bis-Tris, pH 7.2, followed by Sephadex G-25 chromatography to separate
the Hb from the low molecular weight reagent.
Carboxypeptidase A-digested Hb A0 (HbCPA) was prepared by
treating the CO derivative of Hb A0 with carboxypeptidase A
(Sigma, type 1, diisopropyl fluorophosphate) at an enzyme-to-protein
ratio of 1:50. The mixture was incubated at 37 °C for 2 h and
then dialyzed at 4 °C against 0.05 M Tris buffer, pH
8.3. This enzymatic digestion under the conditions employed removes the
C-terminal His and Tyr of the chains as verified by electrospray
ionization mass spectrometry.
Cu(II)-metHb complexes were made by adding CuSO4 to metHb at indicated ratios of copper to heme. Hb samples in some cases were treated with 5-fold excess of Cu(II) and then stripped of unbound or loosely bound copper by passage through a Chelex resin column. In other experiments, the copper-Hb complexes were made by addition of copper at the desired ratio of copper to heme without subsequent Chelex treatment. Elemental analysis of samples by atomic absorption spectroscopy (Perkin-Elmer 5000) following copper treatment validated the copper:iron ratios used and demonstrated that our Chelex chromatography of samples treated with a 5-fold excess of copper typically reduced the copper:heme ratio to about 0.6:1, consistent with retention of copper at unreactive high affinity sites and partial retention of copper at the lower affinity sites associated with electron transfer to the heme. Prolonged exposure to Chelex eventually removes all Cu.
Incubations under CO were done with chemically pure grade (99.5%
purity) CO gas provided by National Welders, Inc. For visible-region spectroscopy of copper-Hb complexes, CO gas was added to tonometers containing thoroughly deoxygenated metHb samples. Deoxygenation was
accomplished by multiple stages of sequential evacuation and purging
with argon or nitrogen to ensure that no oxygen was present in the
samples before CO incubation was begun. For EPR measurements, incubations of samples under CO were done for time intervals indicated under "Results" and immediately frozen in liquid nitrogen in EPR tubes for assays. Aqueous CuSO4 in the absence of Hb or
other proteins was incubated in MES buffer with IHP at pH 6, 20 °C. Sodium dithionite (2 mg/ml) was added after variable periods of incubation under CO to generate the spectra of the fully reduced proteins.
EPR spectra of Hbs at about 0.5 mM in heme were recorded on
a hybrid machine consisting of a Varian E109E spectrometer console used
to provide the field modulation to a Bruker B-E 25 magnet, with an ER
082 power supply and a B-H 15 field controller, plus a Varian E102
microwave bridge coupled to a V-453.3 cylindrical cavity. Temperature
control was effected by means of an Oxford Instruments ESR 900 liquid
helium flow cryostat and accessories. The g values of
samples were measured with reference to diphenylpicrylhydrazyl. Spin
concentrations were determined by double integration using Cu(II)-EDTA,
equine ferricytochrome c, and a quantitatively high spin
methemoglobin sample as integration standards for the g = 2, 3, and 6 regions of spectra, respectively.
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RESULTS |
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Visible Spectroscopy of the CO/H2O-driven Reduction of
Cu(II)-MetHb Complexes--
Heme reduction brought about by incubation
under CO allows for binding of CO, so that time courses show a
disappearance of the spectral features of metHb and an appearance of
the features of CO-Hb. There are generally good isosbestic points for
the transition, with the exception of experimental conditions described
later. Cu(II) addition significantly enhances the rate of metHb
reduction, with enhanced rates evident only for the chains of the
Hb tetramer.
Specificity for chains in Cu(II)-enhanced rates of reduction is
supported by the fact that there are similar reaction kinetics for
half-reduction of a Cu(II)-metHb complex,
(III)2
(III)2, and for full reduction of a
half-metHb preparation,
(II)2
(III)2, with
the same ratio of Cu(II) to heme. Fig. 1
shows the spectral changes in these two samples brought about by
incubation under CO. Good isosbestic points between the met and CO
derivatives are observed in both cases. The fully oxidized Hb becomes
half-reduced in about 10 h, with a half-time of
chain
reduction of about 4.5 h. For the copper-Hb complex with only the
chains oxidized, the half-time for reduction is also about 4.5 h. We conclude that it is the reduction of the
chain hemes that is
appreciably increased in rate by copper addition and that the oxidation
state of the partner chains is not a major determinant of the reduction
rate.
