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INTRODUCTION |
Cell-free hemoglobins, chemically altered or genetically expressed
in microbial host systems, have been developed as oxygen-carrying therapeutics. Site-directed modifications are introduced and serve to
stabilize the protein molecules in their tetrameric, functional forms
(1). Animal studies as well as recent clinical studies have shown that
these proteins probably deliver sufficient oxygen to tissues, but
concerns still persist regarding the spontaneous oxidation
(autoxidation) of hemoglobin and its redox reactions with tissue
oxidants that may potentially impede its clinical usefulness (2).
Vascular endothelium, a source of a number of oxidants, has emerged as
the primary target of hemoglobin-based toxicity due to its proximity to
the circulating protein. Hemoglobin, unlike red cells, can diffuse
through the endothelial barrier lining the vessel wall, where it can
potentially reach nitric oxide (NO), the endothelial derived relaxing
factor (1, 3). Reactions of NO with hemoglobin result in the diversion
of NO away from the smooth muscle target enzymes, leading to the loss of NO-dependent responses such as vasodilation.
Hypertension is a common phenomenon associated with the infusion of
hemoglobins in animal models as well as in humans (4-7). The ensuing
oxidative reactions of hemoglobin with peroxynitrite
(ONOO
), the product of the reaction of NO with superoxide
(O
2), hydrogen peroxide (HOOH), or lipid peroxide in the
vasculature may exacerbate tissue damage (2).
Under normal conditions, the interplay between NO, O
2, and
ONOO
in the vasculature is a delicate balance that must
be kept between the pro- and antioxidant processes (8). This balance
between NO and O
2 can however, be disrupted in favor of
ONOO
and HOOH under a variety of nonphysiological
conditions (8, 9). Additionally, increased HOOH production is thought
to occur under conditions of reperfusion with oxygenated media, in this case hemoglobin (10). Cell culture studies have identified several potential candidates for the agent(s) responsible for the observed cytotoxicity. The oxidative reactions of hemoglobin with HOOH produced largely from endothelial cells are believed to play an important role in hemoglobin-mediated tissue damage (2). Peroxide can
induce rapid oxidation of oxyhemoglobin (Fe2+) to
methemoglobin (Fe3+). The toxicity of hemoglobin
preparations in endothelial cell culture was shown to be dependent on
the rate of autoxidation, and it correlated with time in culture
and the presence of iron chelators (11).
Hemoglobin induces cellular protective mechanisms and genes that
protect against oxidative damage, such as the production of
transferrins and heme oxygenase. This phenomenon underscores the
importance of oxidative stress as a mediator of cell damage (12). The
reaction between the oxy or the ferric forms of hemoglobin with HOOH is
known to proceed via the formation of the highly reactive oxyferryl
complex, Fe(IV)=O detected by optical spectroscopy and a
globin-associated free radical detected by EPR (13). These species have
been implicated in the mechanism of ischemia/reperfusion injury and
most recently were detected in normal human and animal blood (14, 15).
A high uncontrollable rate of autoxidation of hemoglobin and
vasoconstriction due to the scavenging of nitric oxide were shown to be
among the primary constraints in demonstrating the efficacy of some
hemoglobin solutions used as blood substitutes (16). The reactions
between hemoglobin and tissue oxidants are clearly complex phenomena
that must be well understood in order to resolve fully some of the
hemoglobin-mediated toxicities.
Strategies to combat oxidation reactions of hemoglobins are evolving.
Chemical polymerization of the tetrameric molecule or encapsulation of
hemoglobin to inhibit extravasation into the endothelium are among the
most commonly used approaches for NO scavenging and unfavorable
oxidative side effects. Site-directed mutagenesis of the distal pocket
of myoglobin constructs has provided a simple model for the engineering
of hemoglobin subunits (17). Both myoglobin and hemoglobin constructs
have been produced in which the rates of autoxidation and NO-induced
oxidation have been substantially inhibited (18, 19). This result is
achieved by filling the distal pocket with larger, apolar residues at
position 29(B10) and/or 68(E11). The oxygen affinities of some mutants have also been varied by altering hydrogen bonding to the E7 residue and/or interfering sterically with bound ligand (17, 18). Recently,
Doherty et al. (20) have shown that the rate of reaction with nitric oxide determines the hypertensive effect of cell-free hemoglobin. In rats, infusion of recombinant hemoglobins with decreased
NO-scavenging activities causes corresponding decreases in the
hypertensive responses compared with those of hemoglobins with
unchanged NO oxidation rate constants (20).
Here we present data on the interactions of HOOH with oxy and ferric
forms of sperm whale myoglobins and the effects of single, double, and
triple amino acid mutations at positions His-E7, Leu-B10, and Val-E11
on these reactions. We also identify some of the structural features
that regulate the pseudoperoxidase activity of myoglobin that may be
relevant to the design of a stable hemoglobin-based blood substitute.
