(Received for publication, August 18, 1994; and in revised form, October 28, 1994)
From the
Environmental changes of tyrosine and tryptophan residues of
hemoglobin (Hb) upon its T to R transition of quaternary structure were
investigated with ultraviolet resonance Raman (UVRR) spectroscopy
excited at 235 nm. DeoxyHb A (T-form) showed a UVRR spectrum distinctly
different from those of the ligated Hbs (R-form) including oxyHb, COHb,
and metHb A, whereas the ligated Hbs exhibited similar UVRR spectra
irrespective of the ligand species and the oxidation state of the heme.
To characterize the spectral change of Trp-37 at the
interface due to the quaternary
structure transition, the UVRR spectra of Hb A were compared with the
corresponding spectra of Hb Hirose (Trp-
37
Ser). A
difference spectrum between deoxyHb A and deoxyHb Hirose showed only
Trp resonance Raman (RR) bands, which were reasonably ascribed to
Trp-
37 in deoxyHb A. RR bands at 873 cm
(W17)
and at 1360 and 1343 cm
(W7, Fermi doublet)
indicated that the indole ring of Trp-
37 in deoxyHb A formed a
strong hydrogen bond at the N
H site in hydrophobic
environments. Tyr residues in deoxyHb Hirose seemed to be in the same
environments as those of deoxyHb A. In contrast, the difference
spectrum between Hb A and Hb Hirose in the ligated state displayed
peaks for RR bands of both Trp and Tyr. The difference spectra were
unaltered by the addition of 5 mM inositol hexaphosphate. This
means that the differences were not caused by the tetramer to dimer
dissociation but by a conformation change within a tetramer. Comparison
of the Hb A-Hb Hirose difference spectra in the oxy and deoxy states
revealed that the oxygenation-induced changes of Trp RR bands arose
mostly from Trp-
37 with the small portion of remaining changes
coming from Trp-
15, demonstrating that Trp-
37 plays a pivotal
role in the quaternary structural change in Hb A.
Hemoglobin (Hb) is an allosteric protein showing cooperativity in oxygen binding, and the elucidation of a structural mechanism of allostery has been a major subject of Hb studies. X-ray crystallographic studies have shown that the deoxy and ligated forms of Hb possess two distinct quaternary structures named T- and R-states, respectively(1, 2, 3, 4, 5) . Cooperative oxygenation to Hb has been explained in terms of a reversible transition between the two quaternary structures upon partial ligation of the four hemes(6) .
Transmission of
ligation-induced conformational changes (tertiary structural changes)
from the heme of the subunit to the heme of the
subunit (or from the heme of the
subunit to the
subunit) must be responsible for
the cooperative oxygen binding, although its pathway has not been
clarified yet. Comparisons of the atomic coordinates of deoxy and
ligated forms of Hb have indicated that ligation-induced rearrangements
of atoms at the
interface are
essential to trigger the cooperativity(2) .
H NMR
studies have suggested that ligation-induced conformational changes are
propagated from the proximal His residue of the
heme
to the heme vicinity of the
subunit through
alteration of the subunit arrangements at the
(or
) interface(7) . However,
there is still no direct information about the rearrangements of the
interface.
Ultraviolet excitation
of Raman scattering has recently attracted the attention of
biochemists, because this technique can selectively probe the molecular
vibrations of aromatic side chains of proteins beside the amido modes
of the main
chain(8, 9, 10, 11, 12, 13) .
Upon excitation at around 230-240 nm, resonance enhancements of
Raman intensity were observed for Trp and Tyr
residues(8, 9, 10, 11) . Rodgers et al.(14) and Jayaraman et al.(15) applied this technique to study the quaternary
structure of Hb and detected changes of Raman bands arising from Tyr
and Trp residues located at the interface upon the quaternary structure change. They ascribed the
ligation-induced change of a Trp band around 1550 cm
(W3) to Trp-
37 on the basis of the spectrum of Hb Rothschild
(Trp-
37
Arg) and ascribed the ligation-induced changes of
Tyr bands to Tyr-
42(14) , although their interpretation of
the Tyr bands in terms of a change of hydrogen bonding of Tyr-
42
is somewhat controversial considering a more recent UVRR study of metHb
fluoride by Asher and co-workers(16) . It seems quite important
to correlate the quaternary structure-induced UVRR spectral changes to
a status change of a specific residue unequivocally.