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Isolated chains were prepared as described under "Experimental
Procedures." After regeneration of SH groups, the
chains spontaneously form tetramers. The
4 tetramers do
not show an increase in the rate of
CO/H2O-driven reduction when observed at a copper:heme
ratio of 0.5 (Table I). However,
Cu(II)-enhanced rates of reduction are observed when the
copper:heme ratio is raised to 2. Rates of reduction under CO at this
Cu(II) level are appreciably enhanced over controls. Rate estimates at
this Cu(II) level are not given in Table I because of uncertainties associated with some sample precipitation during these experiments. The
requirement for higher Cu(II) levels in
4 tetramers can
be explained by the fact that each
chain has a high affinity
copper-binding site at the
2 position that would be expected to be
ineffective in electron transfer. The failure to see copper-enhanced
rates at ratios below 1 copper per heme is thus an indication that
Cu(II) has limited access to effective electron transfer sites in the
4 tetramers. We conducted an additional experiment to
determine if the requirement of high copper:heme levels for reduction
of
4 tetramers was due to some artifactual modification
of the
chains during the isolation procedure. We found that the
chains do become reduced under CO at a copper:heme ration of
0.5 when present in half-met
(II)2
(III)2
tetramers that were made by 1:1 mixing of the isolated
chains with
reduced
chains. This experiment with reconstituted half-met
tetramers gave results similar to those shown in Fig. 1B.
This confirmed that the isolated
chains were selectively reduced
under CO after copper addition.
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The effects of varied levels of Cu(II) on the time courses of heme
reduction under CO were explored. As shown in Fig.
2, with no copper present there is very
little reduction of metHb A0 or metHb Deer Lodge after a
24-h incubation under CO (data shown only for Hb A0,
open circles). The small extent of reduction of the
copper-free metHb A0 control is consistent with our
previous work that established the half-time for metHb A0
reduction under CO to be about 1000 h at 1 atm pressure. When the
process is carried out with a copper:heme ratio of 0.25, the process is
only slightly increased in rate for Hb A0 but appreciably
increased in rate for Hb Deer Lodge (
2His
Arg). The
CO/H2O-driven reduction of metHb A0 occurs more
quickly at elevated Cu(II) levels. As shown, at a copper:heme ratio of
0.7, approximately one-half of metHb A0 becomes reduced at
a copper-enhanced rate, attributable to preferential reduction of the
chains (see above). The remainder of the reaction is ascribed to
the reduction of the
chains. This much slower phase is less
sensitive to copper addition. No efforts were made to investigate
copper-Hb interactions at ratios above 1.0 copper:heme, since ratios
greater than 0.5:heme can result in gradual protein precipitation (1)
and disulfide formation (2).
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The time courses of heme reduction observed over a 24-h period were
generally well fit by assuming that they are composed of two
exponential phases. Apparent rate constants for the two phases,
obtained under varied conditions, are given in Table I. The apparent
first-order rate constant of the first phase
(k1) reflects the Cu(II)-sensitive rate of chain reduction. The apparent first-order rate constants for the second
phase (k2) are like those of the copper-free controls.
The copper:heme ratios indicated in Table I were established by Cu(II) addition to thoroughly deoxygenated metHb samples (exceptions are indicated). Multiple stages of sequential de-aeration and purging with argon or nitrogen ensured that no oxygen was present in the samples before CO incubation was begun, generally about 20 min after copper addition. That reactive oxygen species are not involved in these reactions was verified by observing similar time courses of CO/H2O-driven reduction in the presence of added catalase and/or superoxide dismutase. Greatly enhanced rates of heme reduction for Cu(II)-metHb complexes under CO relative to samples without copper were observed in the Soret region with dilute samples (about 10 µM in heme) and in the 500-650-nm wavelength region with much more concentrated samples (about 500 µM in heme) in 2-mm path length cells. The phenomena observed are not artifacts of dissociation, as verified by observing similar behavior over a wide protein concentration range and use of high pressure liquid chromatography that showed no experimentally detectable subunit dissociation in the high concentration samples.