The studies detailed in this report were carried out at concentrations
of hydrogen peroxide that are comparable with the levels that are found
in vivo. The concentration of peroxide in normal human
plasma is between 4 and 5 µM (21). It is not known how
much peroxide is actually produced in inflammatory lesions in
vivo. However, endothelial cells lining the blood vessels form the
initial point of contact with extracellular oxygen carriers and have
been shown to be a major source of hydrogen peroxide under both normal
conditions and during reperfusion after ischemia (22). Stimulation of
various cellular components (platelets, neutrophils and macrophages)
attracted to these sites can also contribute to the peroxide pool in
the extracellular space. For example, it has been shown that activated
polymorphonuclear leukocytes alone can generate up to 200 µM HOOH in vitro (23). Moreover, under
conditions of ischemia reperfusion, high levels of nitric oxide have
been shown to inhibit glutathione peroxidase, a scavenger of peroxides,
leading to accumulation of higher levels of this oxidant (24).
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EXPERIMENTAL PROCEDURES |
Myoglobin Mutagenesis--
Recombinant sperm whale myoglobins
were constructed, expressed, and purified as described previously by
Carver et al. (25), Springer et al. (26), and
Egeberg et al. (27), using the recombinant gene constructed
by Springer and Silgar (28).
Myoglobin Derivative Preparations--
Myoglobin
(Mb)1 in the ferric (metMb)
and ferryl (ferryl-Mb) forms was prepared according to established
procedures. Briefly, metMb was prepared by the addition of a 1.2-fold
excess of potassium ferricyanide to oxymyoglobin (oxy-Mb). Unreacted
ferricyanide and its reaction products were removed by a two-step gel
filtration procedure on Sephadex G-25. The first filtration step (high
pH, high salt) was done in 50 mM phosphate (pH 8.3), 1 M NaCl. The following step (neutral pH, low salt) was done
in 50 mM phosphate (pH 7.0) with no added NaCl. Ferryl-Mb
was prepared by treating oxy-Mb with a 5-fold molar excess of HOOH in
50 mM phosphate buffer, pH 7.0. Excess peroxide was removed
by passing the myoglobin solution through a Sephadex G-25 column
(29).
Autoxidation Experiments--
The in vitro rates of
autoxidation of native sperm whale myoglobin and the double mutant
(H64Q/L29F) were studied in the absence of antioxidative enzymes (30).
Oxy-Mb samples (20 µM in heme) were incubated in air
equilibrated with 50 mM phosphate buffer (Chelex®-pretreated), pH 7.0, at 37 °C in a thermostated
spectrophotometric cell, in a Hitachi spectrophotometer (U-2000).
Spectral changes in the visible region were followed to near completion
for both proteins. The proportions of oxy-Mb, metMb, and ferryl-Mb at
various time points were estimated by multicomponent analysis using
published extinction coefficients (31). First order autoxidation rate constants were derived from the plot of percentage of oxy-Mb
versus time and fitted to a single exponential expression
using a nonlinear squares curve fitting routine.
Kinetic Measurements: Slow Processes--
The slower kinetic
processes apparent after mixing oxy-Mb (20 µM heme) with
hydrogen peroxide (in a molar ratios of 1:1, 1:2.5, 1:5, and 1:10) were
monitored by mixing substrates in the thermostated cell of a rapid
scanning diode array spectrophotometer (HP-8453) and collecting spectra
as a function of time. All experiments were run at 37 °C, in 50 mM phosphate buffer (pH 7.0) that had been previously
treated with Chelex® resin. Initial multicomponent analysis of
myoglobin oxidation products was performed according to Whitburn (31)
in order to calculate the percentages of oxy-Mb, metMb, and ferryl-Mb
at successive stages of the oxidation. Independent estimation of these
intermediates along the reaction coordinates and estimation of the rate
constants of the interconversions were performed as described below.
Model for Kinetic Processes--
Estimates for the rates of the
slow kinetic processes discussed above were made based on a simple
reaction scheme (Scheme 1), which
reflects hydrogen peroxide oxidation of both oxyferrous and ferric
myoglobins and the autoreduction of the oxyferryl species back to the
ferric state.
k1 describes the initial oxidation of ferrous
iron by hydrogen peroxide. Previous work (32-34) has indicated that
the reactive iron species is probably the unliganded (deoxygenated Mb)
species, and k1 in the scheme above is not a
simple second order rate constant but reflects this competition between
O2 and HOOH for the ferrous iron site. The individual
reactions are described as follows.
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(Eq. 1)
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(Eq. 2)
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The saturation function for hydrogen peroxide binding can be
described as follows.
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(Eq. 3)
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(Eq. 4)
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Here Q is the total concentration of ferrous species
in the initial equilibrium, KHOOH and
KO2 are the binding constants
for the competing species, and P (the binding polynomial) is
Q normalized to the concentration of the unliganded ferrous
species. At constant oxygen activity, the saturation function for HOOH
is as follows.
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(Eq. 5)
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The apparent rate of ferrous iron disappearance
(k1) can now be described as the product of the
hydrogen peroxide saturation function and the intrinsic rate constant
for heterolytic cleavage of bound peroxide with subsequent ferryl
formation as follows.