According to
x-ray crystallographic studies(2, 3) , Trp-37
forms a hydrogen bond through the indole N
H proton with the
carboxylate of Asp-
94, which has been thought to be one of the
most important hydrogen bonds for stabilizing the T-state(17) .
Hb Hirose is another Trp-
37 mutant, in which Trp-
37 is
replaced by Ser and is known to have high oxygen affinity and low
cooperativity (n = 1.5)(18, 19) . For
further characterization of the contribution from Trp-
37 to the
UVRR spectrum of Hb A, we examined the UVRR spectra of oxyHb, COHb, and
deoxyHb Hirose in a frequency region from 850 to 1700 cm
in comparison with those of Hb A.
Samples for the UVRR measurements were prepared as follows.
OxyHb samples were extensively dialyzed against 0.05 M phosphate buffer, pH 7.0, to remove carrier Ampholite and to
equilibrate with the same buffer, and approximately 0.1 ml of the
solution (400 µM in heme) was brought into a spinning cell
made of a synthetic quartz ESR tube (diameter = 5 mm). DeoxyHb
was prepared by adding sodium dithionite (1 mg/ml) to oxyHb in the
Raman cell after replacement of the inside air with N. Just
before the measurement, Na
SO
was added to the
samples with the final concentration of 0.2 M as an intensity
standard for Raman spectrum. The addition of sulfate did not cause any
apparent Raman spectral change for the states examined in this study,
although its use in Raman experiments was warned against due to
possible tertiary structure change(20) .
The spectral width
observed on one exposure was approximately 700 cm in
the present dispersion, and, therefore, recordings of UVRR spectra were
divided into two regions: a high frequency region from 1200-1700
cm
and a low frequency region from 800-1300
cm
. The two spectra were scaled by the intensity
ratio of the Trp W7 band (
1360 cm
) to the Tyr
Y9a band (
1180 cm
) of deoxyHb A on every
measurement of proteins and by the intensity ratio of the W7 (
1360
cm
) to W16 (
1011 cm
) bands
for free Trp solution. One spectrum is composed of the sum of 400
exposures, each exposure accumulating the data for 0.8 s. The Raman
spectra represented in the figures were derived from an average of
8-10 spectra. The Raman shifts were calibrated with cyclohexane.
Denaturation of the samples due to exposure to the laser light was carefully checked by inspecting the change in the visible absorption spectrum before and after the measurements of the UVRR spectrum. If some spectral changes were observed, the Raman spectrum was discarded. DeoxyHb, COHb, and metHb could be kept in the laser beam up to 1 h without apparent denaturation, whereas oxyHb was oxidized about 10% after 30 min of exposure to the beam. To keep the oxidation less than 5%, the oxyHb sample was replaced with a fresh sample after every two spectra (about 11 min), and the deoxyHb, COHb, and metHb samples were replaced after every four spectra (approximately every 22 min). Visible absorption measurements were carried out with a Hitachi 220S spectrophotometer.
Fig. 1compares the visible absorption spectra of the
oxyHb and deoxyHb Hirose with those of Hb A. As illustrated in the inset, the absorbance between 270 and 290 nm of Hb Hirose is
substantially diminished compared with that of Hb A due to replacement
of Trp-37 by Ser.
Figure 1: Absorption spectra of Hb A (A) and Hb Hirose (B). Absorption spectra of deoxyHb (- - -) and oxyHb (-) (70 µM in heme) in 0.05 M phosphate buffer, pH 7.0, were recorded with a Hitachi U-3210 spectrophotometer using a Thunberg type cell of a 2-mm light path. DeoxyHbs were prepared by adding sodium borohydride after removing the air from the cell. After the recording of the spectrum of deoxyHb, oxyHbs were obtained by introducing air into the cell. The inset shows the direct comparison of oxyHb A (-) and oxyHb Hirose(- - -) in an expanded scale.
