Functional and structural significance of the
"module" in proteins has been investigated for globin proteins. Our
previous studies have revealed that some modules in globins are
responsible for regulating the subunit association and heme
environmental structures, whereas the module substitution often induces
fatal structural destabilization, resulting in failure of functional regulation. In this paper, to gain further insight into functional and
structural significance of the modular structure in globins, we focused
upon the "pseudo-module" in globin structure where boundaries are
located at the center of modules. Although the pseudo-module has been
supposed not to retain a compactness, the 
(PM3)-subunit, in which
one of the pseudo-modules, the F1-H6 region, of the
-subunit is
implanted into the
-subunit, conserved stable globin structure, and
its association property was converted into that of the
-subunit, as
the case for the module substituted globin, the 
(M4)-subunit.
These results suggest that modules are not unique structural and
functional units for globins. Interestingly, however, the recent
reconsideration of the module boundary indicates that the modules in
globins can be further divided into two small modules, and one of the
boundaries for the new small modules coincides with that of the
pseudo-module we substituted in this study. Although it would be
premature to conclude the significance of the modular structure in
globins, it can be safely said that we have found new structural units
in globin structure, probably new modules.
 |
INTRODUCTION |
Recent structural studies of proteins have revealed that many
protein structures are constructed by the compact structural unit,
"modules," which correspond to the exons on the gene structure (1,
2). The gene of globin is made up of three exons interrupted by two
introns, and exons 1-3 correspond to the modules M1, M2 + M3, and M4,
respectively. The correlation of globin structure and function with its
modular structure was exemplified by the observation that specific
functions of globins are attributed to the specific modules (3). As
found in Fig. 1A, the amino acid residues associated with the heme contacts and the
1-
2 contacts are concentrated in module M2 + M3, whereas the
1-
1 contact cluster is located in module M4.

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Fig. 1.
A, module and pseudo-module boundaries
and residues with well defined functional roles in hemoglobin subunits
proposed by Eaton (3). B, amino acid sequence for hemoglobin
- and -subunits in the pseudo-module PM3 and the module M4. The
identical residues are expressed in bold type. The
underlines indicate the residues contributing to 1- 1
or 1- 2 contacts. The proximal histidine (F8) is
marked by an asterisk.
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To gain insights into the structural and functional significance of the
module in globins, we have prepared a variety of the module substituted
globins (4-6). The module M4 substituted hemoglobin
-subunit
(
(M4)-subunit), in which the module M4 was replaced by that of
the
-subunit, exhibited the native
-subunit-like heme
environmental structure, while it preferentially associated with the
native
-subunit and not with the
-subunit (4). These findings
indicate that the module substitution can convert the association
property of the hemoglobin subunit without substantial structural
changes in the heme vicinity, suggesting that the module is a
structural and functional unit (4). However, the counterpart chimeric
globin, the 
(M4)-subunit, of which module M4 was derived from
that of the
-subunit, was quite unstable, and the association property was not affected by the module substitution (5). Such destabilization in globin structure was also encountered for other module substituted globins (6), which leads us to reconsider the
structural and functional significance of the modular structures in
globins and examine effects of "non-module substitution" on globin
structure and function to compare with those of the module substitution.
In the present study, we have focused upon the "pseudo-module" in
globin structure. The pseudo-module is defined as a segment starting at
the center of one module and ending at the center of the adjacent
module (Fig. 1A) and is supposed not to form a compact
structural unit (7). Since the pseudo-modules do not statistically
coincide with exons (8), they would have neither evolutionary nor
functional meanings (9, 10). Herewith, we have prepared a pseudo-module
substituted globin, the 
(PM3)-subunit, as illustrated in Fig.
2 and compared its structural and
functional properties with those of the corresponding stable module
substituted globin, 
(M4)-subunit. We are concerned here with the
effects of the pseudo-module substitution on the globin structure.
Furthermore, we have paid attention to the association property of the
chimeric globins, since the amino acid residues contributing to the
1-
1 contact are concentrated in the pseudo-module PM3 as well as
in the module M4 (Fig. 1A). The heme environmental structure
and oxygen affinity for the chimeric globin have been also examined by
using various spectroscopic methods to describe the functional and
structural effects of the pseudo-module substitution in globins.