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(Eq. 6)
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Under conditions of high [HOOH] and low [O2],
-YHOOH in Equation 6 approaches unity, and the
following is true.
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(Eq. 7)
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Note that this limit is valid even when [O2] = 0.
Likewise, under conditions of high [O2] and low [HOOH],
-YHOOH approaches a limiting value of
KHOOH[HOOH]/KO2[O2],
and thus the following is true.
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(Eq. 8)
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The experiments detailed in this work were all carried out at
relatively low [HOOH]/[O2], approximated by Equation 8
above. Thus, mutation-induced changes in k1 can
be affected by 1) changes in the relative affinities of the protein for
the two competing ligands, 2) a change in the first order rate of
ferryl iron formation from the peroxide-liganded species, or 3) a
combination of these effects.
The process defined by k2 in the cyclic
reaction, Scheme 1, involves the autoreduction of the ferryl
iron species back to the ferric state. The mechanism of this process is
not well understood, and the identity of the electron donor is unknown.
k2 serves as a useful indicator of the lifetime
of the ferryl species under the experimental conditions and in the
absence of a known exogenous electron donor.
k3 is the second order rate constant for the
reaction of the ferric heme protein with HOOH producing a ferryl heme
and a protein radical (35, 36). The lifetime of the protein radical has been reported to be very short with respect to the lifetime of the
ferryl heme group (13). Analogous to the interpretation of the reaction
of MbO2 with HOOH, evidence suggests that the oxidation of
ferric myoglobin (k3 in Scheme 1) also consists
of three discrete steps: 1) dissociation of coordinated water, 2) simple HOOH binding, and 3) heterolytic cleavage of the bound HOOH
(33). At millimolar hydrogen peroxide concentrations (high [HOOH]/[O2]), spectral, kinetic, and EPR evidence for
the formation of a ferric myoglobin-HOOH complex can be detected
in vitro for some mutant myoglobins with apolar replacements
of the distal histidine, His-64 (37). The population of this species in
reactions with the native protein is apparently too low for detection
by these methods, particularly at low [HOOH] used in our studies.
In combination, the steps characterized by k2
and k3 form a loop, which we refer to as a
peroxidase cycle. The rate-limiting step for catalysis is determined by
peroxide concentration. Under conditions of relatively high [HOOH],
which were not employed in this work, k3[HOOH]
will be large with respect to k2, and
autoreduction of ferryl myoglobin will be the rate-limiting step. At
low [HOOH], the flux of [HOOH] through the loop will be determined
by k3[HOOH]. Throughout the major portion of
the process, the Fe(IV)=O complex will be the predominant spectral
species until all HOOH is consumed, at which time the oxidation state
of the iron will revert to Fe3+.
When [HOOH] is low, the extent and apparent rate of oxidation of
oxymyoglobin by peroxide cannot be properly estimated without taking in
account consumption of HOOH by the peroxidase loop. First, both
processes compete for hydrogen peroxide and lower its concentration at
different rates. Second, ferric iron formed by the initial oxidation
reaction forms the reactive iron species that is active in the
peroxidase loop. Estimates of k1 made without taking into account the effects of the peroxidase loop on [HOOH] underestimate the true values. In this work, estimates of
k1 were made utilizing a global analysis fit of
spectral data to the kinetic model shown above.
Spectral data were analyzed by singular value decomposition (SVD) and
nonlinear least squares of the numerical model (30, 38) defined by the
following set of rate equations.
|
(Eq. 9)
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(Eq. 10)
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(Eq. 11)
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(Eq. 12)
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Stopped-flow Kinetic Measurements: Fast Processes--
Rate
constants for the oxidation of ferric myoglobin by HOOH
(k3) were estimated by directly mixing ferric Mb
and hydrogen peroxide in an Applied Photophysics stopped-flow
spectrophotometer. Reactions were monitored at either 416 or 400 nm at
25 °C in the presence of 25 mM phosphate buffer (pH
7.0). A minimum of 200 data points per experiment were collected and
analyzed. Peroxide:myoglobin ratios in these experiments were as low as
(1:1) with the maximum value being 10:1. These data were fitted to
either a single or double exponential function using the
Marquardt-Levenberg fitting routine included in the Applied
Photophysics software. In some experiments, data were analyzed by
singular value decomposition and global exponential fitting routines
using the Applied Photophysics photodiode array accessory and its
relevant software package, Glin.
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RESULTS |
Reaction of HOOH with the Oxy Forms of Recombinant Myoglobins
(His-64)--
Spectral changes accompanying the addition of HOOH to
sperm whale oxy-Mb in a molar ratio of 1 heme:2.5 HOOH are shown in Fig. 1 and are similar to those reported
by Shikama and co-workers (32, 33). The initial oxidation of the
ferrous heme can be seen by the loss of the typical
and
bands
of oxy-Mb at 577 and 541 nm and subsequent formation of a transient
ferryl intermediate characterized by weaker visible absorption peaks at
545 and 580 nm. This ferryl transient spectrum changes with time to a
spectral species with peaks at 510, 550, and 630 nm, characteristic of high spin ferric heme. One factor not accounted for explicitly in our
kinetic model is a spectral change caused by the modification of the
heme after the initial reaction of HOOH with ferrous myoglobin (36).
