(Received for publication, November 20, 1995; and in revised form, March 7, 1996)
From the
Cr(III)-Fe(II) hybrid hemoglobins,
(Cr)
(Fe) and
(Fe)
(Cr), in which hemes in either
the
- or
-subunits were substituted with chromium(III)
protoporphyrin IX (Cr(III)PPIX), were prepared and characterized by
oxygen equilibrium measurements. Because Cr(III)PPIX binds neither
oxygen molecules nor carbon monoxide, the oxygen equilibrium properties
of Fe(II) subunits within these hybrids can be analyzed by a two-step
oxygen equilibrium scheme. The oxygen equilibrium constants for both
hybrids at the second oxygenation step agree with those for human adult
hemoglobin at the last oxygenation step (at pH 6.5-8.4 with and
without inositol hexaphosphate at 25 °C). The similarity between
the effects of the Cr(III)PPIX and each subunits' oxyheme on the
oxygen equilibrium properties of the counterpart Fe(II) subunits within
hemoglobin indicate the utility of Cr(III)PPIX as a model for a
permanently oxygenated heme within the hemoglobin molecule.
We found
that Cr(III)-Fe(II) hybrid hemoglobins have several advantages over
cyanomet valency hybrid hemoglobins, which have been frequently used as
a model system for partially oxygenated hemoglobins. In contrast to
cyanomet heme, Cr(III)PPIX within hemoglobin is not subject to
reduction with dithionite or enzymatic reduction systems. Therefore, we
could obtain more accurate and reasonable oxygen equilibrium curves of
Cr(III)-Fe(II) hybrids in the presence of an enzymatic reduction
system, and we could obtain single crystals of
deoxy-(Cr)
(Fe) when grown in low salt
solution in the presence of polyethylene glycol 1000 and 50 mM dithionite.
Human adult hemoglobin (Hb A) ()cooperatively binds
four oxygen molecules via a complex sequence of intermediate oxygenated
states. Information about the intermediate species is required to
understand the cooperative mechanism of Hb A, yet little is known about
such intermediates because the equilibrium concentrations of the
intermediates under any conditions are markedly reduced by the
cooperativity of Hb.
Cyanomet valency hybrid Hbs have been frequently used for studying the oxygenation intermediates of Hb A(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) . Structural and functional studies on cyanomet valency hybrids have suggested that cyanide-bound ferric heme mimics natural oxyheme, thus deoxy-cyanomet valency hybrid Hbs have been used as models for the intermediate species formed during the cooperative oxygenation process(13, 14, 15, 16, 17, 18, 19, 20) . However, since conventional met-Hb reducing reagents and enzymatic reduction systems reduce the cyanomet heme, it is very difficult to carry out the experiments using deoxy-cyanomet valency hybrids under anaerobic conditions. Thus, there have been no reports on x-ray crystallography of cyanomet valency hybrids because of this difficulty.
During recent years, we have investigated the properties of
metal-substituted hybrid Hbs, (M)
(Fe)
and
(Fe)
(M), using the first
transition metal ions (M). This metal substitution method is the most
suitable modification of Hb A for studying the relationships between
the functional states of Hb A and globin-metalloporphyrins interaction.
In our systematic investigations, we have observed a wide variation of
oxygen affinities in these hybrids, as a result of the variation in the
configuration of 3d electrons of the porphyrin metal. With
respect to the oxygen affinity of metal-Fe(II) hybrids, we can classify
these hybrids into following four groups: (i) hybrids showing oxygen
affinities as high as oxy-Hb A, (ii) hybrids showing intermediate
oxygen affinities, between oxy-Hb A and deoxy-Hb A, (iii) hybrids
showing affinities as low as deoxy-Hb A, (iv) hybrids showing lower
oxygen affinities than deoxy-Hb A. To represent the oxygenation
intermediates of Hb A, the hybrids in group (i) and (iii) are
particularly important. In our recent series of studies, Ni(II)-Fe(II)
hybrid Hbs in group (iii) have been used successfully to investigate
the structures and functions of the intermediates appearing in the
first half oxygenation of Hb
A(21, 22, 23, 24, 25, 26, 27, 28) .