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Fig. 2.
Novel globin subunits synthesized in this
study. Restriction enzyme sites used in these preparations are
noted in parentheses.
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EXPERIMENTAL PROCEDURES |
Expression Vector Construction--
The expression vectors of
the 
(M4)- and 
(PM3)-subunits were constructed as
illustrated in Fig. 2. The N-terminal valine residue was replaced by a
methionine to initiate the peptide elongation for the module and
pseudo-module substituted subunits. To obtain the genes of the

(PM3)-subunit, KpnI (GGTACC) and MluI sites (ACGCGT) were introduced at the start and end of the PM3, respectively, by polymerase chain reaction with silent mutation.
Protein Preparation--
All of the module and pseudo-module
substituted subunits were purified as previously reported for
recombinant Hb (4, 11-13). We confirmed the correct expression of the
desired subunits by fast atom bombardment-mass spectroscopy (data not
shown) (14), and no additional mutations were detected. We also
synthesized "wild-type"
-subunit1 that has a
methionine residue at the N-terminal instead of the valine residue as a
reference and confirmed that the structural properties of the wild-type
-subunit are virtually the same as those of "native"
-subunit
isolated from human red blood cells.
Circular Dichroism Spectra--
CD spectra of the cyano-met
subunits in far UV region were measured with Jasco J-760. Concentration
of the samples was 5 µM, and the light path of the cell
was 1 mm. The buffer used in the measurements was 20 mM
sodium phosphate containing 0.1 M NaCl and 5 mM
NaCN, pH 7.4, at room temperature.
Urea Denaturation Curves--
Reaction solutions contained 20 mM Tris, pH 7.4, 1 mM NaCN, and various
concentrations of urea. Sample concentration was 5 µM.
The ellipticity at 222 nm was monitored by Jasco J-760 CD spectrometer
after ~10 h equilibration at room temperature. Cyano-met derivatives
are used for the measurements to avoid aggregation of heme and reduce
the irreversible
denaturation2 (15). The
fractional denatured population (fD) for various urea concentrations was estimated by Equation 1.
|
(Eq. 1)
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where [
]222, N, [
]222, D,
and [
]222 represent ellipticities at 222 nm in the
native (N) and denatured (D) states and in each urea concentration,
respectively. The free energy of denaturation,
G, was
calculated by Equation 2 (15).
|
(Eq. 2)
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When
G varied linearly with urea concentration,
[urea],
GH2O,
extrapolated
G at [urea] = 0, can be estimated by the
following equation:
|
(Eq. 3)
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where murea is the slope of the linear
relation between
G and [urea].
Gel Chromatogram--
Gel filtration measurements were performed
by using a Sephacryl S-200 HR column (0.8 × 62 cm) at 4 °C.
The buffer used for the chromatography was 50 mM Tris, in
the presence of 0.1 M NaCl, and 1 mM
Na2EDTA, pH 7.4, and the flow rate was 7 ml/h. The eluted fractions were monitored by absorption at the Soret band (16, 17).
Dimer-tetramer dissociation constant of the samples was determined by
concentration dependence of the centroid elution volume over the range
from 0.5 to 200 µM (16, 17). The following functional
dependence of the elution volume (Ve)
versus protein concentration (CT) allows
us to determine the dimer-tetramer equilibrium constants for the
samples (16), see Equation 4.
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(Eq. 4)
|
where Vj is the elution volume for the
individual species pertaining to the various aggregates
(j-mers), and the (mj) term represents
molar concentrations for the respective species.
NMR Spectra--
1H NMR spectra at 500 MHz were
recorded on Bruker Avance DRX 500. We used a WaterGate pulse sequence
for the diamagnetic region to minimize the water signal in the sample.
For the measurements of the hyperfine-shifted proton resonances, we
utilized a LOSAT pulse sequence. The probe temperature was controlled
at 290 ± 0.5 K by a temperature control unit of the spectrometer.
The volume of the NMR sample was 500 µl, and the concentration was
600 µM on the heme basis. Proton shifts were referenced
with respect to the proton resonance of
2,2,-dimethyl-2-silapentane-5-sulfonate.
Oxygen Equilibrium Curves and Analysis--
Oxygen equilibrium
curves were measured by using an improved version (18, 19) of an
auto-oxygenation apparatus (20). The wavelength of the detection light
was 560 nm, and the protein concentration was 60 µM on
the heme basis. The temperature of the sample in the oxygenation cell
was constant within ±0.05 °C. The hemoglobin reductase system (21)
was added to the sample before each measurement to reduce oxidized
subunits. To minimize the autoxidation of the sample during the
measurements, catalase and superoxide dismutase were added to the
sample, and the concentration was 0.1 µM (22, 23). The
oxygenation data were acquired by use of a micro-computer (model
PC-98XA, Nippon Electric Co., Tokyo), which was interfaced to the
oxygenation apparatus (24).
 |
RESULTS |
Circular Dichroism Spectra--
As reported by our previous
studies (5, 6), the module substitutions often induced severe
destabilization in globin structure, which is characterized by the
prominent decrease of the negative ellipticities at 222 and 208 nm in
the CD spectra (5). In Fig. 3, however,
the pseudo-module substituted globin, 
(PM3)-subunit, exhibits two
broad and large negative peaks around 222 and 208 nm characteristic of
the
-helical structure. These negative peaks were also observed for
the module substituted 
(M4)-subunit and native hemoglobin (25),
indicating that the secondary structure for the
-subunit is almost
insensitive to the substitution of the pseudo-module PM3 as the
case for the M4 substitution.