Fig. 2shows the 235-nm excited UVRR
spectra of deoxy- (a), oxy- (b), CO- (d),
and met-forms (f) of Hb A and the deoxy-ligated difference
spectra (c, e, and g). The
982-cm band of SO
ions was used to normalize the spectrum for subtraction. In the
spectrum of deoxyHb A, RR bands of Tyr are seen at 1619.5 (Y8a), 1209
(Y7a), and 1180 cm
(Y9a) and have been assigned by
Rava and Spiro (22) . RR bands of Trp are observed at 1556
(W3), 1360 and 1343 (W7, combination of two out-of-plane modes), 1012
(W16), and 874 cm
(W17), and their vibrational modes
have been elucidated by Harada and Takeuchi(9) . The bands of
Trp around 1360 and 1340 cm
are called a Fermi
doublet, the relative intensity of which reflects the environments
around Trp residues(9) . As seen in the difference spectrum,
ligation to the heme iron yields detectable changes for these RR bands.
It is noted that the difference features of oxy- (c), CO- (e), and met-forms (g) bear close resemblance to each
other, indicating that the protein conformation of the oxy-, CO-, and
met-forms can be categorized as R-state irrespective of a ligand
species and an oxidation state of the heme, in agreement with the
conclusion from x-ray crystallography of Hb A(4, 5) .
Although the relative intensities of these RR bands of Hb A upon 235-nm
excitation are somewhat different from those upon 230-nm excitation,
the essential features of the spectra of deoxyHb A and COHb A in Fig. 2are in agreement with those reported by Rodgers et
al.(14) .
Figure 2:
235-nm excited UVRR spectra of deoxyHb A (a), oxyHb A (b), COHb A (d), and metHb A (f) and the difference spectra between deoxy- and oxy
-(c), deoxy- and CO (e), and deoxy- and met-forms (g). Hb samples are equilibrated with 0.05 M phosphate buffer, pH 7.0, containing 0.2 M NaSO
. Hb concentration was 400 µM in heme. Each spectrum is an average of 8 spectra. The ordinate
scale of the computer-subtracted deoxyHb - ligated Hb difference
spectrum is expanded by a factor of 2.
Fig. 3shows UVRR spectra of deoxyHb
Hirose (a), oxyHb Hirose (b), and their difference
(deoxy - oxy) (c). The Tyr bands of deoxyHb Hirose,
observed at 1208 (Y7a) and 1179 cm (Y9a), are
similar in both frequency and intensity to those of deoxyHb A, but the
band at 1618 cm
(Y8a) is slightly shifted to a lower
frequency than that of deoxyHb A. On the other hand, the Trp bands of
deoxyHb Hirose differ markedly from those of deoxyHb A in frequencies
and intensities. The RR bands of Trp are found at 1560 (W3), 1357 and
1338 (W7, Fermi doublet), and 1011 (W16) cm
, and all
of them exhibit distinct decreases in intensity (
70% of those for
deoxyHb A). A Raman band arising from W17 is not seen around 880
cm
.
Figure 3:
UVRR spectra of deoxyHb Hirose
(-, a) and oxyHb Hirose (- - -, b). Experimental conditions are the same as those in Fig. 2. Each spectrum is an average of 8 spectra. The ordinate
scale of the difference spectrum (c) is expanded by a factor
of 2. The asterisk denotes a Raman band of
SO.
These Trp bands arise solely from the two Trp
residues, 14 and
15, reflecting their environments. The
frequency of indole ring vibration, W3, is known to change as a
function of the torsional angle (
) of the
C
-C
-C
-C
linkage; W3 shifts to higher frequencies when the
angle increases. On the basis of the frequency-
correlation proposed by Miura et al.(23) , the
observed frequency suggests that the
angles of
Trp-
14 and Trp-
15 in deoxyHb Hirose are
120°. It is
evident in Fig. 3that oxygenation produces small but
discernible changes in the W3 and W16 bands for Hb Hirose, suggesting
that Trp-
14 and/or Trp-
15 also contribute to the
ligation-induced UVRR spectral changes of Hb A.
