From the Biological Institute, Graduate School of Science, Tohoku
University, Sendai 980-8578, Japan
Human oxyhemoglobin showed a biphasic
autoxidation curve containing two rate constants, i.e.
kf for the fast autoxidation due to the
chains, and
ks for the slow autoxidation of the
chains,
respectively. Consequently, the autoxidation of the HbO2
tetramer produces two different curves from the pH dependence of
kf and ks. The analysis of
these curves revealed that the
chain of the HbO2
tetramer does not exhibit any proton-catalyzed autoxidation, unlike the
chain, where a proton-catalyzed process involving the distal
histidine residue can play a dominant role in the autoxidation rate.
When the
and
chains were separated from the HbO2
tetramer, however, each chain was oxidized much more rapidly than in
the tetrameric parent. Moreover, the separated
chain was recovered
completely to strong acid catalysis in its autoxidation rate. These new
findings lead us to conclude that the formation of the
1
1 contact produces in the
chain a
conformational constraint whereby the distal histidine at position 63 is tilted away slightly from the bound dioxygen, preventing the
proton-catalyzed displacement of O
2 by a solvent water
molecule. The
chains have thus acquired a delayed autoxidation in
the HbO2 tetramer.
 |
INTRODUCTION |
The reversible and stable binding of molecular oxygen to the heme
iron(II) is the basis of hemoglobin function. Consequently, much
attention has been directed to the elucidation of the molecular mechanism of cooperative oxygen binding to the hemoglobin tetramer (1).
However, the oxygenated form of hemoglobin, as well as of myoglobin, is
known to be oxidized easily to the ferric(III) met form, which cannot
bind molecular oxygen and is therefore physiologically inactive, with
generation of the superoxide anion (2-5).
To this autoxidation reaction, it has been widely accepted that
hemoglobin is much more resistant as compared with myoglobin. Unlike
myoglobin, Mansouri and Winterhalter (6) reported that oxyhemoglobin
(HbO2) showed a biphasic autoxidation reaction with a fast
and a slow component. They also demonstrated that the
chain was
oxidized more rapidly than the
chain in hemoglobin tetramer. At the
same time, however, there have been a number of reports that such a
rate difference was not observed between the
and
chains in the
autoxidation reaction of HbA (7-9). Rather, Zhang et al.
(9) showed that the rate of autoxidation was markedly enhanced when the
HbO2 tetramer dissociates into 
dimers. To clarify
these discrepancies, we have recently examined systematically the
effect of hemoglobin concentration on the autoxidation rate at several
different values of pH, and found that human HbO2 exhibits
a biphasic autoxidation curve only in the pH range from neutral to
acidic (5). By dissociation of tetramers into 
dimers, the rate
of autoxidation for the fast component (due to the
chain) was also
found to increase markedly at the acidic pH, but the addition of
2,3-diphosphoglyceric acid offered no significant effect on the
increment of the autoxidation rate (5).
In the present paper, we have made, for the first time, a full
comparison between tetrameric HbO2 and the separated
and
chains in terms of a detailed pH dependence for the
autoxidation rate over the wide range of pH 5-11. On the basis of
these kinetic results, our present goal is to solve the most important
questions: whether each of the separated
and
chains has its own
different stability, and, if not, what the origin is of such a
nonequivalence of the chains in the autoxidation reaction. This
information is essential, not only for a full understanding of the
molecular nature of hemoglobin autoxidation, but also for planning new
molecular designs for synthetic oxygen carriers that are highly
resistant against the heme oxidation in protic, aqueous solution under
physiological conditions.
 |
MATERIALS AND METHODS |
Chemicals--
Sephadex G-25 was a product of Amersham Pharmacia
Biotech. CM-cellulose (CM-32) and DEAE-cellulose (DE-32) were purchased from Whatman. Sodium p-hydroxymercuribenzoate
(p-MB)1 was an
Aldrich product. Mes, Pipes, Mops, Hepes, Tris, Taps, and Caps for
buffer systems, 2-mercaptoethanol and all other chemicals were of
reagent grade from Wako Pure Chemicals, Osaka, Japan. Solutions were
made with deionized and glass-distilled water.
Oxyhemoglobin Preparation--
Human hemoglobin A was prepared
from freshly drawn blood (of 30 ml each time) by the method of Williams
and Tsay (10). After the addition of 4 ml of CPD solution (CPD: 90 mM sodium citrate, 15 mM citric acid, 15 mM sodium biphosphate, and 130 mM glucose in
1% NaCl solution), the blood was centrifuged at 5000 rpm for 10 min.
The packed red cells (~5 ml) were then washed three times with five
volumes of 1% NaCl solution; 2 ml of the cells, the other portion
being kept at
80 °C until further use, were hemolyzed with four
volumes of cold distilled water. Stroma were removed by centrifugation
at 18,000 rpm for 30 min. For the most part, all procedures were
carried out at low temperature (4 °C). The hemolysate was then
fractionated with ammonium sulfate between 20% and 80% saturation at
pH 7.0. The hemoglobin precipitate was centrifuged down at 18,000 rpm
for 30 min and dissolved in a minimum volume of 5 mM Hepes
buffer (pH 7.9). The solution was then dialyzed against the same buffer
and applied on a DEAE-cellulose column (2.5 × 9 cm) equilibrated
with 5 mM Hepes buffer, pH 7.9. After passing a minor
component (HbA2) through the column with 10 mM Hepes, the major band of HbA was eluted out completely with 20 mM Hepes at the same pH of 7.9. The HbO2
solution (approximately 25 ml) thus obtained was condensed by
centrifugation in a Centriprep-10 tube (Amicon), and kept at low
temperature (4 °C) until use. The concentration of hemoglobin (in
heme) was determined, after conversion into cyanomet form, using the
absorption coefficient of 10.4 mM
1
cm
1 at 540 nm. This value was obtained on the basis of
the pyridine hemochromogen method (11).
