©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ultraviolet Resonance Raman Studies of Quaternary Structure of Hemoglobin Using a Tryptophan 37 Mutant (*)

(Received for publication, August 18, 1994; and in revised form, October 28, 1994)

Masako Nagai (1) Shoji Kaminaka(§) (4) Yuzo Ohba (2) Yukifumi Nagai (3) Yasuhisa Mizutani (4) Teizo Kitagawa (4)(¶)

From the  (1)Biological Laboratory, Kanazawa University School of Allied Medical Professions, Kanazawa 920, the (2)Department of Clinical Science, Yamaguchi University School of Medicine, Ube 755, the (3)Biological Laboratory, Fukui Medical School, Fukui 910-11, and the (4)Institute for Molecular Science, Okazaki National Research Institutes, Okazaki 444, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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-beta37 at the alpha(1)beta(2) interface due to the quaternary structure transition, the UVRR spectra of Hb A were compared with the corresponding spectra of Hb Hirose (Trp-beta37 Ser). A difference spectrum between deoxyHb A and deoxyHb Hirose showed only Trp resonance Raman (RR) bands, which were reasonably ascribed to Trp-beta37 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-beta37 in deoxyHb A formed a strong hydrogen bond at the N(1)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-beta37 with the small portion of remaining changes coming from Trp-beta15, demonstrating that Trp-beta37 plays a pivotal role in the quaternary structural change in Hb A.


INTRODUCTION

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 alpha(1) subunit to the heme of the beta(2) subunit (or from the heme of the beta(1) subunit to the alpha(2) 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 alpha(1)beta(2) interface are essential to trigger the cooperativity(2) . ^1H NMR studies have suggested that ligation-induced conformational changes are propagated from the proximal His residue of the alpha(1) heme to the heme vicinity of the beta(2) subunit through alteration of the subunit arrangements at the alpha(1)beta(2) (or alpha(2)beta(1)) interface(7) . However, there is still no direct information about the rearrangements of the alpha(1)beta(2) 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 alpha(1)beta(2) interface upon the quaternary structure change. They ascribed the ligation-induced change of a Trp band around 1550 cm (W3) to Trp-beta37 on the basis of the spectrum of Hb Rothschild (Trp-beta37 Arg) and ascribed the ligation-induced changes of Tyr bands to Tyr-alpha42(14) , although their interpretation of the Tyr bands in terms of a change of hydrogen bonding of Tyr-alpha42 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-beta37 forms a hydrogen bond through the indole N(1)H proton with the carboxylate of Asp-alpha94, which has been thought to be one of the most important hydrogen bonds for stabilizing the T-state(17) . Hb Hirose is another Trp-beta37 mutant, in which Trp-beta37 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-beta37 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.


EXPERIMENTAL PROCEDURES

Hemoglobins

Hb A was prepared from fresh human blood in the following way. The blood cells were separated by centrifugation in 0.9% NaCl solution and were hemolyzed by adding distilled water. After membrane fragments were centrifuged out, oxyHb A was purified from the supernatant by a preparative isoelectric focusing electrophoresis on a Sephadex G-75 superfine gel flat bed. The flat bed was prepared by pouring 100 ml of 5% Sephadex G-75 superfine slurry containing 5% Ampholine (4 ml, pH 6-8 and 1 ml, pH 7-9) into the tray (10.5 times 24 cm^2, depth = 0.5 cm) followed by evaporation of excess water. Hb Hirose was purified from patient hemolysate by preparative isoelectric focusing electrophoresis on a Sephadex G-75 superfine gel flat bed containing 5% Ampholine, pH 7-8. Electrophoresis was carried out for 10 h at 8 °C. Hb A was focused on pI 7.34, well separated from Hb A(2) (pI = 7.76) and other minor components. Hb Hirose was focused on pI 7.52.

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(2). Just before the measurement, Na(2)SO(4) 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) .

Reagents

Inositol hexaphosphate (IHP) (^1)was purchased from Sigma, Ampholine solution was from Pharmacia Biotech Inc., and sodium sulfate (Suprapur) was from Merck.