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Fig. 3.
CD spectra in far UV region of the cyano-met
native, module, and pseudo-module substituted globin subunits.
Lines correspond to hemoglobin (---),  (M4)-subunit (- - -), and
 (PM3)-subunit ( - ).
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Urea Denaturation Curves--
Alterations in equilibrium stability
upon the module and pseudo-module substitutions were quantified by the
urea-induced denaturation experiment. As clearly delineated in Fig.
4A, the transition curve for
the urea denaturation in the 
(PM3)-subunit is quite similar to
that for the native
-subunit, indicating that the globular structure
of the 
(PM3)-subunit is as stable as that of the native
-subunit. For the 
(M4)-subunit, the denaturation curve was shifted to the right side from that of the
-subunit and almost superimposed on that of tetrameric native hemoglobin. In Fig. 4B, the free energy of denaturation (
G) was
plotted against urea concentrations, and a linear fitting procedure
using Equation 3 determined the extrapolated
G in the
absence of urea,
GH2O, and the
slope of the linear relation (i.e.
d
G/d[urea]), murea (Table
I). Although the substitution of the
module M4 increases
GH2O by about 25 kJ/mol and murea by 2.3 kJ/mol per M
(M, urea concentration), the corresponding parameters for
the substitution of the pseudo-module PM3 are almost unchanged from
those for the
-subunit. The effects of the pseudo-module PM3
substitution on the protein stability of the
-subunit are minimal,
compared with those of the module M4.

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Fig. 4.
A, urea-induced denaturation curves for
hemoglobin ( ), -subunit ( ), -subunit ( ),
 (M4)-subunit ( ) and  (PM3)-subunit ( ). Molecular
ellipticities in the native and completely denatured states are
normalized to 0 and 1, respectively. Experimental conditions were as
follows: 20 mM Tris, 0.1 M NaCl, 5 mM NaCN, pH 7.4, at 290 K. Sample concentration was 5 µM on the heme basis. B, relationship between
G and [urea] for hemoglobin ( ), -subunit ( ),
-subunit ( ),  (M4)-subunit ( ), and  (PM3)-subunit
( ). They were calculated from the respective urea denaturation
curves in A.
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Table I
Parameters of urea denaturation for tetrameric Hb, isolated -,
-,  (PM3)-, and  (M4)-subunits
GH2O and
murea were determined by fitting the
G-[urea] relations to Equation 3.
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Association Properties--
However, the association property of
the pseudo-module substituted globin, 
(PM3)-subunit, clearly
differs from that of the wild-type
-subunit. Fig.
5A illustrates gel
chromatogram of the carbonmonoxy chimeric globins in the presence and
absence of the native subunits, and the centroid elution volumes of the
samples as a function of protein concentration (16, 17) are shown in
Fig. 5B. Under the condition employed here, the mixture of native
- and
-subunits forms a tetramer, whereas the isolated
-subunit remains in a monomer (17). The wild-type
-subunit is in
the equilibrium between monomers and tetramers (17). The position of
the elution peak for the 
(PM3)-subunit is also between those of a
tetramer and a monomer, but the peak position at 22.5 ml was
significantly deviated from that of the wild-type
-subunit at 22.0 ml. Since this elution peak was independent of the sample concentration
from 5 to 80 µM (Fig. 5B), the

(PM3)-subunit forms a stable homodimer, not an equilibrium state
between tetramers and dimers or monomers.