Isolation of Mercuribenzoated
and
Chains--
All
separations were carried out with HbO2 at low temperature
(0-4 °C) by a two-column method. The procedure was essentially the
same as described by Geraci et al. (12) and by Turci and McDonald (13). p-MB (100 mg) was dissolved in 2 ml of 0.1 M NaOH and neutralized with 1 M
CH3COOH. This was reacted with 10 ml of HbO2
solution (4-5 mM in heme) in 30 mM phosphate
buffer, pH 6.0, and in the presence of 0.1 M NaCl. The
solution was left overnight, and the flocculent precipitate was removed
by centrifugation. To obtain
p-MB chains, the
mercurated HbO2 solution was adjusted to pH 8.2 by
filtration through a Sephadex G-25 column (2.5 × 40 cm)
equilibrated with 15 mM Hepes buffer (pH 8.2). The resultant solution was then passed through a DEAE-cellulose column (3 × 12 cm) equilibrated with the same buffer. Under these
chromatographic conditions, the
p-MB chains were
readily eluted out, whereas the
chains and unsplit HbO2
were retained on the top of the column. To obtain
p-MB chains, on the other hand, the mercurated
HbO2 solution was adjusted to pH 6.7 by filtration through
Sephadex G-25 in 10 mM phosphate buffer, pH 6.7. The
resultant solution was then applied to a CM-cellulose column (3 × 12 cm) equilibrated with the same buffer. The unreacted
HbO2 and the mercurated
chains were retained on the top
of the column at this pH, and only the
p-MB
chains were eluted out completely.
Removal of Mercuribenzoate from
and
Chains--
p-MB was removed from the
chains by
incubating with 15 mM 2-mercaptoethanol in 10 mM Pipes buffer (pH 6.5) for 10 min at 0 °C. The mixture
was then applied to a CM-cellulose column (2.5 × 6 cm), which had
been equilibrated with 10 mM Pipes buffer, pH 6.5. The
column was washed first with the same buffer containing 15 mM mercaptoethanol for 30 min at a flow rate of 70 ml/h.
The mercaptoethanol was then removed by washing with the buffer alone for another 1 h, and the regenerated
chains were finally
eluted in the oxy form with 30 mM Hepes buffer at pH 8.2. For
p-MB, the chains were incubated with 20 mM 2-mercaptoethanol in 5 mM phosphate buffer
(pH 8.8) for 10 min at 0 °C. The solution was then placed on a
DEAE-cellulose column (2.5 × 6 cm) equilibrated with 5 mM phosphate buffer, pH 8.8. The column was washed with the
same buffer containing 20 mM mercaptoethanol over a period of 3 h at a flow rate of 100 ml/h. The mercaptoethanol was then removed by washing with 10 mM Hepes buffer (pH 8.2) for
another 3 h, and the regenerated
chains were finally obtained
by changing the buffer concentration to 50 mM at the same
pH. The
and
chains thus separated were kept stably in the oxy
form in liquid nitrogen until use. The concentration of each chain was
determined on the basis of the pyridine hemochromogen method (11).
Determination of SH Groups--
According to the method of Boyer
(14), sulfhydryl groups were titrated spectrophotometrically at 250 nm
with p-hydroxymercuribenzoate at pH 7.0. In the separation
procedure employed above, the regenerated
and
chains gave
uniformly 1.0 (1.05 ± 0.08) and 2.0 (2.01 ± 0.08) free SH
groups, respectively (15).
Autoxidation Rate Measurements--
The autoxidation rate of HbA
was measured in 0.1 M buffer over a wide range of pH at
35 °C according to our standard procedure. A 0.5-ml solution
containing 0.2 M appropriate buffer was placed in a small
tube and incubated in a water bath maintained at 35 (± 0.1) °C. The
reaction was started by adding the same volume of fresh
HbO2 solution (180-300 µM). For
spectrophotometry, the reaction mixture was then quickly transferred to
a quartz cell (of 1-mm path length) held at 35 (± 0.1) °C. The cell
was sealed with a piece of Sealon film (Fuji) to prevent evaporation,
and changes in the absorption spectrum from 450 to 700 nm were recorded on the same chart at measured intervals of time. For the final state of
each run, the hemoglobin was completely converted to its ferric met
form by the addition of potassium ferricyanide. For separated
and
chains, the rate measurements were carried out in a 1-cm cell with
10 µM solution (in heme), and in the presence of 20%
(v/v) glycerol. The addition of such a protein stabilizer was not
essential, but was effective in preventing precipitation during the
course of the autoxidation reaction over a long period of time at
35 °C.