Although the Ni(II)-Fe(II) hybrid system has brought much structural
and functional information about the initial-half oxygenated
intermediates, this approach could not be extended to studies on the
latter-half oxygenation of Hb A. Stable hybrids in group (i) were
required to model the intermediates appearing in the oxygenation of the
last two sites of Hb A.
This paper reports the preparation and
oxygen equilibrium properties of Cr(III)-Fe(II) hybrid Hbs. It also
shows that these Cr(III)-Fe(II) hybrids are an excellent model for the
intermediates appearing in the oxygenation of the last two sites of Hb
A. The influence of Cr(III)PPIX on the oxygen equilibrium properties of
ferrous subunits within Cr(III)-Fe(II) hybrid Hbs results in
quantitative similarities with oxygenated heme under various
conditions. This view is reinforced by other structural results, namely
(i) similar porphyrin geometry and metal-histidine bonds between
Cr(III)PPIX and natural oxyheme, and (ii) the occupation of the sixth
coordination position of Cr(III)PPIX by HO (or
OH
) so that the substitution of oxyheme with
H
O bound Cr(III)PPIX may not significantly alter the
heme-pocket structure. With respect to the ease of experiments under
anaerobic conditions, Cr(III)-Fe(II) hybrid Hbs have several advantages
over cyanomet valency hybrid Hbs as stable and useful models for the
oxygenation intermediates.
The first and second intrinsic Adair constants, K (i = 1 and 2; in torr
), is written as:
p is the partial pressure of oxygen. i/(2
- (i - 1)) is a statistical factor, because the
binding of O to Hb(O
)
is statistically enhanced by a factor of the number of empty
sites, 2 - (i - 1), and the release of O
from Hb(O
)
is statistically enhanced by a
factor depending on the number of filled sites, i. Fractional
saturation with oxygen is expressed as Y:
is the Adair equation used to analyze the data. The
best fit values of the first and second intrinsic Adair constants, K, were obtained by fitting a two-step Adair
equation to each deoxygenation curve through a least-squares procedure (38) .
Fig. 1presents the isoelectric focusing of
Cr(III)-Fe(II) hybrid Hbs in CO form and Hb A in CO form and CrHb.
(Mn
)
(Fe-CO) (42) and
chain are shown as controls. Each hybrid Hb
appears as a nearly single band. CrHb and the hybrid Hbs migrate toward
the higher pH region compared with Hb A, due to the presence of
trivalent Cr(III)PPIX.
Figure 1:
Isoelectric focusing of
(Cr)
(Fe-CO),
(Fe-CO)
(Cr),
(Mn
)
(Fe-CO), Hb A,
and
chain in CO forms, and CrHb. Lanes 1 and 8,
chain; lanes 2 and 6, Hb A; lane 3,
(Cr)
(Fe-CO); lane 4,
(Fe-CO)
(Cr); lane 5,
(Mn
)
(Fe-CO); lane 7, CrHb.
The pH dependence of the absorption spectrum of CrHb is presented in Fig. 2A. Upon raising pH from 6.5 to 8.4, the Soret peak shifted from 445 to 439 nm, and a visible peak at 764 nm shifted to 752 nm. In the range of pH 6.5-8.4, the isosbestic points were reproducibly observed at 748, 720, 441, and 407 nm.
Figure 2:
pH dependence of absorption spectra of
CrHb (A),
(Cr)
(Fe-O
) (B), and
(Fe-O
)
(Cr) (C)
in 50 mM bis-Tris or Tris buffer with 100 mM chloride
at 25 °C. Arrows indicate the absorbance change upon
raising the pH: 6.5, 7.4, 8.4 (for A, pH = 6.5, 7.4,
8.0, 8.4).
mM is a millimolar extinction coefficient on a
metal basis.