At low levels of copper (0.25 copper:heme), the rate and extent of
reduction of metHb Deer Lodge (closed squares in Fig. 2) are
clearly more affected by copper addition than reduction of metHb
A0 (closed circles). Horse Hb, also lacking
His-
2 residues, behaves like Hb Deer Lodge in having an appreciable
copper-enhanced phase of CO/H2O-driven reduction at 0.25 copper:heme (Fig. 2 and Table I). These results mirror the EPR results
presented in the next section. The structural explanation for the
difference in copper sensitivity exhibited by metHb A0,
horse metHb, and metHb Deer Lodge is that the His-
2 residues of Hb
A0 are key residues in high affinity copper-binding sites
that are ineffective in electron exchange with the heme.
Although there are theoretically two high affinity copper-binding sites
per tetramer (0.5 copper per heme) in Hb A0, significant enhancement of its reduction rate under CO occurs at Cu(II) levels above 0.25 copper:heme, indicating a distribution of copper to the
sites responsible for CO/H2O-driven reduction before all
"high affinity" sites are filled. A similar copper concentration
dependence was reported by Louro and co-workers (19) in their studies
of copper-induced oxidation of the chains of Hb.
Sample treatment can alter the time courses of CO-driven reduction by
altering the distribution or access of copper to the "effective"
copper-binding sites. As illustrated in Fig.
3, significant inhibition of the copper
effect occurs when copper-binding sites near the 93 sulfhydryl
groups are blocked by treatment with N-ethylmaleimide (NEM).
NEM treatment also results in a slight increase in the rate of
reduction of the copper-free control. Variable time courses, presumably
reflecting variable distribution of Cu(II) among the binding sites, can
also be achieved by variations in sample handling, as illustrated by
the two lower (faster) time courses of Fig. 3 where samples gave
significantly different time courses of reduction as a consequence of
different sample handling procedures.
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The rates and time courses (within experimental variability) of CO/H2O-driven reduction are unchanged when Cu(II)-metHb complexes are incubated under progressively lower CO levels over a range of solution concentrations from 1 to 0.05 mM CO. Within this range of CO concentrations, good isosbestic points are typically observed between the met and CO derivatives of Hb, and the decreases in absorbance at 406 nm (metHb disappearance) and the increases in absorbance at 419 nm (CO-Hb appearance) have the same time dependence. The CO concentration independence and the wavelength independence of the process together indicate that the Fe(III) (met) and CO-Hb forms are the only significantly populated species and that something other than CO concentration is rate-limiting in the transition toward CO-Hb. Intermediate species are, however, apparent when incubation is carried out at low CO levels and IHP is present. Under progressively lower CO concentrations (<0.25 mM) in the presence of IHP, the time courses observed at 406 and 419 nm become increasingly distinct, and the isosbestic point between met and CO-Hb is not maintained.
We examined the CO/H2O-driven reduction of Cu(II) complexes of metHb A0 under a number of experimental conditions, with results summarized in Table I. Many of our experiments were performed at relatively low protein concentration (10-15 µM in heme) where the concentration of dimeric forms of metHb would be appreciable. Remarkably, there are no systematic differences in reduction rates when the heme concentration is varied over the range of 10 to 500 µM. This is evidence that the internal electron transfer responsible for heme reduction is governed by tertiary rather than quaternary conformational states. This conclusion was also supported by studies reported below, using T-state stabilization by IHP and R-state stabilization by digestion with carboxypeptidase A.
Strong allosteric effectors such as inositol hexaphosphate (IHP) can
shift R-state metHb toward the T-state (20). IHP addition results in an
approximately 2-fold decrease in the half-time for the rapid ( chain) phase of reduction at pH 8.3. This suggested that
copper-enhanced reduction of metHb might be slower in its T-state
conformation. We then looked for possible pH effects on the IHP-induced
changes in rate, since IHP stabilization of the T-state would be
expected to be more evident at low pH. However, the rate of reduction
in the presence of IHP is appreciably faster at pH 7 than at
pH 8.3. The pH shift alone caused only a minor alteration in reduction
rate. Lower rates of reduction would have occurred at pH 7 if the IHP
effect was due to a T-state shift. The altered efficacy of Cu(II) in
the presence of IHP thus does not follow the pattern expected for R
T shifts of quaternary conformation.