The buffers used were Mes, maleate, Pipes, Mops, phosphate, Hepes,
Tris, Taps, bicarbonate, and Caps. The pH of the reaction mixture was
carefully checked, before and after the run, with a Hitachi-Horiba pH
meter (model F-13).
Spectrophotometric Measurements--
Absorption spectra were
recorded in a Hitachi two-wavelength double-beam spectrophotometer
(model 557 or U-3210) equipped with a thermostatically controlled cell
holder. Temperature was controlled by a water bath (Advantec,
thermocool LCH-190) maintained to within ±0.1 °C.
EPR Measurements--
Electron paramagnetic resonance (EPR)
spectra were recorded in a Varian EPR spectrometer (model 112)
operating at 9.0-9.2 GHz. Experiments were carried out with ferric
protein solution (~600 µM in heme) in 10 mM
maleate buffer (pH 6.2), and in the presence of 50% (v/v) glycerol
over a magnetic field of 0-500 mT at 8.0 K. An Oxford flow cryostat
(ESR-900) was used for liquid helium temperature measurements.
Curve Fittings--
Biphasic curves for the autoxidation
reaction were analyzed as described previously (5). The curve fittings
for a plot of log(kobs) versus pH
were made by an iterative least-squares method on a personal computer
(NEC PC-9821 V12) with graphic display, according to our previous
specifications (16, 17).
 |
RESULTS |
Biphasic Nature in the Autoxidation Reaction of
HbO2--
In air-saturated buffers, the oxygenated form of
HbA is oxidized easily to its ferric met form (metHb) with generation
of the superoxide anion (2, 18) as shown by Reaction 1.
In this reaction, kobs represents the
first-order rate constant observed at a given pH in terms of each
subunit. Fig. 1 shows such an example for
the spectral changes with time when fresh HbO2 (10 µM) was oxidized in 0.1 M Mes buffer (pH 6.5)
at 35 °C and in the presence of 1 mM EDTA. The spectra
evolved to the final state, which was identified as acidic (or aquo)
metHb, with a set of isosbestic points at 524 and 591 nm. This process
of autoxidation was therefore followed by a plot of experimental data
as
ln([HbO2]t/[HbO2]0) versus time t, where the ratio of
HbO2 concentration after time t to that at time
t = 0 can be monitored by the absorbance changes at 576 nm (the
-peak of human HbO2).

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Fig. 1.
Spectral changes with time for the
autoxidation reaction of human HbO2 in 0.1 M
Mes buffer at pH 6.5 and 35 °C. Scans were made at 270-min
intervals after 0.5 ml of the fresh HbO2 solution (20 µM) was added to the same volume of 0.2 M Mes
buffer in the presence of 1 mM EDTA. The final spectrum was
that of the acidic met form.
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Fig. 2 shows first-order plots for the
autoxidation reaction of HbO2 tetramer at two different
values of pH. At pH 6.5, HbA showed a biphasic autoxidation curve that
can be described completely by first-order kinetics containing two rate
constants as follows.
|
(Eq. 1)
|
In this equation, kf and ks
represent first-order rate constants for the fast and slow autoxidation
processes, respectively, and P is the molar fraction of the
rapidly reacting hemes. By iterative least-squares procedures inserting
various values for kf and ks, the
best fit to the experimental data was obtained as a function of time.
In these computations, as described previously (5), the initial value
for each of the rate constants was taken from the corresponding slope
of a biphasic curve, and was refined by the step sizes of 0.01 h
1 to 0.001 h
1 to find the best values of
kf and ks. The value of
P was also allowed to vary a large range (from 0.40 to 0.60) in all cases. In this way, the following parameters in Equation 1 were
established: kf = 0.078 (± 0.007) h
1,
ks = 0.011 (± 0.001) h
1, and
P = 0.48 (± 0.04) in 0.1 M Mes buffer at
pH 6.5 and 35 °C. In the autoxidation reaction at pH 8.0, however,
the process could be described completely by a single first-order rate
constant of 0.008 (± 0.001) h
1 (i.e.
kf
ks, P = 0.50),
and the final state of the run was identified as hydroxide-metHb.

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Fig. 2.
First-order plots for the autoxidation of
human HbO2 in 0.1 M buffer at 35 °C. At
pH 6.5, HbA showed a biphasic autoxidation curve that can be described
by a first-order kinetics containing two rate constants,
kf for the initial fast oxidation and
ks for the second slow oxidation, respectively. At
pH 8.0, however, the autoxidation of HbO2 was monophasic.
The rate measurements were carried out in the presence of 1 mM EDTA with 10 µM HbO2 in 0.1 M Mes buffer at pH 6.5, and with 15 µM
HbO2 in 0.1 M Mops buffer at pH 8.0.
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|
As for the biphasic nature of HbA, we have confirmed that the
chain
is oxidized more rapidly than the
chain, for instance, by a factor
of not less than 3 at pH 6.0 and 35 °C. This examination was made by
the rapid chain separation of partially (30%) oxidized HbO2 on a 7.5% polyacrylamide gel (19). Our result is in
good accord with that of Mansouri and Winterhalter (6). Therefore, we
can conclude unequivocally that the rate constant of
kf is due to the autoxidation of the
chain,
while the value of ks is for the
chain of the
HbO2 tetramer. When HbO2 is placed in dilution,
on the other hand, the tetrameric species is known to dissociate into

dimers (20). To keep the tetramer concentration as high as
possible, the following experiments were therefore carried out with
90-150 µM HbO2 using a quartz cell of 1-mm
path length. In such concentrated solutions, the equilibrium fraction
of tetrameric species was estimated to be more than 90% (19). Under
these conditions, a pair of the observed first-order rate constants
involved in Equation 1 was determined by a least-squares fitting to
each of the reaction curves obtained at more than 75 different values
of pH.