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Fig. 5.
A, chromatography of carbonmonoxy form
of the  (PM3)-subunit on a Sephacryl S-200 HR column. Experimental
conditions were as follows: 50 mM Tris, 0.1 M
NaCl, pH 7.4, at 277 K. Sample concentration was 20 µM on
the heme basis. B, centroid elution volumes of hemoglobin,
the isolated  (PM3)-subunit, and the mixture of the - and
 (PM3)-subunits as a function of protein concentration. Symbols
correspond to hemoglobin ( ),  (PM3)-subunit ( ), and the
mixture of the - and  (PM3)-subunits ( ). Experimental
conditions were as follows: 50 mM Tris, 0.1 M
NaCl, pH 7.4, at 277 K.
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In the chromatogram for the mixture of the 
(PM3)- and
-subunits, two peaks were observed, each of which coincides with the
peak for the isolated 
(PM3)- and
-subunits, indicating no
association of the 
(PM3)-subunit with the
-subunit. On the other hand, the peak for the mixture of the 
(PM3)- and
-subunits showed a single broad peak, and the elution pattern for
the mixture was not a simple addition of those of the corresponding
isolated subunits. These elution patterns imply that the

(PM3)-subunit preferentially binds to the
-subunit, not to the
-subunit, and the association property of the 
(PM3)-subunit
corresponds to that of the
-subunit as the case for the

(M4)-subunit (4). It should be noted here that the elution peak
for the mixture was detected at the middle of those for tetrameric
native Hb A and dimeric 
(PM3)-subunits, suggesting that the
complex of the 
(PM3)- and
-subunits is in the
equilibrium between a heterodimer [
(PM3)]
and a
heterotetramer [
(PM3)2]
2. As shown
in Fig. 5B, the elution peak for the mixture of the

(PM3)- and
-subunits depends on the protein concentration, and
the fitting curve of the mixture shifts to the right side, compared
with that of native hemoglobin tetramer, indicating that the
dissociation into dimers was enhanced in the complex of the

(PM3)- and
-subunits. The tetramer-dimer dissociation
constants, KD, were estimated as 1.4 (16) and 11 µM for Hb A and the complex of the 
(PM3)- and
-subunits, respectively.
Subunit Interface Structures--
To gain further insights into
the subunit interface structure for the pseudo-module substituted
globins and its complex of the 
(PM3)- and
-subunits, we have
measured the 1H NMR spectra in the hydrogen-bonded proton
region for the carbonmonoxy and deoxy forms (Fig.
6, A and B).
Although no exchangeable proton signals were observed in the downfield
region from 10 to 15 ppm for the isolated carbonmonoxy
-subunits, a
broad proton signal at 12.4 ppm was detected for the

(PM3)-subunit. This peak disappeared in 100% D2O
(data not shown). Such an exchangeable proton signal in the downfield
region was also encountered for carbonmonoxy Hb A, which has been
assigned to the hydrogen bonds in the subunit interface (26-28).
Similarly, the resonance at 12.4 ppm can be assignable to the hydrogen
bond at the subunit interfaces of the homodimer,
[
(PM3)]2. In the presence of the native
-subunit, another exchangeable proton resonance appeared at 10.4 ppm
(Fig. 6A), which would also originate from a hydrogen-bonded
proton in the subunit interface of the complex of the 
(PM3)- and
-subunits.