We further examined the possible effect of Hb conformation on the
reductive process by monitoring the CO/H2O-driven reduction of metHb after modification by digestion with carboxypeptidase A
(HbCPA). This digestion removes the C-terminal His and Tyr residues of
the chains, strongly stabilizes the R-state conformation, and
inhibits the IHP-induced shift toward the T-state (21, 22). HbCPA
controls behaved like the undigested protein, so that in the absence of
copper metHbCPA became reduced under CO with an apparent rate constant
similar to that of metHb A0 (k2 = 0.006 h
1). In the presence of Cu(II) at 0.5 copper:heme,
half-reduction of metHbCPA was enhanced in rate over that of the
copper-free controls. The rate enhancement was relatively small, so
that
chains became reduced at a rate about half that characteristic of the undigested protein. Constraining the protein to the R-state by
this modification thus caused a decreased rate of reduction. Addition of IHP increased the rate to somewhat greater than observed for undigested protein in the absence of IHP, with a half-time for
reduction of the fast phase of about 1 h. The rate increase brought about by IHP on the reduction of metHbCPA is in marked contrast
to results obtained with metHb A0, where the reductive process is slowed by the presence of IHP (see Table I). We conclude from these comparative studies that the rate of reduction is governed by tertiary effects on the internal electron transfer pathway and not
by the pattern of T
R shifts of quaternary conformation that govern
oxygen binding.
The rate of the reductive process is clearly affected by the level of Cu(II) that is bound at a site that is effective in internal electron transfer. In our studies with metHbCPA, we noted that the magnitude of the fast phase of reduction is not as great as that for metHb A0 at a comparable copper:heme ratio. The reduced magnitude of the fast phase of reduction is probably due to sequestration of copper in non-reactive sites. The magnitude of the fast phase of reduction after addition of the allosteric effector IHP is not reduced, suggesting that IHP binding alters the copper distribution toward sites that are effective in electron transfer.
Visible spectroscopy using the Cu(I)-dependent absorption
changes of bathocuproin at 483 nm (23) demonstrated that significant amounts of Cu(II) are reduced to Cu(I) when protein-free
CuSO4 is incubated under CO. EPR measurements described
below confirmed that incubation of Cu(II) under CO led to its
reduction. Since Cu(I) is a potential heme reductant, we investigated
the possibility that addition of Cu(I) would bring about the reduction
of chain hemes in Hb. For this study thoroughly degassed solutions
of Cu(I) as CuCl at a ratio of 0.5 copper:heme were added to
deoxygenated metHb in tonometers. No appreciable spectral shifts were
observed. We then added 1 atm CO. There was no spectral indication of
heme reduction after 24 h (data not shown), indicating that under
these conditions, free Cu(I) does not directly contribute to reduction of metHb. Exposure to air gave Cu(II)-metHb complexes equivalent to
ones prepared by our normal procedures that were susceptible to
CO/H2O-driven reduction.
EPR Studies of the CO/H2O-driven Reduction of Cu(II)-MetHb Complexes-- Experiments described here demonstrate the crucial role of copper in the mechanism of reduction of Cu(II)-metHb complexes. As reported below, both copper and heme reduction occur to an appreciable extent when Cu(II)-metHb complexes are incubated under CO. We also found that maintaining various preparations of aqueous Cu(II) (in the absence of protein) overnight under 1 atm CO leads to the disappearance of at least 20% of the initial Cu(II) EPR signal as a result of CO/H2O-driven Cu(II) reduction. Thus, Cu(II) in the absence of heme is susceptible to reduction under CO.
The EPR spectrum of human metHb A0 at pH 8.3 typically
contains two distinct sets of signals, as shown in Fig.
4A, that arise from high spin
ferric hemes (g = 5.9 and 2.0) and low spin ferric hemes (gzyx = 2.59, 2.18, and 1.83).
These two kinds of signals correspond to situations in which the sixth
ligand to iron is H2O and OH respectively
(24, 25).
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EPR signals, attributable to Cu(II) in a Cu(II)-metHb complex, appear
following the addition of 0.5 eq (per heme) of CuSO4 to
anaerobic solutions of metHb, as shown in Fig. 4B. These
signals were first observed by Bemski and co-workers (26, 27) and have
since been confirmed by several other groups for the complex of Cu(II)
with Hb A0. Within our experimental uncertainty, the EPR
signals we observed after Cu(II) addition to oxidized Hb A0 are indistinguishable from those previously reported where Cu(II) was
added to the deoxy derivative. Fig.