If the values of kf and ks are
plotted against the pH of the solution, we can obtain a pH profile for
the stability of HbO2. Fig. 3
shows such a profile for both of the
and
chains in the
HbO2 tetramer over the wide range of pH 5-11, under
air-saturated conditions in 0.1 M buffer at 35 °C. This
graph clearly demonstrates the biphasic nature emerged in the
autoxidation reaction of HbO2. In the acidic range of pH 7 to 5, the logarithmic values of ks increased rapidly with increasing hydrogen ion concentration, but much less so than for
kf. The latter has a value of n =
1 for the slope against the pH, while the former shows a value close
to n =
0.6. In the range higher than pH 8.0, on the
other hand, practically no difference was found between the values of
kf and ks, indicative of the
reaction being monophasic. Nevertheless, it is also true that both
graphs depend strongly upon the pH of the solution, having a parabolic
part with a minimum rate appearing at pH 8.5.

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Fig. 3.
Differential pH dependences of
kf and ks for the
autoxidation reaction of human HbO2 in 0.1 M
buffer at 35 °C. The rate measurements were carried out with
fresh HbO2 solution (90-150 µM) in the
presence of 1 mM EDTA. A pair of the observed first-order
rate constants, kf ( ) and ks
( ), was obtained by a least-squares fitting to each of the reaction
curves at different values of pH. In the acidic range of pH 7 to 5, the
logarithmic values of ks increased rapidly with
increasing hydrogen ion concentration, but much less so than for
kf, the latter having a value close to
n = 1 for the slope against the pH of the
solution.
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|
A Kinetic Formulation for the Autoxidation Reaction of the
HbO2 Tetramer--
In the autoxidation reaction, pH can
affect the rate in many different ways. Recent kinetic and
thermodynamic studies of the stability of mammalian oxymyoglobins have
shown that the autoxidation reaction is not a simple, dissociative loss
of O
2 from MbO2, but is due to a nucleophilic
displacement of O
2 from MbO2 by a water molecule
or a hydroxyl ion that can enter the heme pocket from the surrounding
solvent (3, 21-23). The iron is thus converted to the ferric met form,
and the water molecule or the hydroxyl ion remains bound to the Fe(III)
at the sixth coordinate position to form aqua- or hydroxide-metMb,
respectively. Even the complicated pH profile for the autoxidation rate
can thereby be explained primarily in terms of the following three
types of displacement process (3, 17, 21, 24).
In these reactions, k0 is the rate constant
for the spontaneous displacement by H2O,
kH is the rate constant for the proton-catalyzed displacement by H2O, and kOH is the
rate constant for the displacement by OH
. The extent of
contribution of these elementary processes to the observed autoxidation
rate, kobs in Reaction 1, can vary with the
concentrations of H+ or OH
ion. Consequently,
the stability of MbO2 shows a very strong pH dependence
having a parabolic shape. The reductive displacement of the bound
dioxygen as O
2 by H2O can proceed without any
protonation, but it has been clearly shown that the rate is enormously
accelerated with the proton assistance by a factor of more than
106/mol, as formulated by Reaction 3. In this proton
catalysis, the distal histidine, which forms a hydrogen bond to the
bound dioxygen (25), appears to facilitate the effective movement of a
catalytic proton from the solvent to the bound dioxygen via its
imidazole ring by a proton-relay mechanism (3). In fact, such a
proton-catalyzed process can never be observed in the autoxidation
reaction of myoglobins lacking the distal histidine residue, such as
Aplysia Mb where the His(E7) is replaced by Val (17,
24).
On the basis of this view, we have investigated two different types of
pH dependences emerged from the autoxidation of the
and
chains
in the HbO2 tetramer in 0.1 M buffer at
35 °C. To know definitely the kinetic and thermodynamic parameters
contributing to each kobs versus pH
profile, we have proposed some mechanistic models for each case. The
rate equations derived therefrom were tested for their fit to the
experimental data with the aid of a computer.
For the
chain, its pH dependence curve has thus been described in
terms of an "acid-catalyzed two-state model". In this model, it is
assumed that a single, dissociable group, AH with pK1, is involved in the reaction. Consequently,
there are two forms of the oxygenated
chain, represented by A and
B, at molar fractions of
and
, respectively, which are in
equilibrium with each other but which differ in dissociation state for
the group AH. These forms can be oxidized to ferric met form by
displacement of O
2 from the FeO2 center by an
entering water molecule or hydroxyl ion. Using the rate constants
defined above, therefore, the reaction scheme may be written as shown
below.