This modification does not significantly change the rate of the ferric
heme/HOOH reaction in subsequent reactions of the cycle (data not
included), i.e. the second order rates of the reaction of
the native and the modified ferric species with hydrogen peroxide are
similar. The "ferric" species in the model corresponds to the sum
of all ferric species including those with irreversible modifications
to the heme.

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Fig. 1.
Spectral changes during the oxidation of
sperm whale myoglobin by HOOH. Myoglobin (20 µM in
heme) was incubated with 50 µM HOOH at 37 °C in 50 mM phosphate buffer, pH 7.0. Spectra were collected every
minute for 1 h in a fast scanning diode-array spectrophotometer.
The oxidation of myoglobin occurs immediately, as evidenced by the loss
of absorption at the 577- and 541-nm bands that are typical of
oxyferrous myoglobin, yielding a final spectrum of ferrimyoglobin with
characteristic absorportion peaks at 510, 550, and 630 nm. The
disappearance of oxymyoglobin occurred through the formation of a
ferryl intermediate as can be seen by the appearance of the spectrum
with peaks at 545 and 580 nm.
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Table I shows the results of the analysis
of the oxidation profiles of sperm whale myoglobin and a number of
distal pocket mutants using low levels of hydrogen peroxide. Spectral
time courses were analyzed by singular value decomposition with
subsequent nonlinear least squares fitting to the model discussed under
"Experimental Procedures." The estimates of
k1 (oxidation of the ferrous to the ferryl
species) and k2 (autoreduction of the ferryl
species back to ferric) shown in Table I were estimated in this manner. The values for k3 (the second order oxidation of
the ferric myoglobins with hydrogen peroxide) given in Table I were
determined independently by stopped-flow analysis of the reaction of
the ferric proteins with hydrogen peroxide. With the exception of the
double mutant L29F/V68F, k3 does not appear to
be sensitive to the configurations of the distal pocket for the mutants
investigated. Only the rate constant for the L29F/V68F double mutant is
significantly different, 3-fold greater than the average estimated for
the other proteins. Curiously, the k3 values for
the L29F and the V68F mutants are both roughly equal to that for
wild-type and native sperm whale myoglobin. Thus, the effects of the
single mutations on k3 are not additive in the
double mutant, presumably because of the close proximity of the two
large benzyl side chains to each other and to the active site.
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Table I
Peroxide oxidation, autoxidation, and oxygen affinity of sperm whale
myoglobin and its site-directed mutants
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The Leu-29 to Phe mutation markedly decreases the initial oxidation
rate of the oxyferrous species (k1) while having
little effect on the rate of autoreduction of the resulting ferryl
species (k2). Insertion of valine at this
position (L29V) results in a modest increase in
k1 and also a 2-fold increase in
k2. Although it appears that
k1 is inversely correlated with the size of the position 29 residue, decreasing the empty volume further back in the
distal pocket by substitution of Val-68 with Phe or Leu has the
opposite effect, causing a modest 30% increase in
k1. It is therefore reasonable to conclude that
both the size and the location of the residue in the heme pocket
determine these effects. In addition, the estimate of
k2 for V68F is significantly larger than that
for the wild type protein. In this case, the double mutant (L29F/V68F)
displays a combination of the kinetic properties exhibited by single
mutants with a slow k1 (like L29F) and an
increased k2 (like V68F) (Table I).
Fig. 2 elaborates on the analysis of wild
type sperm whale myoglobin and three of the mutants included in Table
I. These three mutants include the two phenylalanine replacements (L29F and V68F) and the double mutant (L29F/V68F). The curves shown in the
figure are the model-dependent estimates of the
distributions of the ferrous, ferryl, and ferric species as a function
of time after mixing with a modest 2.5-fold excess of hydrogen
peroxide. Several features illustrate the relationship of the initial
rate of oxidation of the ferrous protein (k1) to
the rate of the peroxidative loop, which is governed primarily by
k2 under conditions where [HOOH] is moderate,
making k3[HOOH] > k2.
Both L29F and L29F/V68F have a significant amount of ferrous heme
remaining after the complete removal of hydrogen peroxide, and both are
characterized by a relatively high ratio of
k2/k1, i.e.
high peroxidative capacity for removing HOOH from solution in relation
to ferrous oxidation (Table I).

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Fig. 2.
Fractional changes of oxy-Mb, metMb, and
ferryl-Mb during the oxidation of sperm whale myoglobin and its mutants
with HOOH (solid, dotted, and dashed
lines, respectively). Normalized time courses
for the reaction of 20 µM (heme) native, L29F, V68F, and
L29F/V68F recombinant sperm whale myoglobins with 50 µM
HOOH at 37 °C in 50 mM phosphate, pH 7.0.
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These properties are further emphasized in Fig.
