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Changes of tyrosine and tryptophan residues in human hemoglobin by oxygen binding: near- and far-UV circular dichroism of isolated chains and recombined hemoglobin
Journal of Inorganic Biochemistry 82 (2000) 93–101 www.elsevier.nl / locate / jinorgbio
Changes of tyrosine and tryptophan residues in human hemoglobin by oxygen binding: near- and far-UV circular dichroism of isolated chains and recombined hemoglobin R. Li, Y. Nagai, M. Nagai* School of Health Sciences, Kanazawa University Faculty of Medicine, Kodatsuno 5 -11 -80, Kanazawa 920 -0942, Japan Received 7 March 2000; received in revised form 17 July 2000; accepted 19 July 2000
Abstract In order to assign the circular dichroism (CD) spectral change in the region between 280 and 300 nm of human adult hemoglobin (Hb A) upon the quaternary structure transition induced by oxygen binding, the near- and far-UV CD spectra of the isolated chains and the recombined hemoglobin were examined. Deoxygenation made the negative CD band at 290 nm of oxy-a chain deeper. On the other hand, positive CD bands of oxy-b chain at the 280|300 nm became negative upon deoxygenation. These changes were interpreted as being due to environmental alterations of tyrosine (Tyr) and / or tryptophan (Trp) perturbed by tertiary structural changes from the oxy to deoxy form in isolated chains, referring to the CD spectra of model compounds. From the difference between CD bands of the arithmetic mean of deoxy isolated chains and the CD band of deoxyHb tetramer, the contribution of tertiary structural change to the negative CD band of deoxyHb A at 287 nm was estimated to be 50%. This finding has revealed that the net contribution of quaternary structure transition to the negative band is 50%. In far-UV CD spectra, the environmental changes of aromatic residues upon the quaternary structure transition were also detected as a negative band at 225 nm. 2000 Elsevier Science B.V. All rights reserved. Keywords: Hemoglobin; Near-UV CD; Far-UV CD; Isolated chains; Recombined Hb
1. Introduction Human adult hemoglobin (Hb A), a well known allosteric protein, is a tetramer composed of two a and two b subunits. With an increase in the number of bound oxygens, the quaternary structure transition of Hb A occurs from the low-affinity deoxy, or T (tense) to the highaffinity oxy, or R (relaxed) form. This allosteric effect in Hb A has made it one of the most important proteins for studying the relation between structure and function. The a1b2 subunit interface is considered to play a pivotal role in the quaternary structure transition [1,2]. Aromatic residues such as a42Tyr, a140Tyr, b37Trp, and b145Tyr are located at the a1b2 subunit interface and undergo changes of environment with the quaternary structure transition from T to R [1,2]. All these aromatic residues remain invariant throughout the evolution of a and b chains [1], implying their indispensability for the mainte*Corresponding author. Tel.: 181-76-265-2581; fax: 181-76-2344360. E-mail address: [email protected] (M. Nagai).