For the mechanism delineated in Scheme 1, the observed rate
constant, kobsf (
kf) in h
1, for the autoxidation of the
chain can be reduced to the following equation,
|
(Eq. 2)
|
where
|
(Eq. 3)
|
and
|
(Eq. 4)
|
By iterative least-squares procedures inserting various values for
K1, the adjustable parameter in Equations 3 and
4, the best fit to more than 75 values of
kf was obtained over the whole range of pH 5-11, as
demonstrated in Fig. 4. As a reference, Fig.
5 represents the sum of the squared
residuals as a function of pK1 values inserted
in this computation. In this way, the rate constants and the acid
dissociation constant involved in the autoxidation reaction of the
chain were established in 0.1 M buffer at 35 °C, and are
summarized in Table I.

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Fig. 4.
A plot of log(kf)
versus pH for the autoxidation of the chain of
HbO2 tetramer in 0.1 M buffer at 35 °C.
The computed curve (------) was obtained by a least-squares fitting to
the experimental data ( ) over the whole range of pH 5-11, based on
Equation 2, derived from an acid-catalyzed two-state model (see
"Results"). In this procedure, three kinetic parameters for state B
were first established so as to best cover a parabolic part of the pH
profile, since its component (- - -) was manifested by the term
kB = k0B[H2O] + kHB[H2O][H+] + kOHB[OH ] in the
alkaline range, where the molar fraction of approaches unity in
Equation 2. HbO2 concentration, 90-150
µM.
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Fig. 5.
A graphic representation of sum of the
squared residuals (SSR) as a function of
pK1 values inserted into Equation 2.
After the values of k0B,
kHB, and
kOHB had been fixed in Equation 2, iterative least-squares procedures inserting various values for
K1, the adjustable parameter in Equations 3 and
4, were carried out so as to obtain the best fit to more than 75 values
of kf over the whole range of pH 5-11. At the
minimum of the sum of the squared residuals (SSR), the
conjugate values of k0A and
kHA, as well as the value of
pK1, are established. The resulting kinetic
constants and acid dissociation constant are summarized in Table
I.
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Table I
Rate constants and acid dissociation constants obtained from the pH
dependence curves for the autoxidation rate of HbO2 tetramer in
0.1 M buffer at 35 °C
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From these results, it becomes evident that the proton-catalyzed
processes with the rate constants
kHA and
kHB promote the autoxidation of
the
chain above the spontaneous processes in water with the rate
constants k0A and
k0B. In fact, the catalytic
proton enhances the rate enormously, by a factor of 5.6 × 106 mol
1 for state A and by a factor of
3 × 107 mol
1 for state B. In this
proton catalysis, the distal histidine at position 58 (the dissociable
group AH with pK1 = 6.2) appears to be involved
by the same mechanism as proposed previously in mammalian oxymyoglobins
(3, 17, 23, 24).
In sharp contrast to the
chain, the rate of autoxidation of the
chain in the HbO2 tetramer exhibited a saturation behavior below pH 5. Unfortunately, in more acidic pH range, data points could
not be obtained due to the denaturation of the protein. Nevertheless,
we could finally establish the best fit to more than 80 values of
ks by a "two-state model" in a quite acceptable
way, as shown in Fig. 6. In this
mechanism, we assume that a single, dissociable group (AH with
pK1) is also involved in the reaction. Employing
the same notations defined above, the autoxidation reaction of the
chain may therefore be written as shown by Scheme 2.
For this reaction, the observed rate constant,
kobss (
ks) in h
1, is given by Equation 5,
|
(Eq. 5)
|
where
|
(Eq. 6)
|
and
|
(Eq. 7)
|
By the same fitting procedures as for the
chain, the rate
constants and the acid dissociation constant involved in the autoxidation reaction of the
chain were established over the whole
range of pH 5-11 in 0.1 M buffer at 35 °C, and are also summarized in Table I.

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Fig. 6.
A plot of log(ks)
versus pH for the autoxidation of the chain of
HbO2 tetramer in 0.1 M buffer at 35 °C.
The computed curve (------) was obtained by a least-squares fitting to
the experimental data ( ) over the whole range of pH studied, based
on Equation 5, derived from a two-state model (see "Results"). The
resulting kinetic constants and acid dissociation constant are listed
in Table I. HbO2 concentration, 90-150
µM.
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|
In these kinetic formulations, one of the most remarkable features is
that the
chain does not show any proton-catalyzed process having
the term of
kH[H2O][H+], such as
the one that can play a dominant role in the autoxidation reaction of
the
chain, involving the distal histidine as its catalytic residue.
Instead, the
chain contains a dissociable group (AH) with
pK1 = 5.1 in 0.1 M buffer at 35 °C.
Although it is hazardous to identify a dissociable group only by its
pKa value, we suggest that the most probable
candidate for the group AH in Scheme 2 is the distal histidine residue
at position 63. In the
chain, however, this residue seems to be
less accessible to protons, titrating at a lower pH by a factor of
almost one pH unit compared with the value of
pK1 = 6.2 for the
chain. At the same time,
this residue would probably be located apart from the bound
O2, since a lack of hydrogen bonding with the terminal oxygen atom could reduce the autoxidation rate of the
chain. As
mentioned in Reaction 3, the proton transfer from the distal histidine
to the bound, polarized dioxygen can facilitate displacement of
O
2 as the hydroperoxyl radical HO2. Therefore, our
next step was to examine whether each of the separated
and
chains has its own different susceptibility to aqueous
autoxidation.