3, which compares the relative integrated
areas under the ferryl curves as a qualitative comparison of the
"dose" or time persistence of the ferryl species for the four
proteins under the given reaction conditions. Estimates of the amount
of ferryl species generated are in the following order: native sperm
whale > L29F > V68F > L29F/V68F. Under the conditions
in Fig. 3, the reactive ferryl species in the double mutant is cleared
from the solution by both the high rate of autoreduction
(k2) and the high rate of HOOH consumption
(k3) in the peroxidative cycle. The presence of
the V68F mutation appears to be responsible for this property. Also shown in Fig. 3 are the relative proportions of ferrous protein remaining after depletion of hydrogen peroxide from the reaction mixture when the original HOOH:heme ratio was 2.5:1 (50 µM
HOOH, 20 µM heme). It is apparent that, for the four
proteins examined, the presence of the L29F mutation in the protein
significantly preserves the amount of "functional" protein
remaining after the oxidation reaction due to the lower initial rate of
reaction of the ferrous mutant with HOOH. Fig. 3 also emphasizes that
these two functional properties, persistence of the ferryl intermediate and stability of the functional ferrous oxidation state, can be altered
in an independent fashion depending on the configuration of the
residues at positions 29 and 68.

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Fig. 3.
Relationship between oxygen binding capacity
and the levels of the ferryl species during the oxidation of sperm
whale myoglobin and its mutants with HOOH. Black bars,
percentage of ferrous (unoxidized) myoglobin remaining after depletion
of the starting HOOH from the reaction mixtures described above.
Cross-hatch bars, comparison of the "dose" or time
persistence of the ferryl species for the four myoglobins for HOOH
oxidations with an initial HOOH:heme ratio of 50 µM:20
µM. Estimates represent the numerically integrated areas
under the ferryl curves shown in Fig. 2.
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Reaction of Peroxide with Sperm Whale Myoglobin Mutants Containing
a Distal Glutamine (Gln-64)--
Experiments were carried out by
reacting hydrogen peroxide with myoglobin mutants in which the distal
histidine was replaced by either the polar amino acid glutamine or the
apolar amino acid leucine. With respect to the native protein, the H64Q
mutation shows approximately 3-fold increases in both
k1 (initial oxidation of ferrous protein) and
k2 (autoreduction of the ferryl intermediate) with no corresponding increase in k3 (oxidation
of the ferric protein). The increase in k1 and
decrease in oxygen affinity
(KO2), are returned to wild
type levels upon formation of the double mutant H64Q/L29F (Table I). A
high rate of ferryl autoreduction (i.e. large
k2) preserves a more efficient pseudoperoxidase
cycle. Like H64Q, the apolar H64L mutation causes a 3-fold increase in the rate of the oxidation of the ferrous protein by HOOH. Values of
k2 and k3 are not
available from our data. However, Brittain et al. (37) have
reported rate constants for the reactions of HOOH with a series of
apolar 64 mutants of metMb. The values of k3 for
H64V and H64F metMb were 3.0 and 1.5 M
1
s
1, respectively. These rates are significantly slower
than those we have estimated for myoglobins with a polar residue in the
distal position (Table I, k3 = 600 M
1 s
1) and those measured by
Brittain et al. (37) for wild-type and H64Q metMb
(k3 = 200-600 M
1
s
1). Assuming that the H64L mutant also shows a reduced
reaction rate with HOOH, the situation is analogous to the slow rate of HCN binding to ferric apolar 64 mutants. In both cases, it is almost
certainly the anions, CN
or HOO
, that bind,
and as a result the apolar group is needed to facilitate deprotonation,
either directly or through distal pocket water (39).
The distal pocket configuration (H64Q/L29F) corresponds to the in
vivo situation seen in the Asian elephant (40), which is one of
the few, apparently successful, naturally occurring replacements of the
distal histidine in vertebrate myoglobins. It is interesting to note
that the addition of the V68F replacement to H64Q/L29F myoglobin
reverses the effect of the L29F mutation, restoring the value of
k1 to the high value seen with the H64Q single
mutant. However, the relative effects on k1 and
k2 of the addition of the V68F substitution to
L29F background are the same whether the distal position (E7) is
occupied by histidine or glutamine.
Fig. 4 shows reconstructed spectra of the
intermediates in the oxidation of myoglobins that have either histidine
or glutamine at the distal position (E7). These representations are the
averaged spectra of the individual proteins shown in Table I, grouped to show the general differences in the E7 configuration. The ferrous oxymyoglobin (initial spectra) are similar, with some possible differences in the relative heights of the
and
bands. The spectra of the intermediate species (ferryl in the model) differ substantially between His-64 and Gln-64 myoglobins. In the case of
His-64, this spectrum shows the profile characteristic of the ferryl
iron for native myoglobin (31), whereas the Gln-64 series of mutants
have an intermediate spectrum with small absorbance bands in the region
of the ferrous
and
bands and a flattened band at 630 nm. The
final spectrum (ferric) for the His-64 series of mutants has the basic
character of high spin hemin. As pointed out earlier, superimposed on
this spectrum is the evidence of heme modification during the oxidation
process (36). The final reconstructed spectrum in the case of the
Gln-64 series of proteins is generally flattened and without detail in
the visible region. This final model-dependent spectrum
looks very much like the actual spectra of the oxidation products of
the Gln-64 series of mutants. It is likely that significant heme loss
and breakdown and globin precipitation take place for these mutants
even when oxidized with the moderate ratio of peroxide:heme utilized in
these experiments. This is consistent with the normal tendency of the
Gln-64 proteins to lose heme at an accelerated rate and precipitate as
apoglobin is formed (41).