nance of structure and function of Hb A. In fact, all mutant Hbs at these aromatic residues, recombinant Hb (rHb) (a42Tyr→His) [3], Hb Rouen (a140Tyr→His) [4], Hb Hirose (b37Trp→Ser) [5] and Hb Fort Gordon (b145Tyr→Asp) [6], have an increased oxygen affinity and decreased or no cooperativity. Circular dichroism (CD) is a sensitive physical technique for determining structures and monitoring structural changes of biomolecules. The far-UV CD spectra of proteins are highly sensitive toward protein structure, especially useful for determining the changes of protein secondary structure. On the other hand, the near-UV CD spectra of proteins reflect contributions of aromatic side chains and disulfide bonds [7]. The studies on CD spectra of Hb A have shown that both the tertiary structural change and the quaternary structure transition contribute to the spectral change upon oxygen binding [8–10] and that the CD band at the 280|290-nm region is ascribable to the aromatic residues [11–13]. It has been shown that the far-UV CD spectra in the 200|250-nm region also give information on aromatic residues [14]. Hb A shows a pronounced change of the CD spectra in
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the 280|300-nm region upon the T→R transition. The change of CD band in the 280|290-nm region from small positive peaks in oxyHb A to a distinct negative trough in deoxyHb A was suggested to be due to environmental alterations of Tyr or Trp residues located at the a1b2 subunit interface [11–13]. This spectral change has been thought to be due to b37Trp [15,16]. However, assignment of this spectral change to specific aromatic residues remains incomplete. Our recent CD study on Hb Hirose (b37Trp→Ser) has demonstrated that the contribution of b37Trp to the negative CD band in deoxyHb A is only 26%, indicating possible involvement of other aromatic residues at the a1b2 subunit interface [17]. We suspected that the environment of aromatic residues might be influenced by the tertiary structure change upon oxygen binding, resulting in the CD spectral changes in the 280| 300-nm region. Isolated a chains are monomeric with high oxygen affinity like myoglobin [18]. In contrast, isolated b chains aggregate to form a tetramer (b 4 ) without cooperativity [18]. Isolated chains enable us to distinguish the contribution of tertiary structural changes to the CD spectra on oxygen binding in the far- and near-UV region. In the present study, the near- and far-UV CD spectra of isolated chains and recombined Hb were examined to estimate the contribution of aromatic residues to the spectra due to the tertiary structural change, referring to the spectra of model compounds such as N-acetyltyrosineamide (NAc-Tyr-amide) and N-acetyltryptophanamide (NAc-Trp-amide).
2. Materials and methods
2.1. Isolation of a and b chains After fresh human blood cells were washed with 0.9% NaCl solution and hemolyzed, a five-fold excess of phydroxymercuribenzoate ( p-HMB) was added to hemoglobin solution containing 0.1 M NaCl and adjusted to pH 5.8, which was allowed to stand overnight at 48C [19,20]. a-HMB and b-HMB chains were separated by isoelectric focusing electrophoresis on a Sephadex G-75 superfine gel bed containing 5% ampholine, pH 3.5–9.5. The p-HMB of a and b chains were removed by adding 3 mg dithiothreitol (DTT) / ml in 0.1 M Tris–HCl buffer, pH 8.2, and passage through a Sephadex G-25 column. Regeneration of sulfhydryl (SH) groups of a and b chains was confirmed by SH-titration with p-chloromercuribenzoate. The concentration of hemoglobin was determined after conversion to pyridine hemochrome using emM (557 nm)534. The recombined Hb was prepared by mixing a chains and b chains at equimolar concentration just before the CD measurements.
2.2. CD measurements CD measurements were performed with a Jasco J-725
spectropolarimeter at 258C. Spectra were measured several times for each sample using different preparations, recorded as an average of 40 (far-UV region) or 20 scans (near-UV region). The solvent spectrum obtained under the same conditions was subtracted to obtain the actual sample spectrum. Hbs used were 100 mM (in heme) for the 240|340-nm region and 10 mM for the 200|250-nm region in 0.1 M phosphate buffer, pH 7.0, respectively. The path length of the cell used was 2 mm. The optical density of the Hb samples was maintained below 2.0 for CD measurements in all regions. DeoxyHb was prepared by adding small quantities of sodium dithionite powder to the oxyHb. As excess amounts of sodium dithionite used for deoxygenation interfered with the measurement of CD in the UV region by increasing the noise, we usually measured the CD spectra of the samples with OD less than 0.5 at 310 nm. (1)-10-Camphorsulfonic acid was used for the calibration of the instrument. CD spectra were expressed as molar CD, De (M 21 cm 21 ).