Stability Properties of the Separated
and
Chains--
In
separated chain solutions, the protein is known to exist in an
equilibrium of
2 or
4,
respectively. Under our experimental conditions, the monomeric form
(87%) was predominant in the
chains, while the tetrameric form
(99%) was predominant in the
chains. This estimation was made on
the basis of the results by McDonald et al. (26). Compared
with the HbO2 tetramer, the separated
and
chains
were both oxidized much more rapidly over the whole range of pH 5-10,
according to a simple, first-order kinetics based on Reaction 1.
Fig. 7 shows such pH dependences of the
observed rate constants, kobs
and kobs
, for the autoxidation
reaction of the separated
and
subunits in 0.1 M
buffer at 35 °C. Surprisingly, it became thus evident that the
separated
chain by itself does not show any saturation behavior in
its rate at low pH. Rather, the rate increased rapidly with increasing
hydrogen ion concentration, exhibiting a value close to
n =
1 for the slope against the acidic pH.
Consequently, each pH profile could be almost superimposed on the
other, except for the rate minimum region where
chains became
rather susceptible to autoxidation. We have therefore established
the best fit to more than 60 experimental points, for each of
kobs
and
kobs
, as a function of pH
by the same mechanism as delineated in Scheme 1.
|
(Eq. 8)
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Table II summarizes the kinetic and
thermodynamic parameters involved in the autoxidation reaction of
separated
and
chains.

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Fig. 7.
pH profiles for the autoxidation rate of
separated and chains in 0.1 M buffer at
35 °C. The logarithmic values of the observed first-order rate
constants, kobs ( ) and
kobs ( ), for the
autoxidation of separated and chains are plotted against the pH
of the solution. For each chain, the rate measurements were carried out
with 10 µM solution (in heme) in the presence of 1 mM EDTA. Both of the computed curves (------) were obtained
by a least-squares fitting to the experimental points over the whole
range of pH studied, based on Equation 8. The resulting kinetic
constants and acid dissociation constant are summarized for each chain
in Table II.
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Table II
Rate constants and acid dissociation constants obtained from the pH
dependence curves for the autoxidation rate of separated and
chains in 0.1 M buffer at 35 °C
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These results show that both
and
chains are inherently quite
susceptible to autoxidation over the whole range of pH studied. For
example, the values of k0B are
even higher (by a 2.5-4.5-fold) than that of bovine MbO2 (k0B = 0.17 × 10
3 h
1 M
1) in 0.1 M buffer at 35 °C (27). According to the view described already, our present results suggest that both of the heme pockets in
and
chains are sufficiently open to allow easier attack of the
solvent water molecule on the FeO2 center, with a
consequent very rapid formation of the ferric species. Moreover, the
chain tetramer can manifest proton-catalyzed processes with the
rate constants kHA and
kHB, if it is placed free from
the
chain. In this proton catalysis, the distal histidine at
position 63 (the dissociable group AH with pK1 = 6.1) appears to participate by recovering a hydrogen bond with the
bound dioxygen.
Another important feature of the separated
chain was seen in the
hemichrome formation. As already shown in Fig. 1, when the proteins
exist in HbO2 tetramer, both of the
and
chains were
oxidized to its ferric met form, accompanying typical spectral changes
due to the acid-alkaline transition with pKa = 7.9. However, things were quite different with the separated
chain, for
its oxidation product carried with it a hemichromogen spectrum over the
wide range of pH 5-10. Fig. 8 shows such
an example for the spectral changes with time when the separated
chain (10 µM) was oxidized in 0.1 M maleate
buffer, pH 6.2, at 35 °C. The final spectrum was not that of the
acidic (or aquo) met form, but for its admixture of the hemichrome with
a peak at 530 nm and a shoulder near 560 nm. In the framework of the accepted mechanisms both of the autoxidation reaction (3, 24) and the
hemichrome formation (28), our present results imply that a
nucleophilic displacement of O
2 from the
chain by an entering water molecule or hydroxyl ion is the rate-limiting step, and
that the subsequent conversion of the met form into a hemichrome must
proceed very quickly. Therefore, the autoxidation of separated
chains can be described by the following scheme at a neutral pH.
X represents a heme ligand endogenous to the protein, with the
kinetic relationship of kX
k0. In separated
chains, the distal
histidine residue at position 63 is the most probable candidate for X,
and the observed first-order rate constant,
kobs
, corresponds to the
rate-limiting step for the reaction leading to the formation of a
hemichrome.

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Fig. 8.
Spectral changes with time for the
autoxidation of separated chains in 0.1 M maleate
buffer at pH 6.2 and 35 °C. Scans were made at 40-min intervals
after 1.0 ml of the oxygenated chains (20 µM) was
added to the same volume of 0.2 M maleate buffer at pH 6.2 in the presence of 1 mM EDTA and 20%(v/v) glycerol. The
final spectrum was not for the acidic met form, but an admixture with
hemichrome. Over the wide range of pH 5-10, such a hemichrome
formation was not observed in separated chains, as well as in
HbO2 tetramer (see Fig. 1).
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In respect to the hemichrome formation, an EPR analysis of the ferric
derivatives may provide us with more detailed information. Fig.