UVRR Measurement

The UVRR spectra were excited by a XeCl excimer laser-pumped dye laser (EMG103MSC and FL2002, Lambda Physik), dispersed with an asymmetric double monochrometer (Spex 1403) in which the gratings in the first and second dispersion steps are 2400 grooves/mm (holographic) and 1200 grooves/mm (machine-ruled, 500 nm blaze), respectively, and detected by an intensified photodiode array (PC-IMD/C5222-0110G)(21) . The 308-nm XeCl excimer laser was operated at 100 Hz to pump coumarin 480 laser dye, and the 470-nm output was frequency-doubled with a beta-BaB(2)O(4) crystal to generate 235-nm pulses. The Raman excitation light (3-4 mJ/cm^2) was introduced to the sample with a rectangular prism from the front of the bottom. The spinning cell was moved vertically 1 mm for every spectrum (every 5 min) to shift the laser illumination spot on the sample. Details of this sample illuminating system are described elsewhere. (^2)The temperature of the sample solution was kept at 10 °C by flashing with cooled N(2) gas against the cell. The scattered light was collected with a Cassegrainian optics with f/1.1.

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.


RESULTS

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-beta37 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(4) 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 Na(2)SO(4). 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(4).



These Trp bands arise solely from the two Trp residues, alpha14 and beta15, reflecting their environments. The frequency of indole ring vibration, W3, is known to change as a function of the torsional angle () of the C(2)-C(3)-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-alpha14 and Trp-beta15 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-alpha14 and/or Trp-beta15 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-beta37 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(4).



The W3 frequency (1548 cm) indicates that the angle of Trp-beta37 is 90°(23) . The Fermi doublet is thought to reflect the hydrophobicity of the environments around the indole ring of Trp-beta37; 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-beta37 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(1)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-beta37 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-beta37 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-beta37 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 (circle) and oxyHb A (bullet) with and without IHP. 150 µl of Hb (1 mM in heme) solution was applied to a 1 times 87 cm column of Sephadex G-75 equilibrated with 0.05 M phosphate buffer, pH 7.0, containing 0.2 M Na(2)SO(4). 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(r) 45,000; chymotrypsin, M(r) 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(4).



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-beta37.


DISCUSSION

The Role of Trp-beta37 in the Quaternary Structural Changes of Hemoglobin

On the basis of x-ray crystallographic analysis, Baldwin and Chothia (2) have suggested that ligation-induced rearrangement of the alpha(1)beta(2) interface is an essential component of the cooperative mechanism. There are two important contacts, that is the contact between alpha(1)FG and beta(2)C (and between alpha(2)FG and beta(1)C) and the contact between alpha(1)C and beta(2)FG (and alpha(2)C and beta(1)FG). The former is called the ``flexible joint'' because this region allows the quaternary structural change to occur. The latter contact is designated as a ``switch'' because the side chains of residues in this region take two alternative positions depending upon the quaternary structure. The x-ray study (2) pointed out the importance of the hydrogen bonds between Tyr-alpha42 and Asp-beta99 in the switch region and between Trp-beta37 and Asp-alpha94 in the flexible joint region, which have been thought to be important for stabilizing the T-structure of deoxyHb A. These hydrogen bonds are broken upon ligation. ^1H NMR studies demonstrated that these hydrogen bonds were actually present in deoxyHb A(26, 27, 28) . The resonances at 9.4 and 6.4 ppm, which are characteristic of the T-state, have been assigned to the OH proton of Tyr-alpha42 hydrogen-bonded to Asp-beta99 and the N(1)H-proton of Trp-beta37 hydrogen-bonded to Asp-alpha94, respectively(26, 27, 28) . These ^1H NMR signals disappear upon ligation(26) . In the present UVRR study, we could selectively identify the RR spectral feature of Trp-beta37 of Hb A from the Hb A - Hb Hirose difference spectrum (Fig. 4b). Fig. 7compares the UVRR spectrum of Trp-beta37 in deoxyHb A with that of free tryptophan in water. Free tryptophan gave UVRR bands at 1619.5 (W1), 1552 (W3), 1360 and 1343 (W7, Fermi doublet), 1011 (W16), and 877 cm (W17). There are great differences between Trp-beta37 in deoxyHb A and free tryptophan with respect to both the intensity and the frequency of RR bands. The intensity ratio of the Fermi doublet indicates that Trp-beta37 is placed in hydrophobic environments in deoxyHb A(24) . The intensity of the 873 cm band (W17) suggests that Trp-beta37 makes a strong hydrogen bond in the deoxy-form (25) . These characteristics of Trp-beta37 seem to disappear upon ligation (Fig. 4d). On the other hand, ligation does not alter the angle (90°) of Trp-beta37, because the W3 frequency that is sensitive to the angle (23) remained unchanged upon ligation. The torsional angle of Trp-beta37 deduced from the frequency- correlation (23) is in agreement with the results of x-ray analysis: 92° in oxyHb A (5) and 94° in deoxyHb A(3) .


Figure 7: Comparison of UVRR spectrum of Trp-beta37 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(2)SO(4). The spectrum of Trp-beta37 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(4).