The absorption spectrum of CrHb was not affected by the presence
of either 50 mM dithionite under anaerobic condition or the
enzymatic reduction system of Hayashi et al.(37) ,
indicating that Cr(III)PPIX in CrHb was not reduced to Cr(II)PPIX by
these reductants. Thus, the oxygen equilibrium curves of Cr(III)-Fe(II)
hybrid Hbs could be measured with enzymatic reduction system in order
to reduce the met-heme contents to a minimal level. Moreover,
absorption spectra of deoxygenated Cr(III)-Fe(II) hybrid Hbs under
anaerobic condition in the presence of 50 mM dithionite did
not change remarkably for 96 h at 20 °C except decreasing of
dithionite absorption (Fig. 3). After these measurements, CO
could reasonably bind to the ferrous subunits of Cr(III)-Fe(II) hybrid
Hbs (Fig. 3). Autooxidation rate of ferrous subunits of
(Cr)
(Fe-O
) and
(Fe-O
)
(Cr) were
measured by detecting 410-nm absorbance change in air equilibrated
buffer, 37 °C condition (Fig. 4). Precipitate did not
appear. The autooxidation rates of Cr(III)-Fe(II) hybrid Hbs were
comparable with that of Hb A under the same condition (Fig. 4),
meaning that ferrous subunits of Cr(III)-Fe(II) hybrid Hbs were as
stable as those of Hb A against autooxidation. Thus, we conclude that
Cr(III)-Fe(II) hybrid Hbs have enough stability for our oxygen
equilibrium and crystallization experiments. The sum of the absorption
spectra of
(Cr)
(Fe-O
) and
(Fe-O
)
(Cr) was
almost identical to that of oxy-Hb A and CrHb. (Fig. 2, B and C). Oxy-deoxy difference spectrum of each
Cr(III)-Fe(II) hybrid Hb agrees closely with that of isolated
or
chain. These findings mean that Cr(III) subunits do not bind
oxygen molecules and that the absorption spectra of Cr(III) subunits
are not affected by the ligation state of the corresponding ferrous
subunits within Cr(III)-Fe(II) hybrid Hbs.
Figure 3:
Absorption spectra of
(Fe)
(Cr) in the presence of
dithionite. Deoxy-
(Fe)
(Cr) in the
presence of 50 mM dithionite (a), after 48 h (b), after 96 h (c), spectrum after 96 h measurement
followed by exposure to CO (d). Conditions are as follows:
protein concentration, 170 µM (on a metal basis);
temperature, 20 °C; buffer condition, 50 mM Tris with 100
mM chloride, pH 7.4; under N
atmosphere, except
CO-bound condition; 1 mm light path cell was
used.
Figure 4:
Autooxidation rates of Hb A,
(Cr)
(Fe-O
), and
(Fe-O
)
(Cr).
Absorbance change per 1 µM heme at 410 nm are plotted
against time: Hb A (
);
(Cr)
(Fe-O
), (
); and
(Fe-O
)
(Cr) (
).
Conditions are as follows: protein concentration, 200 µM (on a metal basis); temperature, 37 °C; buffer condition, 50
mM Tris with 100 mM chloride, pH 7.4, air
equilibrated buffer.
In the absence of IHP, Cr(III)-Fe(II) hybrid
Hbs showed very high affinity for oxygen molecules. At pH 8.4, both
hybrids bound oxygen noncooperatively (n = 1.0-1.1) with very high affinity comparable with
that of isolated
or
chain, while they exhibited n
values significantly higher than unity (n
= 1.2-1.3) at pH 6.5. In both
hybrids, Hill coefficients became larger as pH decreased. There were
slight differences between the cooperativity of
(Cr)
(Fe) and that of
(Fe)
(Cr). The n
values of
(Cr)
(Fe) (n
= 1.1-1.2) were slightly smaller
than those of
(Fe)
(Cr) (n
= 1.1-1.3) at all pH values
examined, and the latter hybrid exhibited a slightly larger Bohr effect
than the former. The oxygen equilibrium properties of both hybrids were
significantly affected by the addition of IHP. The oxygen affinities
were reduced, and the cooperativity and the number of released Bohr
protons was increased (Fig. 5). It is important to note that the
extent of the IHP effect on the K
values of both
hybrids was very similar to those on the K
value
of native Hb A (43) (
)(see Fig. 5). These
results suggest that Cr(III)PPIX behaves like a permanent oxy-heme.