9 shows the 8 K EPR spectrum for the
oxidation product of separated
chains in 10 mM maleate
buffer at pH 6.2. In addition to a high spin EPR spectrum attributed to
the usual aqua-met species with g values of 5.86 and 1.99, the
chain exhibited a low spin spectrum with
g1 = 2.77, g2 = 2.27, and
g3 = 1.68, which differentiates this species
from that of the hydroxide-type complex. According to Rifkind et
al. (28), such low spin complexes characterized by the highest
g value in the range of 2.83-2.75 and the lowest g value in the range of 1.69-1.63 have been designated as
complex B, indicating crystal field parameters of the reversible
hemichrome. They also suggest that the bis-histidine complex B may
still have water retained in the heme pocket, and therefore in solution
it is in rapid equilibrium with the high spin aquo complex (29). In the oxidized
chain, in fact, the molar fraction of the
hemichrome (complex B) was estimated to be 85% at pH 6.2. These EPR
interpretations agree very well with our present results
concerning the lability of the distal histidine residue in the
chain.

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Fig. 9.
The 8 K EPR spectra for the oxidation product
of separated chains in 10 mM maleate buffer at pH
6.2. In addition to a typical high spin aqua species with
g values of 5.86 and 1.99, the oxidized chain exhibited
a low spin spectrum with g values of 2.77, 2.27, and 1.68, which can be attributed to a hemichrome complex. These EPR signals are
completely coincident with the results from optical absorption in Fig.
8.
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 |
DISCUSSION |
It has been widely accepted that hemoglobin tetramer is
considerably more resistant to autoxidation than myoglobin. However, it
becomes evident that the separated
and
chains are oxidized more
easily than most other mammalian oxymyoglobins. Enhancements in the
oxidation rate of MbO2 or HbO2 have been
frequently attributed to the increased concentration of the
deoxygenated species, since each deoxy form is certainly the preferred
target for many kinds of oxidant. This mechanism cannot explain the
present results for the enhanced oxidation of the separated chains,
because it has been definitively established that both of the chains
have a much higher oxygen affinity with fewer deoxygenated species compared with the parent hemoglobin. In 0.1 M phosphate
buffer at pH 7.02 and 30 °C, for instance, Tyuma et al.
(30) reported the values of P50 = 1.00 mmHg for
chains and 0.45 mmHg for
chains, while HbA showed a value of
P50 = 16.59 mmHg in the absence of
2,3-diphosphoglyceric acid.
For a very long time, the autoxidation reaction of HbO2 has
been investigated at a single pH or within a limited range of pH.
However, the present study has disclosed that the rate of autoxidation
depends strongly upon the pH of the solution in a very complicated
manner, and that the pH dependence curves can vary significantly among
the separated
and
chains and their tetrameric parent over the
wide range of pH 5-11. On the basis of the molecular mechanism
proposed for the myoglobin autoxidation (3, 24), we have made, for the
first time, a full kinetic formulation for the autoxidation reaction of
human HbO2 in terms of a nucleophilic displacement of
O
2 from the FeO2 center, and revealed that the
chain plays a crucial role in hemoglobin autoxidation. In this
SN
2 mechanism, we did not consider whether
geminate recombination of O
2 plays a role in the reaction; so
this point remains open to future study. However, it should be remarked
that after photodissociation, the geminate rebinding of CO, NO, or
O2 ligand to myoglobin or hemoglobin was observed on the
pico- to nanosecond time scales at room temperature (31-33). It was
also reported that in myoglobin the ligand association and dissociation
rates could be controlled by a small fraction of the "open and
closed" conformations observable for the distal heme pocket on a
microsecond time scale (34). Compared with these processes, the
autoxidation of HbO2 and MbO2 takes place only
on the hour to day time scales.
At any rate, it becomes evident that the separated
chain, although
it exists predominantly as a tetramer, is inherently quite susceptible
to autoxidation. The FeO2 bond is always exposed to the
nucleophilic attack of an entering water molecule with and without
proton catalysis, and to the attack of an entering hydroxide anion.
These nucleophiles can thus cause a strong pH dependence having a
parabolic part. The proton-catalyzed displacement of O
2, in
which the distal histidine at position 63 appears to participate by a
proton-relay mechanism, can also account for most of the enhanced rate
occurring in the acidic pH range. Such properties are essentially the
same as in the separated
chain, except for the formation of
hemichrome as an oxidized product of
chains. The conversion of the
ferric met form into a hemichrome must involve changes of the protein
conformation so that a nitrogenous residue (probably of the distal
histidine at position 63) is coordinated as the sixth ligand of the
ferric heme iron. The spontaneous formation of such a hemichrome was at
variance with the
chain, as well as with the HbO2
tetramer, during the course of the autoxidation reaction over the wide
range of pH 5-11. Therefore, it appears that the distal heme pocket of
the
chains, as well as the
chains in the HbA tetramer, is more
rigid than that of the
chain tetramer.
When HbO2 is placed in dilution, the tetrameric species is
known to dissociate into 
dimers along
1
2 interface, so that the dimers formed
are of the
1
1 type (35, 36). In a
previous paper, we reported that the 
dimers can be oxidized to
the ferric met form without any spectral evidence for the formation of
hemichromes. In the 
dimers, we also found that the slow
component (ks) due to the
chain is quite
resistant to the acidic autoxidation (5). These results imply that the
intrinsic tendency of the
chain to form a hemichrome, as well as to
produce an acid-catalyzed autoxidation, must have been suppressed by
the formation of the
1
1 (or
2
2) contact.