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Fig. 6.
A, NMR spectra in the hydrogen-bonded
proton region for carbon monoxygenated subunits. Proton resonance peaks
for Hb at 10.4, 11.9, and 12.8 ppm have been assigned to the
hydrogen-bonded protons between Asp-94 1 and
Asn-102 2 (27), His-103 1 and
Asn-108 1 (28), and Asp-126 1 and
Tyr-35 1, respectively (28). An asterisk
represents a novel resonance peak which appears upon the mixture of the
- and  (PM3)-subunits. Experimental conditions were as follows:
50 mM sodium phosphate, 0.1 M NaCl, pH 7.4, at
290 K. Sample concentration was 600 µM on the heme basis.
B, NMR spectra in the hydrogen-bonded proton region for
deoxygenated subunits. Proton resonance peaks for Hb at 11.0, 12.2, 12.9, and 13.9 ppm have been assigned to the hydrogen-bonded protons
between Asp-94 1 and Trp-37 2 (27, 30),
His-103 and Asn-108 (28), Asp-126 and Tyr-35 (28), and
Tyr-42 1 and Asn-99 2, respectively (27).
An asterisk represents a novel resonance peak which appears
upon the mixture of the - and  (PM3)-subunits. Experimental
conditions were as in A.
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Structural alterations in the subunit interface by the pseudo-module
substitution are also evident in the NMR spectra of the deoxygenated
state of the chimeric globin. By the dissociation of the ligands from
Hb A, the rearrangements in the
1-
2 subunit interface are
induced (29), which is reflected in the NMR spectra in the downfield
region. For deoxy-Hb A, the characteristic T-state marker resonances
were observed at 13.9 and 11.0 ppm (Fig. 6B) (27, 30),
whereas in the 
(PM3)-subunit, these marker signals were not
detected and a new peak appeared at 11.3 ppm by deoxygenation. Although
the appearance of the resonance at 11.3 ppm by deoxygenation suggests
the rearrangements in the hydrogen bonds at the subunit interface in
the pseudo-module substituted globin, its spectral pattern is quite
different from that of hemoglobin. In addition to the resonance peaks
at 11.4 and 12.4 ppm, the complex of the 
(PM3)- and
-subunits
in the deoxy state exhibited an exchangeable proton signal at 10.7 ppm
(Fig. 6B), which was not observed for the isolated
deoxygenated subunits. The spectral features for the complex are still
quite different between the carbonmonoxy and deoxy states, but the
resonance positions for the complex are not identical with those for
hemoglobin. This implies that the quaternary structural changes
accompanied by deoxygenation for the complex would not fully
correspond to those for native Hb A.
Heme Environmental Structures--
The structural perturbation was
also manifested in the heme environmental structure, as revealed by
1H NMR spectra of the carbonmonoxy and deoxy form of the
chimeric globins (Figs. 7 and
8). In the carbonmonoxy isolated
subunits, a peak from the
1-methyl proton of Val(E11)
appeared at
2.0 and
2.2 ppm for the
- and
-subunit,
respectively, which has served as a marker for the tertiary structure
in the heme vicinity (31, 32). The corresponding signal for the

(PM3)-subunit was detected at the same position as that of the
native
-subunit, implying that the heme environmental structure near
Val(E11) residue was not so perturbed by the pseudo-module substitution
(33). However, the resonance peak for the 
(PM3)-subunit is
significantly broadened and
asymmetric,3 suggesting
conformational changes in the heme vicinity upon the pseudo-module
substitution. Such conformational changes in the heme distal site are
supported by the spectral changes in the spectrum between
0.5 and
1.4 ppm.

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Fig. 7.
Proton NMR spectra (500 MHz) for carbon
monoxygenated subunits. Experimental conditions were as follows:
50 mM sodium phosphate, 0.1 M NaCl, pH 7.4, at
290 K. Sample concentration was 600 µM on the heme basis.
The resonance peak around 2.0 ppm has been assigned to 1-methyl
protons of Val(E11). The signals for the -subunit at 0.9, 0.7,
and 0.6 ppm are from the 2-methyl of Leu(B10),
1-methyl of Leu(FG3), and 1-methyl
protons of Leu(B10) (53). The signals for the -subunit at 1.2,
1.0, and 0.8 ppm are from the 1- and
2-methyls of Leu(H19) and 2-methyl of
Leu(B10) (54).
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Fig. 8.
Proton NMR spectra (500 MHz) for deoxygenated
subunits. Experimental conditions were as follows: 50 mM sodium phosphate, 0.1 M NaCl, pH 7.4, at 290 K. Sample concentration was 600 µM on the heme basis.
I, hyperfine-shifted proton resonances of proximal His
N -H. II, hyperfine-shifted proton resonances
of heme methyl groups.
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Here, noteworthy is that the spectral pattern for the complex of the