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Fig. 4.
Model-dependent reconstructed
spectra of the intermediates in the oxidation of myoglobins that have
either histidine (His-64) or glutamine (Gln-64) at the distal position
(E7). These representations are the averaged spectra of the
individual proteins shown in Table I, grouped to show the general
spectral differences between myoglobins with the two E7
configurations.
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To estimate the degree of oxidation-stimulated heme loss from these
proteins, aliquots of the reaction mixtures of myoglobin and its
oxidation products were analyzed by the use of reverse-phase HPLC (42).
Protein and heme components were detected by their absorbance at 220 and 400 nm, respectively. Fig.
5a shows typical elution
profiles of both the heme and protein fractions of native sperm whale
myoglobin untreated with hydrogen peroxide. Fig. 5b shows
the averaged amount of heme lost, as calculated from the peak areas at
400 nm, from both His-64 and Gln-64 series before and after treatment
with a 2.5-fold molar excess of hydrogen peroxide. There was a dramatic
drop in heme retention (~80% of the control) by the Gln-64 mutants
compared with a modest decrease (~9%) in case of the His-64
mutants.

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Fig. 5.
Reversed-phase HPLC profiles of untreated and
HOOH-treated sperm whale and its His-64 and Gln-64 mutant myoglobins.
a, HPLC profile of untreated sperm whale myoglobin. The
major fraction with absorption at 220 nm corresponds to the protein
(dotted line), whereas the fraction with an absorption of
400 nm corresponds to the heme (solid line). Samples (50 µl) were taken after 1 h from the reaction mixture described in
the legend to Fig. 1 and were analyzed by HPLC on a Vydac C4 reverse
phase chromatography column, as in Ref. 42. b, relative
amounts of heme before and after treatment of His-64 and Gln-64 mutants
with a 2.5-fold molar excess of peroxide. The peak areas at 400 nm were
calculated from HPLC profiles such as those in a. Values are
mean ± S.D. (n = 3) for both His-64 and for the
Gln-64 series. It appears that ~20% of the heme was lost when His-64
mutants reacted with HOOH as compared with ~90% heme loss in the
case of Gln-64 series
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Literature values of the autoxidation rate constants (17, 18) are
plotted versus the initial HOOH oxidation in Fig.
6 for the mutants examined in this study.
It appears that kauto is roughly correlated with
k1 for 9 out of the 10 myoglobins reported in
Table I. There is however, a fair amount of scatter in this correlation
(r = 0.54). We carried out some autoxidation
experiments under more relevant conditions, i.e. under air
at 37 °C, pH 7.4, with no added antioxidant enzymes for two
representative proteins, native and H64Q/L29F sperm whale myoglobins.
The initial rate of autoxidation for sperm whale myoglobin and for
H64Q/L29F were comparable under our conditions (0.109 and 0.106 h
1 for native myoglobin and the double mutant,
respectively).

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Fig. 6.
Plot of the autoxidation rate constant
(kauto) of ferrous Mb, taken from Refs. 17 and
18, versus rate constants of hydrogen peroxide reaction
with ferrous Mb (k1) for nine native and mutant
sperm whale myoglobins at 37 °C, in 50 mM phosphate, pH
7.0. The autoxidation rate constants were measured in air at
37 °C and in the presence of catalase and superoxide
dismutase.
|
|
Long term stability studies in the absence of antioxidant enzymes
showed that the double mutant (H64Q/L29F) is a more stable protein. At
the end of 46 h of incubation, there was 65% metmyoglobin present
in the H64Q/L29F solutions as compared with almost 97% in the native
protein. The reason for this increased stability is the ~4-fold
greater rate of autoreduction of the H64Q/L29F ferryl intermediate
(k2), which enhances the removal of HOOH by the
peroxidative cycle. Although the H64Q/L29F double mutant has a similar
initial rate of autoxidation, the mutant metmyoglobin formed is a
4-fold better catalyst for removing the resultant HOOH, which
ordinarily would accelerate the oxidation process as described
previously (43). Thus, the H64Q/L29F combination produces a myoglobin
that is significantly more resistant to HOOH damage without altering
significantly its oxygen binding properties and initial rate of autoreduction.