In this connection, Borgstahl et al. reported the 1.8 Å structure of carbonmonoxy-
4 (CO
4)
tetramer of human hemoglobin, and compared subunit-subunit contacts
between three types of interfaces (
1
1,
1
2, and
1
2)
of oxyHb and the corresponding CO
4 interfaces (37). In
their examinations, interfaces were defined using a 3.5-Å cutoff and
at least one interaction is <3.5 Å in each case. As a result, they
found that, in contrast to the stable
1
4
interface, the
1
2 interface of the
CO
4 tetramer is less stable and more loosely packed than
its
1
1 counterpart in oxyHb. In
particular, there are significant packing differences at the end of the
B helix between these homologous interfaces; the B helix-H helix contact region is spread apart by ~1 Å in CO
4
relative to oxyHb. Specifically, the CO
4
1
2 interface does not include close
contacts between residues Pro-125(H3) and Val-33(B15), Gln-127(H5) and Val-34(B16), and Ala-128(H6) and Val-34(B16). The side chain disorder also makes the center of the CO
4
1
2 interface packed less tightly. Therefore, the contact sites in the
4 tetramer are
indeed different from the
1
1 contact
sites in the HbA tetramer. This supports explicitly our conclusion
described above. As is evident from Fig. 3, the remarkable stability of
HbO2 tetramer can be ascribed mainly to the delayed
autoxidation of the
chains at acidic pH range. It is also clear
that the
chain has acquired this stability against autoxidation by
blocking out the proton catalysis (Reaction 3) from the aqueous
autoxidation, in a manner that the distal histidine cannot act as its
catalytic residue.
In this regard, Shaanan (38) investigated the stereochemistry of the
iron-dioxygen bond in human HbO2 tetramer by single-crystal x-ray analysis. In the
chain, the distance between N
of His(E7) and the terminal oxygen atom (O-2) is found to be 2.7 Å,
and the geometry favors a similar hydrogen bond as in oxymyoglobin (25). In the
chain, however, N
of His(E7) is located
further both from O-2 and O-1 (3.4 and 3.2 Å, respectively),
indicating that the hydrogen bond, if formed, is much weaker. Our
kinetic results seem to be in good accord with the crystal structural
evidence for the FeO2 bonding in HbO2 tetramer.
Fig. 10 illustrates in a very schematic
way the structure of HbO2 tetramer, as seen in the 
contact leading to the nonequivalence of the chains. The four heme
pockets are all exposed at the surface of the molecule. The distal
histidine can stabilize the bound dioxygen by hydrogen bond formation.
Nevertheless, it is also true that each FeO2 bonding is
always subject to the nucleophilic attack of an entering water molecule
including its conjugate ions OH
and H+. In
this process, the distal histidine is proposed to participate, via its
imidazole ring and a proton-relay mechanism, in facilitating the
effective movement of a catalytic proton from the solvent to the bound,
polarized dioxygen. This proton-catalyzed nucleophilic displacement of
O
2 from the FeO2 center by the entering water molecule, a SN
2 type process with proton
assistance, can account for most of the autoxidation reaction occurring
under physiological conditions. These features are essentially the same
as those in the autoxidation of myoglobin (3, 23, 24).

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Fig. 10.
Schematic representation of human
oxyhemoglobin as seen in the 1 1 contact
to produce tilting of the distal histidine in the chain. In
HbO2 tetramer, the heme pockets are all exposed at the
surface of the molecule. By the formation of the
1 1 contact, the chain is subject to a
structural constraint whereby the distal histidine at position 63 is
tilted away slightly from the bound O2. As a result, the
chain loses a proton-catalyzed process, and HbO2
tetramer can acquire the enhanced stability against the autoxidation
reaction.
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In the hemoglobin molecule, however, there are two types of the 
contacts. One is the
1
1 (or
2
2) contact involving B, G, and H helices
and the GH corner, and other is the
1
2
(or
2
1) contact involving mainly helices
C and G and the FG corner (37, 39). The mechanism whereby
and
chains acquire the enhanced stability against the aqueous autoxidation
must be associated with the formation of the
1
1 (or
2
2)
contact. These packing contacts are also expected to produce in the
chains a conformational constraint, whereby the distal histidine at
position 63 is tilted away slightly from the bound O2 so as
to prevent the acid-catalyzed autoxidation from the
chains in the
HbO2 tetramer. In this way, the hemoglobin molecule seems
to differentiate two types of the 
contacts quite properly for
its own function. The
1
2 and
2
1 contacts are associated with the
cooperative oxygen binding, whereas the
1
1 and
2
2
contacts are used for controlling the stability of the bound
O2. In fact, the former sliding contacts are known to
undergo the principal change when the hemoglobin molecule goes from its
deoxy to oxy configuration during the course of the ligand binding (1,
39). To the latter packing contacts, for the first time, we have
assigned a new role in the stabilization of the HbO2
tetramer against autoxidation.
We thank Dr. Y. Ohba (Institute for Chemical
Reaction Science, Tohoku University) for EPR measurements.