The ligation-induced
spectral changes are also recognizable for Tyr bands at 1618 (Y8a),
1208 (Y7a), and 1179 cm (Y9a). For Hb A the
frequency of Y8a was downshifted to 1618 cm
upon
ligation (Fig. 2), but Y8a of Hb Hirose exhibited no frequency
shift upon ligation. Y8a did exhibit an intensity decrease upon
ligation. On the other hand, Y7a and Y9a bands showed very small
changes upon ligation. Consequently, the deoxy - oxy difference
spectrum of Hb Hirose is distinct from the corresponding difference
spectrum of Hb A (Fig. 2c). These results indicate that
oxygenation induces conformational changes of Tyr residues in Hb Hirose
that are appreciably different from those of Hb A.
Fig. 4shows the UVRR spectra of Hb Hirose (a and c) and the Hb A-Hb Hirose difference spectra (b, d, and e). The difference spectrum between Hb A and
Hb Hirose in the deoxy state (b) exhibits clear peaks for Trp
RR bands but no discernible peaks for Tyr RR bands. A small peak
observed at 1624 cm is presumably attributable to
benzene ring mode (W1) of Trp but not to Y8a of Tyr, because other Tyr
bands are not recognized near 1200 cm
in the
difference spectrum. The absence of difference features for Tyr modes
means that all Tyr residues in deoxyHb Hirose take the same
conformation as those of deoxyHb A. In other words, all the features of
the difference spectrum are attributed to Trp-
37 in deoxyHb A.
Figure 4:
UVRR spectra of Hb Hirose (a and c) and the difference spectra between Hb A and Hb Hirose (b and d). a, deoxyHb Hirose; b,
difference between Hb A and Hb Hirose in the deoxy state; c,
oxyHb Hirose; d, difference between Hb A and Hb Hirose in the
oxy state; e, difference between spectrum b and
spectrum d. The ordinate scale of the difference spectrum is
expanded by a factor of 2. The asterisk denotes a Raman band
of SO.
The W3 frequency (1548 cm) indicates that the
angle of Trp-
37 is 90°(23) . The
Fermi doublet is thought to reflect the hydrophobicity of the
environments around the indole ring of Trp-
37; when the intensity
ratio of the 1360-cm
band to the
1340-cm
band is higher than 1.0, Trp is located in
hydrophobic environments and vice versa(24) . Because the
intensity ratio for Trp-
37 displayed in Fig. 4b is
higher than 1.0, this residue is considered to be placed in hydrophobic
environments inside the protein molecule. The frequency of the W17 band
is known to correlate with the strength of the hydrogen bond at the
N
H proton of the indole ring; a stronger hydrogen bond
induces a shift to a lower frequency with a concomitant increase in
intensity(25) . Accordingly, an intense peak at 873
cm
in Fig. 4b means that Trp-
37
forms a strong hydrogen bond in deoxyHb A.
In contrast, the
difference spectrum between Hb A and Hb Hirose in the oxy state
contains contributions from both Trp and Tyr residues. The raw UVRR
spectrum of oxyHb Hirose and the difference spectrum between oxyHb A
and oxyHb Hirose are shown in Fig. 4(c and d). The difference peaks of the Trp modes are seen at 1547
(W3), 1359 and 1345 (W7, Fermi doublet), and 1008 cm (W16), but the W17 band is very weak, indicating that, in oxyHb
A, Trp-
37 makes no hydrogen bond or a very weak one(25) .
In addition to these Trp bands, peaks of Tyr modes appear at 1621
(Y8a), 1207 (Y7a), and 1175 cm
(Y9a). The difference
spectrum between spectra b and d mainly indicates a
change of Trp-
37 upon oxygenation. Changes of the W3 and W16 bands
of Trp and the Y8a and Y9a bands of Tyr are discernible.