(PM3)- and
-subunits is a simple addition of that for the
corresponding isolated subunits, regardless of rather stable complex
formation of these two subunits. The association of the 
(PM3)-
and
-subunits would not affect the heme distal structure of the
counterpart subunit, which is in sharp contrast to the complex
formation of the native
- and
-subunits.
Fig. 8 shows the 1H NMR spectra for the deoxy state of the
chimeric and native globins. In the NMR spectra of the isolated native
- and wild-type
-subunits, a hyperfine-shifted exchangeable proton resonance was observed at 78 and 88 ppm in the far downfield hyperfine-shifted region (Fig. 8I), respectively, which had
been assigned to the N
-H proton of the proximal
histidine (F8) (34, 35). A couple of hyperfine-shifted resonances in
the region between 12 and 28 ppm (Fig. 8II) originated from
the protons of heme peripheral groups including heme methyl groups
(36). By the substitution of the pseudo-module PM3 in the
-subunit,
the resonance position of the proximal N
-H proton was
obviously shifted to the position for the
-subunit. The spectral
pattern for the resonances of the heme peripheral group is also highly
perturbed by the substitution of the pseudo-module PM3. These prominent
spectral changes suggest that the substitution of the pseudo-module PM3
alters the heme proximal structure of the
-subunit and converts the
coordination structure of the
-subunit into that of the
-subunit.
On the other hand, the substitution of the module M4 induced a much
smaller upfield shift for the resonance of the proximal histidine
N
-H,4 although
the spectral pattern for the heme peripheral groups are rather close to
that of the pseudo-module substituted subunit.
In addition to the isolated native and chimeric subunits, we have
measured the NMR spectra for the complex of the 
(PM3)- and
-subunits in the deoxy state. As shown in Fig. 8, native hemoglobin
subunits show a large upfield shift of the proximal histidyl
N
-H proton by formation of the
2
2 tetramer, indicating that significant structural changes are induced by the tetramer formation of the native
subunits. The resonance positions of the heme peripheral groups in the
NMR spectra of tetrameric hemoglobin are quite different from those of
the two isolated subunits, supporting the structural changes by
formation of the functional tetrameric hemoglobin (36). However, such
remarkable spectral changes were not accompanied by the association of
the 
(PM3)- and
-subunits. Only a slight downfield shift of the
proximal histidyl N
-H proton resonance was detected for
the 
(PM3) subunit. Such a small shift for the resonance positions
of the proximal histidines implies that the subunit association of the

(PM3)- and
-subunits does not induce the large structural
rearrangements around their heme vicinity.
Oxygen Binding Property of the 
(PM3)-Subunit--
To
evaluate the effects of the pseudo-module substitution on the oxygen
affinity and cooperativity for the oxygen binding of globins, oxygen
equilibrium curves for the chimeric and native globins were examined.
Fig. 9A delineates the
oxygenation curves, expressed by saturation versus log
P plots, and the Hill plots for the isolated subunits and
their mixtures are illustrated in Fig. 9B. The
P50 value of the 
(PM3)-subunit was
estimated to be 1.37 mm Hg, corresponding to the lower affinity than
that for the isolated wild type
-subunit (Table
II).

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Fig. 9.
A, Y versus log P
plots of oxygen equilibrium curves. B Hill plots.
Symbols correspond to hemoglobin ( ), -subunit ( ),
-subunit ( ),  (PM3)-subunit ( ), and the mixture of the
- and  (PM3)-subunits ( ). Experimental conditions were as
follows: 50 mM Tris, 0.1 M NaCl, pH 8.4, at
25 °C. Sample concentration was 60 µM on the heme
basis.
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Table II
Oxygen equilibrium parameters for tetrameric Hb, isolated -, -,
 (PM3)-subunits and mixture of the - and  (PM3)-subunits
Experimental conditions are listed in Fig. 9.
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For the complex of the
- and 
(PM3)-subunits, the
P50 value is 0.97 mm Hg, which is middle of
those of the isolated subunits. Since the oxygen curve for the complex
of the
- and 
(PM3)-subunits was not biphasic, the difference
of the oxygen affinities of 
(PM3)- and
-subunits in the
complex are indistinguishable (37). The nmax
value for the complex is unity, implying that it does not show any
cooperative oxygen binding as the case for the isolated chimeric and
native
- and
-subunits (Table II).
 |
DISCUSSION |
Effects of Pseudo-module Substitution on Structure and Function of
Globins--
As illustrated in the CD spectra, the pseudo-module
substituted subunit, 
(PM3)-subunit, can be folded to a stable
globin structure as observed for native globins. These minimal effects of the pseudo-module substitution on the protein structure were also
supported by the urea denaturation curve for the chimeric subunits
(Table I). The free energy change
(
GH2O) for
the denaturation of the 
(PM3)-subunit was minimally affected by
the substitution of the pseudo-module PM3. These findings clearly
indicate that the replacement of the pseudo-module PM3 in hemoglobin
-subunit with that of the
-subunit did not perturb the globin
structure of the native protein.
However, the effects of the pseudo-module substitution on the heme
environmental structure were prominent. The characteristic features of
the heme environment for the 
(PM3)-subunit is that the resonance
of the proximal N
-H proton for the deoxy