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-beta37 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, alpha140, alpha42, and beta35 at alpha(1)beta(2) interface, which are considered possible partners for stacking with Trp-beta37. All these Tyr residues are located within 10 Å from Trp-beta37 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-beta37 of deoxyHb A (A) and Arg-beta37 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).



Conformation of Tyr Residues in Hb Hirose

The present results show that, although Tyr residues of Hb Hirose can adopt the same conformation as those of Hb A in deoxy-form, they take different orientations from those of Hb A in oxy-form. It is evident from a comparison of the UVRR spectrum of oxyHb Hirose with that of oxyHb A that the benzene ring C=C stretch (Y8a) greatly diminishes in oxyHb Hirose, whereas ring C-C (ext) stretch (Y7a) and C-H and C-O-H bending modes (Y9a) show only small changes. Because the RR intensity of the Y8a band of Tyr excited at 229 nm is reported to increase in nonpolar environments(29) , the observed decrease in the intensity of Y8a band of oxyHb Hirose means that at least 1 among 6 Tyr residues is exposed to hydrophilic environments in oxyHb Hirose.

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 alpha and beta subunits. In the R-state, Tyr-alpha140 maintains its intrasubunit hydrogen bond with Val-alpha93. In the transition from the R-state to the R2-state, the C terminus of the alpha subunit becomes more extended as Tyr-alpha140 moves away from Val-alpha93. 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 alpha(1)beta(2) 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-beta37 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 alpha(1)beta(2) interface. Its structure around the Arg-beta37 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-beta35, Tyr-alpha42, and Tyr-alpha140) 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.

Possibility of Conformational Changes of Trp-alpha14 and Trp-beta15

Although most ligation-induced changes of Trp bands for Hb A disappeared in Hb Hirose, small but distinct changes were detected for Trp bands in the (deoxy - oxy) difference spectrum (Fig. 3), indicating that the remaining two Trp residues in Hb Hirose are also involved in those changes. Using x-ray crystallographic coordinates obtained from the Protein Data Bank, we compared the environments around Trp-alpha14 and Trp-beta15 in COHb A (32) and in deoxyHb A(3) . Trp-alpha14 and Trp-beta15 are located on the A helices of the respective chains, and the indole rings of these residues interact with the E helices by donating hydrogen bonds to the OH groups of Thr-alpha67 and Ser-beta72, respectively. These hydrogen bonds are present in both COHb and deoxyHb A, and, accordingly, it is reasonable that UVRR spectral changes of the two Trp residues upon oxygenation are very small. However, there are appreciable differences between alpha14 and beta15 residues. As illustrated in Fig. 9, ligand binding induces a big movement of the side chain of Phe-beta71 against Trp-beta15; the benzene ring of Phe-beta71 faces Trp-beta15 in COHb A, whereas it turns sideways so that its plane is almost perpendicular to that of Trp-beta15 in deoxyHb A. This movement occurs because the Phe-beta71 is located within 4 Å from the heme methyl groups in the ligated form(1) . According to the x-ray study by Silva et al.(30) , Phe-beta71 in the R2-state adopts the same orientation as that in the R-state(5) . The orientation of Phe-beta71 in deoxyHb Rothschild (31) is the same as that in deoxyHb A(3) . Accordingly, the Phe-beta71 in Hb Hirose is also expected to adopt an orientation similar to that in Hb A. Such a change in a nearby aromatic amino acid residue does not occur to Trp-alpha14. Consequently, it is very likely that Trp-beta15 is responsible for the remaining 30% of changes of Trp bands upon ligation that have been identified with Hb Hirose. This change is caused by a conformation change of Phe-beta71.


Figure 9: Orientations of Phe-beta71 in the beta 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.




FOOTNOTES

(^1)
The abbreviations used are: IHP, inositol hexaphosphate; UVRR, ultraviolet resonance Raman; RR, resonance Raman.

(^2)
S. Kaminaka and T. Kitagawa, Appl. Spectrosc., in press.

*
This work was supported in part by Grants-in-aid for Scientific Research 0520920 and 05070116 from the Ministry of Education, Science, and Culture, Japan (to M. N.), by Grant 04NP0301 from the New Program (to T. K.), and by the Joint Program of the Institute for Molecular Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Virology, Kurume University School of Medicine, Asahi-machi 67, Kurume 830, Japan.

To whom correspondence should be addressed: Institute for Molecular Science, Okazaki National Research Institutes, Myodaiji, Okazaki, 444 Japan. Tel.: 81-564-55-7340; Fax: 81-564-55-4639.


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