OxyHb Hirose tends to dissociate into dimers even at high concentrations, whereas deoxyHb Hirose stays on as a tetramer. Hence, difference peaks of Tyr bands in the Hb A - Hb Hirose difference spectrum for the oxy state might reflect the conformational change induced by the dissociation from tetramers to dimers. In order to clarify this argument, we examined the dissociation problem with gel chromatography. Sasaki et al.(19) showed that an allosteric effector, 2,3-diphosphoglycerate, could stabilize deoxyHb Hirose at a low concentration as a tetramer. Because IHP is known as a more potent allosteric effector than 2,3-diphosphoglycerate, we used IHP. Fig. 5shows gel filtration patterns of oxyHb A and oxyHb Hirose without (upper) and with (lower) IHP. In the absence of IHP, more than half of oxyHb Hirose was dissociated into dimers. However, in the presence of IHP, oxyHb Hirose was fractionated at the same position as oxyHb A tetramer, although the Hb concentration of the eluted solution was as dilute as one-tenth of the concentration used for UVRR measurement. These results demonstrate that IHP can effectively prevent oxyHb Hirose from dissociation.
Figure 5:
Sephadex G-75 gel filtration patterns of
oxyHb Hirose () and oxyHb A (
) with and without IHP. 150
µl of Hb (1 mM in heme) solution was applied to a 1
87 cm column of Sephadex G-75 equilibrated with 0.05 M phosphate buffer, pH 7.0, containing 0.2 M Na
SO
. Fractions (one fraction, 1.32 ml)
were collected, and their absorbances at 415 nm are plotted against the
fraction numbers. The column was calibrated using the following
proteins, whose elution positions are marked by arrows:
ovalbumin, M
45,000; chymotrypsin, M
27,000. The void volume was determined using
blue dextran. IHP was added to the sample and elution buffer solution
in a final concentration of 5 mM.
It was noticed that IHP accelerates the oxidation of Hb Hirose into the met-form during UVRR measurement. Because CO produces the R-state without promoting oxidation of the protein, we also examined the spectral difference between the deoxy- and CO-forms instead of the oxy-form. Fig. 6shows the difference spectra between Hb A and Hb Hirose in the deoxy- (b) and CO-bound forms (c) in the presence of 5 mM IHP. RR bands of both Tyr and Trp exhibit the difference peaks very similar to those in the absence of IHP shown in Fig. 4. We confirmed that the same change of RR spectrum as that for CO binding occurs to the oxygen binding in the presence of IHP. These observations exclude the possibility that the differences between the Tyr RR bands of Hb A and Hb Hirose in the oxy state are caused by the dissociation into dimers.
Figure 6:
UVRR spectra of Hb A (a and e) and the difference spectra between Hb A and Hb Hirose (b and c) in the presence of 5 mM IHP. a, deoxyHb A; b, difference in the deoxy state; c, difference in the CO-bound state; d, difference
between spectrum b and spectrum c; e, COHb
A. Each spectrum is an average of 10 spectra. Other experimental
conditions are the same as those in Fig. 2. The ordinate scale
of the difference spectrum is expanded by a factor of 2. The asterisk denotes a Raman band of
SO.
Although the Tyr difference RR bands
gave negative peaks in the deoxy (Hb A - Hb Hirose) - oxy
(Hb A - Hb Hirose) difference spectrum (Fig. 4e),
the difference features of the Trp bands bear a resemblance in both the
frequency and the intensity to those in the deoxy - oxy
difference spectrum of Hb A (Fig. 2c). The intensity
enhancement at 1011 cm accounts for approximately
70% of that in deoxyHb A, indicating that most of the oxygen-induced
spectral changes of Trp in Hb A arise from status changes of
Trp-
37.