(PM3)-subunit was observed at the position for the isolated
-subunit. For the module substituted 
(M4)-subunit, the
coordination structure of the proximal His is still a
-like structure as shown in Fig. 8I (4). Since the pseudo-module PM3 shares the amino acid residues from FG4 to H6 with the module M4,
this different coordination structure between the two chimeric subunits
would originate from the region from F1 to FG3 which is included in PM3
and not in M4. In fact, the amino acid sequence from F1 to FG3
constructs most parts of the heme proximal structure and includes the
proximal histidine, which strongly suggests that this segment regulates
the heme coordination structure.
Contrary to the coordination structure, the structure of the heme
distal site of the chimeric 
(PM3)-subunits is rather close to the
-subunit. The NMR resonance from methyl group of Val(E11) in the
carbonmonoxy form appeared at the position for the native
-subunit
as found for the module substituted 
(M4)-subunit, although minor
conformational changes around Val(E11) were detected in the NMR
spectrum. Based on the amino acid sequences for the
-subunit, the
distal cavity of globins is formed by the B, C, D, and E helices which
are not involved in the pseudo-module PM3 or module M4. Thus, we can
conclude that the substantial structural changes induced by the
pseudo-module PM3 substitution are rather localized in the proximal
site.
The subunit association is another property that depends on the
pseudo-module PM3. As Fig. 5A shows, the 
(PM3)-subunit
can associate with the
-subunit but not with the
-subunit, which also resembles that of the native
-subunit. Such a conversion of the
association property was also experienced for the module substituted
subunit, 
(M4)-subunit (4). As depicted in Fig. 2, the common
region in the pseudo-module PM3 and the module M4 is from FG4 to H6 in
the amino acid sequence, and these amino acid residues would play key
roles in the subunit association of hemoglobin. Inspection of the x-ray
crystal structure also supports the functional role of this region,
since most parts of the
1
1 subunit interface dominating subunit
assembly in hemoglobin consist of these residues as revealed in Fig.
1A.
In contrast to the effects of the PM3 substitution on the coordination
structure of the proximal histidine and on the association property of
the subunits, the oxygen binding properties for 
(PM3)-subunit are
not so simple to interpret. As found in Fig. 9, the isolated 
(PM3)-subunit shows a lower oxygen affinity than the isolated
-subunit. The NMR spectra of its deoxygenated state (Fig. 8) could
also support the lower oxygen affinity of the 
(PM3)-subunit; the
upfield bias of the proximal His N
-H resonance
corresponds to the increased strain of the iron-histidyl bond, which
would be responsible for the decreased oxygen affinity (35). However,
the oxygen affinity of the 
(PM3)-subunit is still lower than that
of the
-subunit in which the resonance position of the proximal
histidine N
-H is the same as that in the

(PM3)-subunit. As discussed in the extensive NMR studies on
natural mutant hemoglobins by Ho and co-workers (38, 39), the
correlation between the position of the hyperfine-shifted NMR signal
from the proximal histidine N
-H and the strain of the
iron-proximal histidine bond (38-41) is not simple, and the resonance
position is also dependent on the subunit interface structure, salt
bridges within the subunits, and conformation of the heme pockets as
well as on the strain imposed on the iron-proximal histidyl bond (38,
39). In the 
(PM3)-subunit, therefore, the amino acid replacements
by the pseudo-module substitution would affect those structural
factors, which might lead to the lower oxygen affinity.
On the other hand, the oxygen affinity of the complex of the