 |
DISCUSSION |
Mechanistic analysis of the reaction of hemoglobins/myoglobins
with HOOH has revealed the formation of a higher oxidation state, the
ferryl heme iron (Fe4+), which can be detected by optical
spectroscopy (30, 32, 34), and a globin-associated free radical, which
can only be detected by EPR spectroscopy (14, 37). Despite its
transient nature and ultimate self-destruction, ferryl hemoglobin and
ferryl myoglobin can peroxidize lipids, degrade carbohydrates, and
cross-link proteins (44). The globin radical of ferryl hemoglobin was
recently detected by EPR in normal human and animal whole blood (13, 14). The source of HOOH in blood was reported to be the product of
dismutation of O
2 produced via the autoxidation of the
intraerythrocytic hemoglobin (14). This reaction, interestingly, occurs
despite the presence of normal blood reducing systems (14). A recent study on the reactivity of hemoglobin toward low density lipoproteins has shown that both the oxidation and the cross-linking of the LDL
proteins can be initiated by the heme-globin radical (45). One concern,
relevant to the use of hemoglobin as an oxygen therapeutic, is that
in vivo production of both globin-based and heme-based radicals can occur under ischemia and reperfusion in patients with
diminished ability to control oxidative reactions of hemoglobin.
Although, no in vivo evidence exists to attribute the
cytotoxicity seen with cell-free hemoglobins directly to the ferryl species, several protective strategies are under consideration to
control or reduce the levels of these highly reactive intermediates. Several chemical strategies are aimed at cycling of ferryl back to
ferric hemes, by attachment of catalase-like activity to hemoproteins using redox-active compounds such as nitroxide (46) or directly cross-linking the red cell antioxidative enzymes superoxide dismutase and catalase to the hemoglobin molecules (47). Other approaches include
site-directed mutagenesis that targets distal heme pocket amino acids
to sterically limit access of oxidants to the heme iron while
maintaining the normal oxygen delivering capabilities of the protein
(17, 20).
In recent years, sperm whale myoglobin has been successfully used as a
subunit protein prototype to engineer safer, second generation
hemoglobin-based blood substitutes (17-20). One approach for
constructing a low oxygen affinity blood substitute prototype is to
weaken the hydrogen bonding between bound O2 and the E7 residue in myoglobin (17-20). Replacement of His-64(E7) with Gln causes a 5-fold reduction in oxygen affinity. Apolar substitutions (i.e. H64F) produce dramatic, 100-1000-fold, reductions in
affinity (18). However, these substitutions at E7 are not useful,
because they dramatically increase the rate of autoxidation, making the protein too unstable for clinical use. On other hand, the Leu-B10
Phe mutation decreases the rate of autoxidation 10-fold, but increases
oxygen affinity by 15-fold (17, 18). The large benzyl side chain
inhibits autoxidation reactions by both stabilizing bound
O2 and by filling the space adjacent to the bound oxygen, preventing protonation by solvent water. The Phe-B10 substitution is
also effective in selectively inhibiting NO-induced oxidation of
oxymyoglobin (19). When Leu is replaced with the even larger Trp
residue (L29W) at the B10-position, steric hindrance becomes the
dominant factor, and both NO binding and NO-induced oxidation of
myoglobin are markedly decreased (19). This approach has been explored
further by examining the effects of large B10, E11, and G8
substitutions on the rates of NO-induced oxidation of the
- and
-subunits in recombinant human hemoglobin (20). A series of
recombinant hemoglobins have been made with varying oxygen binding
kinetics and reactivities with NO with apparent success in some
in vivo models (20). This approach has provided protein engineering strategies for designing hemoglobin-based oxygen-carrying therapeutics that exhibit increased resistance to autoxidation and
decreased reactivity with endothelial derived NO.
In this study, attention was focused on the reactivity of some of these
recombinant sperm whale myoglobin prototypes with HOOH. These
reactions, even at stoichiometric levels, occur much more quickly than
simple autoxidation and represent potentially significant pathways for
hemoglobin degradation and oxidative stress of the vasculature (2, 3).
For the B10 series of single mutants, increasing the residue size
resulted in a progressive decrease in the initial rate of HOOH
oxidation, whereas increasing the size of the E11 residue caused only a
small increase (30%) in k1. The V68F
replacement also caused a significant, ~4-fold, increase in
autoreduction (k2), whereas the L29F mutation
only caused a 40% increase in the rate constant. The two effects,
k1 decrease by L29F and
k2 increase by V68F, appear to operate
independently, so the double mutant has both changes. The decrease seen
in the initial HOOH oxidation rate for both the single and double
mutant is probably a result of the significant increase oxygen affinity caused by L29F replacement.
Evidence has been presented (32) to suggest that the deoxyferrous
species is the form that is oxidized by HOOH, so oxygen affinity is a
major determinant of this rate. At low [HOOH]/[O2], k1 should be universally proportional to oxygen
affinity, as shown in Equation 8. However, the relative magnitude of
this depends on how the substitution affects HOOH binding. For example,
the data show that the L29F mutation, which has 15-fold higher oxygen affinity than the wild type myoglobin, induces only a 3-fold drop in
k1, the apparent rate constant for the oxidation
of the ferrous species. Our analysis (Equation 8) suggests that this
apparent discrepancy can be rationalized by a compensatory increase in KHOOH (the equilibrium constant for hydrogen
peroxide binding to deoxy-Mb) and kferryl (the
rate of heterolytic cleavage of bound HOOH with subsequent ferryl iron
formation). Previously, Carver et al. (25) showed that this
dramatic increase in oxygen affinity of the L29F mutant is due to a
stabilization of the bound oxygen by an interaction with the partially
positive edge of the phenyl ring. Presumably, a similar phenomenon can
occur with bound peroxide anion.