Figure 7:
Comparison of UVRR spectrum of Trp-37
in deoxyHb A (b) with that of free tryptophan in water (a). Tryptophan (2.4 mM) was dissolved in water
containing 0.2 M Na
SO
. The spectrum of
Trp-
37 is obtained by subtracting the UVRR spectrum of deoxyHb
Hirose from that of deoxyHb A (Fig. 4c). The asterisk denotes a Raman band of
SO
.
We have also observed that the W3 and W16 RR bands of Trp are
greatly intensified upon the R T transition, in agreement with
Rodgers et al.(14) and Cho et
al.(16) . We attribute this to a stacking of Trp-
37
with a nearby aromatic amino acid residue in oxyHb A and an unstacking
in the deoxy state. Usually stacking results in hypochromism of the UV
absorption and thus in a reduction of UVRR intensity. Fig. 8A illustrates arrangements of the three nearby
Tyr residues,
140,
42, and
35 at
interface, which are considered
possible partners for stacking with Trp-
37. All these Tyr residues
are located within 10 Å from Trp-
37 and alter their
orientations upon ligation(2) , although we cannot specify the
Tyr residue responsible for the spectral change at this moment.
Figure 8:
Orientations of aromatic amino acid side
chains around Trp-37 of deoxyHb A (A) and Arg-
37 of
deoxyHb Rothschild (B). X-ray crystallographic coordinates are
taken from the Brookhaven Protein Data Bank: 4HHB (deoxyHb A) (3) and 1HBA (deoxyHb Rothschild)(31) . The figures
were generated with Chem3DPlus (Cambridge Scientific
Computing).
Recently Silva et al.(30) proposed the ``R2-state'' for the
quaternary structure of ligated Hb A, which is a stable form but is
located in a R T pathway. Their x-ray data for the R2-Hb A grown
in the low salt and acidic pH conditions indicate that major
differences between the ``R-state'' and
``R2-state'' exist in the conformation of C-terminal residues
of both
and
subunits. In the R-state, Tyr-
140
maintains its intrasubunit hydrogen bond with Val-
93. In the
transition from the R-state to the R2-state, the C terminus of the
subunit becomes more extended as Tyr-
140 moves away from
Val-
93. Silva et al.(30) also suggest that low
concentrations of inorganic anions or low pH may favor the R2-state and
that at least one mutation at the
interface stabilizes a quaternary structure very similar to the
R2-state. RR bands of Tyr in oxyHb Hirose are considerably different
from those of oxyHb A. Accordingly, the quaternary structure of oxyHb
Hirose might be stabilized in the ``R2-state.''
In
contrast, there is no perturbation of Tyr in deoxyHb Hirose compared
with deoxyHb A, because RR bands for Tyr in deoxyHb Hirose were the
same as those of deoxyHb A as shown in Fig. 4. Although x-ray
crystallographic data of deoxyHb Hirose are not available, the crystal
structure of deoxyHb Rothschild, in which the Trp-37 is replaced
by Arg, was recently reported by Kavanaugh et
al.(31) . It has been revealed that in deoxyHb Rothschild
the conformation of the switch region is almost conserved and that a
new anion binding site is generated in the
interface. Its structure around the Arg-
37 residue, obtained
from the Protein Data Bank, is reproduced in Fig. 8B for comparison with that of deoxyHb A (Fig. 8A)(3) . It is apparent from Fig. 8that the orientations of three nearby Tyr residues
(Tyr-
35, Tyr-
42, and Tyr-
140) in deoxyHb Rothschild
closely resemble those of deoxyHb A. If this is also applicable to Hb
Hirose, the UVRR results regarding the close similarity in the Tyr
spectra between deoxyHb A and deoxyHb Hirose are consistent with the
structure.
Figure 9:
Orientations of Phe-71 in the
subunit. X-ray crystallographic coordinates are taken from the
Brookhaven Protein Data Bank: 4HHB (deoxyHb A) (3) and 1HCO
(COHb A) (32) . The figures were generated with Chem3DPlus
(Cambridge Scientific Computing). Broken lines represent
hydrogen bonds.