5A, showing enlarged details of Fig. 4B, illustrates the 9-line superhyperfine pattern
indicative of square-planar coordination of the Cu(II) by four
electronically equivalent nitrogen ligands (27, 28). As reported
previously by Antholine et al. (29), this pattern is the
spectral signature of the high affinity Cu(II) binding site (site 3 in
Antholine's terminology), located near the N terminus and His-2 of
each
chain. Not all Cu(II) sites are occupied at a ratio of
copper:heme of 0.5:1, and most of the Cu(II) binds preferentially to
the high affinity site. Consequently the distinctive signature of
Cu(II) at the lower affinity site at or near the Cys-
93 (site 2 in
Antholine's terminology) is not readily observed in the spectrum. Fig.
5B shows that this superhyperfine pattern is absent in Hb
Deer Lodge which lacks His-
2.
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We fortuitously prepared an uncomplexed metHb sample that contained no measurable low spin component. By using this sample as an integration standard for the g = 6 region, we determined that the heme groups of Fig. 4A were at least 50% high spin. Upon complexation with Cu(II) (Fig. 4B), the heme groups were 30% (±10%) high spin. Apparently all the added Cu(II) contributed to the intensities of resulting spectra so that there was no clear evidence for coupling between the ferric hemes and Cu(II), although we would not have been able to detect low levels (10%, or less) of Cu(I) or anti-ferromagnetically coupled (and thus EPR silent) species.
Fig. 4C shows that there is no significant change in the EPR spectrum if the Cu(II)-metHb A0 complex is incubated at room temperature under 1 atm CO for 1 h prior to freezing in the EPR tube. However, if the CO incubation is continued overnight, some heme and copper reduction takes place as shown in Fig. 4D. In this case about 20% of the low spin heme signal and about 20% of the Cu(II) signal disappears (cf. Fig. 4, C and D) as a result of reduction. This result is consistent with spectral changes indicative of heme reduction observed by visible spectroscopy (see previous section).
Fig. 6A shows that the EPR
spectrum of metHb Deer Lodge prepared at pH 8.3 contains the same high
spin and low spin ferric heme signals as found for metHb
A0, but the ratio of these two components differs in the
two hemoglobins. Typical Hb Deer Lodge samples contained only 15%
(±5%) of the high spin component. Addition of 0.5 eq (per heme) of
CuSO4 leads to the spectrum of Fig. 6B. Due to
the overlapping low spin ferric heme signals in the present spectra, we
cannot determine the EPR parameters for the Cu(II) signal in the Hb
Deer Lodge complex with high precision, but we note that, within our
experimental uncertainty, they are indistinguishable from those
previously reported for a "low affinity" Cu(II)-binding site in
deoxyhemoglobin A0 that is associated with the Cys-93 residue, located less than 10 Å from the
chain heme (30). It is
this copper-binding site that has been associated with internal electron transfer in copper-induced oxidation of heme (19). Moreover,
the Cu(II)-metHb complex of Hb Deer Lodge does not show the 9-line
superhyperfine pattern associated with Cu(II) binding at or near the
His-
2, indicating that this site is absent (Fig. 5B). The
Cu(II) complexes of other Hbs without a His-
2, such as horse
hemoglobin (1) and cat hemoglobin (30), have also been shown to give
EPR spectra that lack the 9-line superhyperfine pattern.
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Incubation of the Cu(II)-metHb Deer Lodge complex under CO leads to
strikingly different results compared with the Cu(II)-metHb A0 complex. After 1 h there is 20-30% reduction of
the hemes and 30-40% reduction of the copper, as shown in Fig.
6C. After 17 h, some 60-70% of both hemes and copper
have been reduced as shown in Fig. 6D. The structural
explanation for the greater extent of reduction in this case is that
copper is more available at the effective site for electron transfer in
Hb Deer Lodge, since its 2His
Arg substitution eliminates the
high affinity copper-binding site found in Hb A0. This
result of heme and copper reduction mirrors previous reports on heme
oxidation (1) where more copper is required for oxidation of the
chains in Hb A0 than in Hbs like Hb Deer Lodge that lack
His-
2 residues.
The site of copper binding clearly determines its ability to
participate in the CO/H2O-driven reduction reaction, as
evidenced by the much slower rate of reduction of Hb A0
relative to Hb Deer Lodge (2His
Arg) at low copper levels (Figs.
4 and 6). Copper is clearly a partner in CO/H2O-driven
reduction of the Cu(II)-metHb complexes since both heme and copper
reduction occurs for Cu(II)-metHb complexes incubated under CO.