(PM3)- and
-subunits was much higher than that of Hb A, and allosteric cooperativity was not detected for the complex. As revealed
by the NMR spectrum for the hydrogen-bonded region, the hydrogen bonds
in the subunit interface of the complex are quite different from that
of native Hb A, and the spectral feature characteristic of the T-state
was missing for the complex. The NMR spectrum in the hyperfine-shifted
region at 12-28 ppm is also suggestive of absence of the specific
interactions between these two subunits (36). On the basis of x-ray
structural studies, two sliding contact regions, C2-CD1 and FG3-G7, are
essential for the allostericity in hemoglobin A (16, 42-48). However,
the C2-CD1 region of the 
(PM3)-subunit is derived from the
-subunit (Fig. 2). The incomplete adjustment of the
1
2 subunit
interface would reduce the subunit interactions in the complex of the
- and 
(PM3)-subunits, resulting in non-cooperative oxygen
binding.
Structural and Functional Significance of the Pseudo-module PM3 in
Globins--
In this study, we have shown that the substitution of the
structural segment other than the module can also produce a stable "chimeric" globin, which leads us to re-examine the structural and
functional significance of the modular structure in globins. Comparison
of the chimeric property of the 
(PM3)-subunits with that of the
native and the module substituted subunit can reinforce the structural
and functional significance of the pseudo-module PM3. One of the
possible reasons for formation of the stable and functional chimeric
globin by the pseudo-module substitution would be high structural
homology between the
- and
-subunits. Although the amino acid
sequence homology is not so high between the two subunits (~40%),
their globin structures are quite similar except for the deletion of
the D helix in the
-subunit. However, our previous study on the
module substituted 
(M4)-subunit, in which the module M4 of the
-subunit was implanted into the
-subunit, showed that the globin
structure of the 
(M4)-subunit is highly perturbed and
destabilized probably due to loss of the stable intramolecular helix
packing (5), concluding that the structural similarity is not enough to
produce a stable chimeric protein. It is thus likely that the PM3
substitution would not accompany substantial failures in the helix
packings. According to the previous study by Jennings and Wright (49),
the packing between the A and H helices is formed in the first step of
the folding process in apo-myoglobin, and these helices are crucial for
protein folding of globins. Since the pseudo-module PM3 contains only a
part of H helix as shown in Fig. 1B and most of the A and H
helices are derived from the parent
-subunit, the PM3 substitution
would not substantially perturb the helix packing.
The stable structure and functional conversion for the

(PM3)-subunit suggest that the pseudo-module PM3 might have some structural significance in the globin structure like the modules, which
leads us to re-examine the boundary of the module in globins. It should
be noted here that the modules in globins, which have been defined on
the basis of the diagonal plots of the inter-C
atom
distances for the main chain (1), are much larger in size than those in
other proteins (50). That is, the modules in globins consist of 30-40
amino acid residues (1), whereas the numbers of the amino acid residues
for the modules in most of other proteins are 10-25 (7, 50). Such a
large modular structure in globins may allow us to infer that the
current modules in globin are not minimal units for the structure and
function of globin and can be further divided into some
"sub"-modules. In fact, this speculation is strongly supported by
the recent revised module assignment with a centripetal profile (7)
which indicates that each of the modules in globins can be decomposed into the two "small" modules, and consequently, globins has eight small modules.5
Interestingly, the new identified module boundaries in the modules M3
and M4 almost coincide with the boundaries of the pseudo-module PM3. In
other words, the substitution of the pseudo-module PM3 might be a type
of module substitution. Although it is still premature to conclude that
the pseudo-module PM3 is a combination of the two small modules, it can
be safely said that the pseudo-module PM3 of globins has some
structural and functional significance.
In summary, the substitution of the pseudo-module PM3 which is
originally supposed to have no structural and functional significance can retain the structural stability comparable to the native globins. Moreover, the structural and functional properties of the PM3 substituted subunit were chimeric between the parent subunits. The
coordination structure of the proximal histidine and the association property for the 
(PM3)-subunit was rather close to that for the
-subunit, whereas the structure of the distal site was still
-subunit like. These findings strongly suggest that the
pseudo-module has some structural and functional significance. Together
with the structural and functional properties of the native and module substituted subunits, we can propose that the former half of the pseudo-module PM3 and the latter one would correspond to the
sub-modules regulating the heme proximal structure and subunit
assembly, respectively. To strengthen our argument on structural and
functional significance of the sub-module in globins, the preparation
and characterization of the novel chimeric globin subunits in which one
of these sub-modules is replaced by that of the partner subunit are now
in progress.
We thank Dr. Yoshinao Wada for fast atom
bombardment-mass spectrometry. We are indebted to the reviewers'
suggestions.