3. Results
3.1. Near-UV CD spectra of isolated chains and recombined Hb CD spectra of the isolated a and b chains in the near-UV region are shown in Fig. 1. As shown by Ueda et al. [8], a distinctive difference in positive band near 260 nm can be seen between the isolated chains. The ellipticity associated with the 260-nm band is strongly influenced by the attached ligand and, thereby, the spin state of the iron atom [21]. A large decrease of the band near 260 nm from oxy to deoxy form was attributed to changes of optical activity of the heme moiety in both a and b chains [15,16]. The CD spectrum in the 280|300-nm region is useful for monitoring the environmental changes of Tyr and Trp residues in the globin [7]. Upon deoxygenation of the a chain, the CD band at 290 nm became more negative and the positive ellipticity at 280 nm increased. On the other hand, the positive CD spectrum at the 280|300-nm region of the b chain became negative upon deoxygenation. These results suggest that the tertiary structural changes upon oxygen binding also induce change of local environment of Tyr and Trp residues in both a and b chains. In order to assess, upon oxygen binding, the above changes of CD spectra of isolated chains to Tyr and Trp residues, we determined CD spectra of NAc-Tyr-amide and NAc-Trp-amide as model compounds in water and in dioxane (Fig. 2). In water, NAc-Tyr-amide showed a negative CD band in the 270|290-nm region that remained negative but red-shifted in dioxane (less polar solvent). NAc-Trp-amide is distinctive in exhibiting two positive bands at 282 and 288 nm and a negative band at 298 nm in water, and changing into negative troughs at 287 and 295 nm in dioxane. Since NAc-Tyr-amide does not exhibit the CD band in the 290|300-nm region, changes of CD bands
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Fig. 1. Near-UV CD spectra of isolated a chains (A) and b chains (B). The dotted line refers to the oxy form and the solid one to deoxy form. The spectra shown here are the mean of two spectra (each spectrum was an average of 20 scans). The Hb solution was 100 mM (in heme) in 0.1 M phosphate buffer, pH 7.0.
Fig. 2. Near-UV CD spectra of N-acetyl-tyrosineamide (A) and N-acetyl-tryptophanamide (B). The dotted line refers to the spectra in water and the solid one to those in dioxane. The spectra are an average of 40 scans with 1 mM NAc-Tyr-amide or 0.33 mM NAc-Trp-amide in 1 cm cell.
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at the 290|300-nm region are ascribable to local environment alteration of Trp residues. On the other hand, two negative CD bands in the 270|290-nm region of NAc-Tyramide are characteristic of Tyr residues but obscured by those of Trp residues. In order to extract, upon oxygen binding, the change in CD bands due to local environment alterations of Tyr and Trp residues induced by tertiary structure changes in a and b chains, we examined the difference spectra (deoxy-minus-oxy) of a and b chains (Fig. 3). The difference spectrum of the a chain displayed a positive peak at 278 nm and a broad negative bands in the 285|310-nm region. In contrast, the b chain exhibited broad negative bands in the 280|300-nm region without distinct peak at 278 nm as in the a chain. These results indicate that oxygen binding in the a chain brings about local environment alteration of both Tyr and Trp residues due to tertiary structure change but in the b chains induces distinctively that of Trp. Fig. 4 shows the CD spectra of recombined Hb from equimolar a and b chains (solid lines) in comparison with that of the arithmetic mean of a and b chains (dotted lines). The CD spectra of the recombined Hb in the 250|270-nm region were very similar to the arithmetic mean of the isolated chains in both the oxy and deoxy form, indicating that the optical activity of the heme in the isolated chains was scarcely influenced by recombination to tetramer. In the 270|300-nm region, the difference between the arithmetic mean and recombined Hb was very Fig. 4. Comparison of CD spectra of the recombined Hb with the arithmetic mean of the spectra of a and b chains in the near-UV region. Recombined Hb was prepared by mixing a chain and b chain at equimolar concentration. The arithmetic mean was calculated from 1 / 2 (CD spectrum of a1CD spectrum of b). The dotted line refers to the arithmetic mean of a and b chains and the solid one to recombined Hb. The spectra shown here are the mean of two spectra (each spectrum was an average of 20 scans). The Hb solution was 100 mM (in heme) in 0.1 M phosphate buffer, pH 7.0.