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DISCUSSION |
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We previously demonstrated that a reaction similar to the
water-gas shift reaction, driven by the oxidation of CO/H2O
to CO2 + 2H+ + 2e (Equation 1),
can drive the reduction of a number of heme proteins, with free energy
changes dependent on the type of heme protein and the experimental
conditions (9). The results presented here show that Cu(II) addition
enhances the rate of reduction of the
chains of metHb under CO,
implicating
heme and Cu(II) as acceptors of the two electrons
generated by Equation 1. The EPR measurements show reduction of Fe(III)
and Cu(II) when metHb is incubated under CO in the presence of Cu(II).
The
chain hemes in Cu(II)-metHb assemblies exhibit rates of
CO/H2O-driven reduction that are roughly 1000-fold faster
than those for Hb in the absence of Cu(II). The fastest
chain
reductions we observed have half-times of about 1 h, only slightly
less rapid than previously reported for the CO/H2O-driven
reduction of heme a3 and copper of the
oxygen-binding site of cytochrome c oxidase (9).
Clearly, the heme and copper together enhance the rate of CO/H2O oxidation that drives the reductive process in Cu(II)-metHb complexes. The rate enhancement makes use of an internal pathway of electronic communication in the Cu(II)-metHb complexes that is functionally similar to but structurally distinct from that of heme a3 and copper in cytochrome c oxidase. The differences are evident in the EPR spectra arising from these two systems, where the Cu(II) in the binuclear site of the oxidase, but not in the Cu(II)-metHb complexes, is EPR silent (31) due to an anti-ferromagnetic exchange interaction with heme a3 (Ref. 32 and references therein).
Scheme A and Scheme B as described below summarize two reaction sequences that are consistent with the available data. They are not mutually exclusive and differ primarily in how CO oxidation is coupled to the electron transfer reaction.
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Scheme A invokes the generation of an intermediate as a result of CO
attack on a metal hydroxide. This process limits the reaction rate at
low CO concentration (less than 0.05 mM) and achieves
saturation and becomes CO independent above 0.05 mM. Subsequently, as CO2 is released, an intermediate species
is formed with two-electron reducing potential that is equivalent to a
transient species with the reducing potential of a metal hydride (shown in quotes). A subsequent internal electron transfer process is postulated to be the rate-limiting step at high CO pressure, supported by the observed Cu(II) dependence of the reaction rate. In Hb, Fe(III)
would serve as M1 and Cu(II) bound at or near Cys-93 would serve as M2. Cu(II) bound at or near Cys-
93 in Hb
is less likely to serve as M1 because this less hydrophobic
environment would be less favorable for generation and distribution of
electrons from the postulated intermediate to heme in the active site.
The presence of a second electron acceptor, M2, in
electronic communication with M1 is ultimately necessary
for facile reduction of both metals and release of protons.
Under some conditions the partial oxidation of Fe(III) by Cu(II) may also play a role in utilizing the electrons provided by CO/H2O oxidation (Scheme B). Since Cu(II) can oxidize the heme Fe(II) of the chains of Hb (1), it is possible that the internal electron exchange pathway can allow for some population of a Fe(IV)-Cu(I) redox pair.
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This report deals exclusively with the Cu(II)-induced enhancement of
the reduction of metHb under CO that is NEM-sensitive and utilizes an
internal electron transfer pathway. Comparative studies revealed that
the rate and extent of reduction of the Cu(II)-metHb complexes under CO
are limited by the availability of Cu(II) at a site that allows for
electron exchange with the chain hemes. The sulfhydryl group at
93 is implicated in the process since Cu(II)-induced rate increases
are inhibited when this residue is blocked by treatment with NEM. The
chain hemes are separated from Cys-
93 by only a single residue,
the proximal histidine. Therefore, this histidine can be presumed to be
part of the pathway between iron and copper bound at or near Cys-
93. It may constitute the entire pathway, since an effective electron transfer pathway would be created if Cu(II) bound at or near the Cys-
93 rearranged its binding site by displacing the N-2 proton of
the imidazole of the proximal histidine. This rearrangement would
create an imidazolate bridge between the heme iron and the bound copper
and provide a short internal pathway for electron transfer. Our data
are unable to support or refute an imidazolate bridge as the sole
member of an internal electron transfer pathway.