Figure 5:
pH dependence of the equilibrium constant
for the last oxygen molecule to bind to Hb. K (torr
) of
(Cr)
(Fe) (
); K
(torr
) of
(Fe)
(Cr) (
); and K
(torr
) of Hb A (
) ((43) ). log K
and log K
values are plotted. Filled symbols indicate the presence
of IHP (2 mM). Data for Hb A with IHP (2 mM) are from
K. Imai (Footnote 3). Conditions for hybrid Hbs are as follows: protein
concentration, 60 µM (on a metal basis); temperature, 25
°C; buffer condition, 50 mM bis-Tris or Tris buffer with
100 mM chloride; wavelength of detection light, 560 nm.
Protein concentration of Hb A is 60 µM (on a metal basis),
and other solution conditions are the same as those for hybrid
Hbs.
We succeeded in obtaining single crystals of deoxy-
(Cr)
(Fe) from solution of 2% protein,
in the presence of 27-28% polyethylene glycol 1000 and 50 mM dithionite under anaerobic condition (Fig. 6). Crystals
were examined by precession photography. The
deoxy-
(Cr)
(Fe) crystals were found to
be isomorphous to the native deoxy-Hb A crystals, which belong to space
group P2
2
2.
Figure 6:
Crystals of
deoxy-(Cr)
(Fe). The crystals were
obtained from solution of 2% protein, 50 mM-sodium phosphate,
pH 7.0, in the presence of 27-28% polyethylene glycol 1000 and 50
mM dithionite, under N
atmosphere.
The pH-dependent spectra of CrHb show well defined isosbestic
points (Fig. 2A), indicating that CrHb exists in a
pH-dependent equilibrium between two alternate states. Since Cr(III)
complexes are almost universally hexacoodinated(44) , and
Cr(III)PPIX prefers counter anion due to an extra positive charge, it
is reasonable to consider that the proximal histidine coordinates to
the Cr(III) ion and that the remaining coordination position is
occupied by a water molecule or a hydroxyl ion, as in the case of
aquomet-Hb. Thus, observed pH-dependent spectral changes may result
from a coordination equilibrium between HO and
OH
in CrHb. Previously, Fiechtner reported that
hemichrome structure is formed in CrHb(45) ; however, their
published absorption spectrum is quite different from ours. Their
OD
/OD
ratio is 1.3, whereas ours is 3.6
at pH 7.4.
The dimer-tetramer association equilibrium constants of
(Cr)
(Fe-CO) and
(Fe-CO)
(Cr), which were
obtained by gel-filtration experiments, are about 5
10
M
and 1
10
M
, respectively. (
)Although part of the Cr(III)-Fe(II) hybrids dissociated
into a
dimers under our experimental conditions used for oxygen
equilibrium experiments, the Hill plots of both Cr(III)-Fe(II) hybrids
exhibited little protein concentration dependence (Table 1). We
calculated the protein concentration dependence of the Hill plots of
Cr(III)-Fe(II) hybrids using estimates of dimerization from
dimer-tetramer association equilibrium constants and the assumption
that a hemoglobin dimer containing Cr(III)PPIX and Fe(II)PPIX exhibits
high oxygen affinity, comparable with that of isolated chains. The
calculations revealed that the Hill plots were only slightly influenced
by dimerization in the concentration range of above 10 µM (on a metal basis). Because the experimental Hb concentrations
which we measured oxygen equilibrium curves were 12, 60, and 240
µM (on a metal basis), the theoretical consideration was
consistent with the experimental results.
K values of both Cr(III)-Fe(II) hybrid Hbs agreed with K
values of Hb A, including both pH and IHP
dependence (Fig. 5). In recent years, both association and
dissociation rate constants for the last step (
- and
-subunit, respectively) in oxygen binding to Hb A were determined
kinetically, and the Adair constants (K
for
subunit
and K
for
subunit, respectively) were calculated from
these rate
constants(46, 47, 48, 49) . In Table 2, we compared the K
values of the
Cr(III)-Fe(II) hybrid Hbs with the kinetically determined K
values of Hb A. The K
values of the Cr(III)-Fe(II)
hybrids were comparable with the K
values of Hb A. These
findings indicate that the influence of Cr(III)PPIX on the oxygen
equilibrium properties of the counterpart ferrous subunits is similar
to that of oxyheme. Stereochemical theory of Hb allostery (50) indicates that the oxygen affinity of Hb is regulated by
the equilibrium position of the central metal with respect to the
porphyrin ring, so that the position of the proximal histidine relative
to the hemeplane is a key determinant of oxygen affinity of Hb. In this
regard, Cr(III)PPIX can be an adequate model for an oxyheme for several
reasons. (i) The Cr(III) ion has an ionic radius of 0.62
Å(51) , which is almost equal to that of low spin Fe(II).