Fig. 3. Deoxy-minus-oxy difference spectra of the isolated a and b chains in the near-UV region. The dotted line refers to the difference CD spectrum between deoxy-a chain minus oxy-a chain, and the solid one to that between deoxy-b and oxy-b chain.
small in the oxy form, but distinct in the deoxy form. Fig. 5 shows the same CD spectra in the 260|310-nm region as Fig. 4 and the difference spectra between the recombined Hb and the arithmetic mean of the isolated chains in an expanded scale. The spectra of the arithmetic mean of the a and b chains reflect only environmental conditions of the aromatic side chains of Tyr and Trp residues in the tertiary structure. But the effects of further changes of environment induced by tetramerization (quaternary structure) are added to the above spectra in recombined Hb. Therefore, difference spectra in both oxy (Fig. 5A) and deoxy (Fig. 5B) forms show the net contribution of quaternary structure to both CD spectra in the 270|300-nm region. The difference spectrum of the oxy form exhibited no apparent CD band. In contrast, the arithmetic mean of deoxy chains gave a negative CD band with a peak at 292 and a shoulder at 287 nm, but recombined Hb gave a single negative CD band at 287 nm and the depth was twice of the arithmetic mean.
R. Li et al. / Journal of Inorganic Biochemistry 82 (2000) 93 – 101
Fig. 5. Expanded CD spectra of recombined Hb and the arithmetic mean of a and b chains and difference between them in the near-UV region in oxy (A) and deoxy form (B). The dotted line refers to the arithmetic mean of the a and b chains and the thin solid line to recombined Hb. The bold solid line designates the difference spectrum (recombined Hb — the arithmetic mean of a and b chains). Experimental conditions were the same as in Fig. 4.
These results reveal that about half of negative CD band of deoxyHb A at 287 nm reflects the tertiary structure change and the other half is due to the quaternary structure transition upon the R→T transition. In other words, the contribution of the quaternary structure transition to the negative CD band at 287 nm is estimated to be 50%. The difference spectrum showed a broad band with peaks at 281 and 285 nm in addition to 287 nm. Judging from their band positions, Tyr residues seem to contribute to the former two peaks.
3.2. Far-UV CD spectra of isolated chains and recombined Hb The far-UV CD spectra give a measure of helical content in the secondary structure of proteins. The in-
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fluence of secondary structure on the CD spectra of hemoglobin was well illustrated by the dramatic changes that occurred upon heme binding to the apoprotein [22]. However, it was also reported that no significant difference was observed between isolated chains and native Hb [8]. Fig. 6 shows the far-UV CD spectra of a chain, b chain and recombined Hb. Oxygen binding did slightly influence the spectra of b chain but increased the intensity of the two negative CD bands at 209 and 222 nm of a chain. On the other hand, deoxygenation brought about a very small change of the CD band at 209 nm but caused an obvious increase in negative intensity at 222 nm in the recombined Hb. The observed change in CD spectra of recombined Hb was the same as that in the CD spectra of native Hb (not shown). The difference between reconstituted Hb and the arithmetic mean of isolated chains in oxy (dotted line) and deoxy (solid line) forms are shown in Fig. 7A. The difference in oxy form exhibited small negative bands at 209 and 222 nm, suggesting that helical content underwent a slight change by tetramerization. In contrast, a distinct negative band at 225 nm was detected in the deoxy form. Since a negative CD band at 209 nm is not observed in the difference spectrum of the deoxy form, it is unlikely that an increase in helical content accompanying by deoxygenation contributes to this band. Fig. 7A shows that the deoxy-minus-oxy difference in the CD band at 225 nm is 77 / M / cm. This difference is attributable to the quaternary structure change upon deoxygenation, as the spectra in Fig. 7A represent ‘recombined-minus-arithmetic mean’. On the other hand, the