We tested the hypothesis that metHb reduction proceeds via CO reduction
of Cu(II), followed by Cu(I) coordination of the sulfhydryl of
Cys-93, followed by electron transfer to the heme group. However, experiments conducted with Cu(I) addition to metHb (with 0.5 Cu(I)/heme) showed no detectable heme reduction before or after
incubation under 1 atm CO. It is possible that strong Cu(I) binding to
Cys-
93 prevents Cu(I) from occupying the site where electron
transfer is effective. There is some experimental basis for this
possibility, since it has been shown that when Cu(II) is added to
oxyHb, the Cu(II) changes its position with respect to Cys-
93 prior
to Cu(II)-induced oxidation of the
chains (30). The alternative
explanation is that Cu(I) does not promote heme reduction because of
unfavorable energetics.
The rate of the process whereby Cu(II)-metHb becomes reduced when
incubated under CO is affected by Hb type, by digestion with
carboxypeptidase A, by IHP binding, and by reaction temperature. We
investigated the possibility that these variations were due to
alterations in allosteric equilibrium between quaternary conformations of high and low oxygen affinity. Although unable to bind oxygen, metHb
is also switchable between these conformational states (20, 24). The
rates observed for CO/H2O-driven reduction of the
Cu(II)-metHb complexes did not, however, correlate with alterations in
the R T equilibrium. Furthermore, the conformational equilibrium is
very sensitive to pH, but the CO/H2O-driven reduction of
Cu(II)-metHb complexes shows no appreciable pH sensitivity. No
systematic changes were noted between experiments conducted at high
concentration (500 µM in heme) where the percentage of
dimers was confirmed to be vanishingly small and at low concentration
(10 µM in heme) where the percentage of dimers was
appreciable. We conclude that while the process is sensitive to protein
conformation, the quaternary R
T equilibrium is not the primary
determining factor that establishes the rate or extent of this
reductive process. The internal pathway may, however, be altered by
structural changes induced by IHP binding, such as the movement of the
A helices of the
chains toward the center of the molecule (34).
There is an increasing awareness that novel internal electron exchange pathways in proteins can be of physiological significance. Copper toxicity associated with Hb oxidation (1) and CO/H2O-driven reduction of metHb demonstrated in this paper are cases in point. We speculate that CO and NO may have parallel biological functions that are dependent upon internal electron exchange pathways in proteins. The pathway shown to be utilized in CO/H2O-driven reduction of metHb may also be involved in NO-driven reduction of metHb. We have shown, for example, that the rate of NO-driven reduction of metHb is, like CO/H2O-driven reduction, dependent on the nature of the Hb being reduced and, also as shown for CO/H2O-driven reduction, the rate of the process is independent of NO concentration over a wide range (35).
The importance of SH groups at 93 is indicated by the fact that they
are highly conserved in mammalian Hbs. Our studies emphasize the role
of these SH groups in the internal electron exchange pathway that links
heme and SH groups and can lead to regeneration of active (Fe(II))
heme. In earlier studies, it was suggested that these SH groups are
protective against Hb oxidation (1-3). Other studies have shown that
the interactions of NO with these SH groups are critical for control of
blood pressure and that formation of SNO-Hb is dependent upon the redox
state of the heme, so that oxy- and metHbs differ in their rates of
SNO-Hb formation (36). This redox dependence may be another consequence
of the existence of the internal electron exchange pathway that allows for Cu(II)-enhanced rates of CO/H2O-driven reduction.
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ACKNOWLEDGEMENT |
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Expert analytical assistance provided by Dr. Robert Stevens of the Duke Mass Spectrometry Facility is gratefully acknowledged.
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FOOTNOTES |
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* This work was supported in part by National Institute of Environmental Health Sciences Center Grant ESO1908 (to C. B.) and National Institutes of Health Grant HL-58248 (to C. B. and A. L. C.).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.
b To whom correspondence should be addressed. Tel.: 252-504-7591; Fax: 252-504-7648; E-mail bona{at}duke.edu.
d Supported by the Tobacco Research Council and Apex Biosciences, Inc.
g National Institutes of Health Postdoctoral Fellow.
i Supported by grants from the American Heart Association (Alabama Affiliate), Biomedical Research Support Grant S07RR7151-14, and National Science Foundation Grant 9506817.
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ABBREVIATIONS |
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The abbreviations used are: Hb, hemoglobin; CPA, carboxypeptidase A; MES, 2-(N-morpholino)ethanesulfonic acid; NEM, N-ethylmaleimide; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)- propane-1,3-diol.
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REFERENCES |
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