(ii) The Cr(III) ion is also expected to lie in the mean porphyrin
plane(52) . (iii) Cr(III) complexes are almost universally
hexacoordinated(44) , and (iv) Cr(III) porphyrin binds ligands
so tightly (52) that the Cr(III)-histidine bond is expected to
be as short as Fe(II)-histidine bond in oxy-Hb. Thus, both the proximal
and the distal environments of Cr(III)PPIX in Hb may be similar to
those of oxy-heme.
Cyanomet valency hybrid Hbs have been widely used
as models for understanding the nature of the intermediate species
formed during the cooperative oxygenation process. There are several
structural and functional reasons for using cyanide-bound ferric heme
as an oxyheme model. (i) The crystal structure of cyanomet-Hb (10) is closely similar to that of oxy-Hb A(53) . (ii)
Like an oxyheme, cyanide-bound ferric heme is low spin, with the ferric
ion firmly anchored in the heme plane (4, 12, 54) . (iii) Cyanomet subunits in
cyanomet valency hybrids give NMR spectra that are very similar to
those observed in cyanomet Hb in the absence of 2,3-diphosphoglycerate
or IHP(5, 7) . (iv) The ferrous subunits of cyanomet
valency hybrids show fast ligand binding kinetics (3, 6) and high affinity for
oxygen(8, 11) . On the basis of these findings,
cyanomet valency hybrid Hbs are generally assumed to be a good model
for the oxygenated
intermediates(13, 14, 15, 16, 17, 18, 19, 20) .
Because the K value of both
(Fe
CN
)
(Fe
)
and
(Fe
)
(Fe
CN
)
agreed well with the K
value of Hb A including pH effect (11) , (
)cyanide-bound ferric heme is a good model
for an oxygenated heme as Cr(III)PPIX. Actually, the oxygen equilibrium
properties of both Cr(III)-Fe(II) hybrid Hbs almost agreed with those
of the corresponding cyanomet valency hybrid Hbs. Yet, it should be
pointed out that cyanomet valency hybrid Hbs have at least one serious
disadvantage. The ferrous subunits of cyanomet valency hybrid Hbs are
unstable against autooxidation. The inevitable autoxidation of ferrous
subunits in these cyanomet hybrids results in asymmetric oxygen
equilibrium curves(11) . Since the addition of the met-Hb
reducing reagent or enzymatic reduction system reduces not only the
oxidized heme but also the cyanomet heme, these standard techniques for
native Hb are not applicable to the cyanomet hybrids.
In contrast to
cyanomet heme, Cr(III)PPIX in Hb is not reduced to Cr(II)PPIX with 50
mM sodium dithionite or with the enzymatic reduction system.
This advantage allows us to measure more accurately and reasonably
oxygen equilibrium curves of Cr(III)-Fe(II) hybrid Hbs in the presence
of the enzymatic reduction system. Moreover, complete deoxygenation of
Cr(III)-Fe(II) hybrids can be easily attained by the addition of sodium
dithionite, so that we can safely carry out the experiments under
anaerobic condition. For example, we actually succeeded in making the
single crystals of deoxy-(Cr)
(Fe)
grow in a low salt solution with the presence of both polyethylene
glycol and 50 mM dithionite and under anaerobic conditions (Fig. 6).
Note that Cr(III)-Fe(II) hybrid Hbs are not damaged by the spectrometer beam for measuring oxygen equilibrium curves. To confirm the stabilities of Cr(III)-Fe(II) hybrid Hbs against monochromatic light in spectrophotometers, we measured the oxygen equilibrium curves of the hybrids twice in succession in the presence of met-heme reduction systems, there was no difference between the two measurements in the range of standard error.