As can be seen from the spectral intermediates derived from the His-64
series in Fig. 4, no single or double mutation at either the
B10- or E11-position shows a large reduction in initial rate of HOOH
oxidation and ferryl heme formation. Mutants in which His-64 was
replaced with Gln singularly or in combination with Phe-B10 show three
common phenomena: 1) a spectral intermediate with less resemblance to
that of traditional ferryl intermediate, 2) significantly enhanced
pseudoperoxidase activity, and 3) enhanced hemin loss or breakdown.
These results agree with the observation made recently by Brittain
et al. (37) that a nonoxyferryl, a low spin ferric heme
intermediate, is generated when ferric H64Q myoglobin is reacted with
millimolar levels of HOOH. Their studies also suggest that His-64 plays
a key role in the production and stabilization of the ferryl iron (37).
Replacement of His-64, by large apolar amino acids prevents
peroxyradical-mediated epoxidation by slowing the rate of ferryl
formation when the mutant sperm whale myoglobin is reacted with
peroxide (48). On the other hand, replacements by smaller residues
(i.e. alanine (H64A) and serine (H64S)) have destabilizing
effects, making the ferryl oxygen more accessible to substrates (49).
Hydrogen bonding by His-64 not only controls ligand binding and ferryl
formation but also restricts the ability of the heme group to undergo
facile electron transfer (50).
Detailed functional studies and high resolution crystal structure data
on distal histidine mutants of sperm whale myoglobin have suggested
that the H64Q mutation is fairly conservative with respect to the size
and polarity of residue 64 (51). This conclusion explains the presence
of Gln-E7 in a few naturally occurring hemoglobins (e.g.
opossum (52), Ascaris (53), and Lucina pectinata
(54) and myoglobins from elephant (40) and shark (55)). Our data clearly demonstrate that combining H64Q with L29F produces a double mutant that is much more resistant toward oxidation than native sperm
whale myoglobin. This H64Q/L29F mutation maintains a normal or slightly
lower oxygen affinity, large association and dissociation rate
constants for O2 uptake and release, and normal resistance to autoxidation in the presence of catalase and superoxide dismutase (56). The enhanced resistance to oxidation in the absence of these
antioxidant enzymes is due to the larger rate of autoreduction of the
ferryl H64Q/L29F intermediate. As a result, the ferric form of the
double mutant is a much better pseudoperoxidase, facilitating the rapid
removal of HOOH before it can react with the remaining reduced
H64Q/L29F oxymyoglobin. Thus, the double substitution produces a
protein with a more effective pseudoperoxidase activity. This
protection against further HOOH oxidation may account for the combined
H64Q/L29F replacement in the elephant myoglobin.
The enhanced enzymatic-like ability of H64Q/L29F to consume HOOH and to
autoreduce the ferryl intermediate back to a less toxic ferric heme is
clearly a desirable property in an oxygen-carrying agent. In
endothelial cell cultures, chemically modified hemoglobin-blood substitutes were less effective in removing HOOH added to the medium
than unmodified hemoglobin. This suppressed pseudoperoxidase activity,
due possibly to chemical modifications, correlated with the formation
of a long lived ferryl hemoglobin that was able to induce apoptotic
cell death (57). We believe that the H64Q/L29F mutation or combinations
similar to it can be developed further as useful blood substitute
prototypes. One problem, however, remains unresolved, and that is how
to overcome the enhanced hemin dissociation and degradation inherent
with replacement of His-64 with Gln, either singularly or in
combination with aromatic residues of the B10 and E11 positions. It is
interesting to note that in the case of opossum hemoglobin (Gln-E7 in
-subunits), the enhanced susceptibility of its hemoglobin toward
oxidative damage by peroxide (58) is counterbalanced by the presence of
a more effective NADH/NADPH-dependent methemoglobin
reductase system than that found within human red cells (59).
In summary, we have shown that replacing Leu-B10 with Phe in myoglobin
generally promotes resistance to HOOH-induced oxidation. The large
benzyl side chain in L29F mutant also serves to protect the
FeO2 complex from direct reactions with NO and from
autoxidation (18, 19). Combining this substitution with a Gln
substitution at the E7-position maintains the size and polarity of
residue 64 and increases the rate of ferryl reduction but has little
affect on O2 affinity. The net result is a blood substitute
prototype with enhanced pseudoperoxidase activity. Consequently, a
small amount of oxidation of H64Q/L29F myoglobin produces a ferric
protein that rapidly removes HOOH, preventing any further oxidation of remaining ferrous myoglobin.