deoxy-minus-oxy difference of recombined Hb in the CD band at 225 nm which includes contribution of both tertiary and quaternary structure change is estimated to be approximately 60 / M / cm from Fig. 6C. In order to examine this discrepancy, we compared the deoxy-minus-oxy difference spectra of the isolated chains and recombined Hb (Fig. 7B). As shown in the figure, the deoxy-minus-oxy difference spectrum in the CD band at 225 nm reflecting the quaternary structure change is estimated to be 79 / M / cm, in good accordance with 77 / M / cm in Fig. 7A. We used 10 mM Hb (in heme) for far-UV CD measurements. As the tetramer–dimer dissociation constant have shown to be 2 mM [23], the majority of the Hb molecule is assumed to stay as tetramer under the present experimental conditions. Fig. 8 shows the far-UV CD spectra of model compounds of Tyr and Trp residues dissolved in either water or dioxane. The CD spectra of aromatic side chains are strongly influenced by the hydrophobicity of solvent. In particular, the positive peak at around 225 nm of both aromatic amino acids in water was greatly diminished upon exposing to hydrophobic environment in dioxane. The negative CD band at 225 nm in the difference spectrum between the recombined Hb and the arithmetic mean of isolated chains in deoxy form is probably attributable to environmental alterations of aromatic residues upon the R→T transition. These results suggest that aromatic
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Fig. 6. Far-UV CD spectra of isolated a chain (A), b chain (B) and their recombination (C). The dotted line refers to the oxy form and the thin solid one to the deoxy form. The bold solid line designates the difference spectrum (deoxy-oxy). The spectra shown here are an average of 40 scans. The Hb solution was 10 mM (in heme) in 0.1 M phosphate buffer, pH 7.0.
Fig. 7. Difference CD spectra between recombined Hb and the arithmetic mean of isolated subunits (recombined — the arithmetic mean) in oxy- and deoxy-forms (A) and the deoxy-minus-oxy difference spectra of the isolated chains and recombined Hb (B) in the far-UV region. (A) The dotted line refers to the oxy form and the solid one to the deoxy form. (B) The dotted line refers to the isolated chains (a), the solid one to the recombined Hb (b) and the dashed line (c) to the difference spectrum between (b)-spectrum minus (a)-spectrum.
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Fig. 8. Far-UV CD spectra of N-acetyl-tyrosineamide (A) and N-acetyl- tryptophanamide (B). The dotted line refers to the spectra in water and the solid one to those in dioxane. The spectra are an average of 40 scans with 1 mM NAc-Tyr-amide or 0.33 mM NAc-Trp-amide in 0.2-cm cell.
residues at the a1b2 subunit interface also contribute to the CD band at the far-UV region upon the quaternary structure transition.
4. Discussion
4.1. Near-UV CD spectra The R and T forms of Hb A differ both in quaternary structure (the relative arrangements of the four subunits) and in tertiary structure (the conformation of the individual subunits) [1]. In particular, aromatic residues (b37Trp, a42Tyr, a140Tyr and b145Tyr) undergo large changes in their environment at the a1b2 subunit interface with the structural transition [1,2]. It is generally accepted that these changes of aromatic residues contribute dominantly to the near-UV CD spectral differences between the R and T forms [15,16,21]. Perutz et al. [15,16] stated that the sharp negative peak in the CD spectrum at 287 nm is characteristic of the change in quaternary structure of the globin, but independent of the ligation of the heme and the accompanying changes in tertiary structure of the subunits. From these results they also suggested that part of the intensity of the band at 287 nm reflected the contribution of the change of the heme from oxy to deoxy form [15]. In the present paper, we compared CD spectra of the arithmetic mean of deoxy-a and deoxy-b chains with that of recombined Hb in the deoxy form (Fig. 5B) to remove the
contribution of the heme. Since both spectra contain the contribution of the deoxy heme, the observed difference between them represents only the contribution of aromatic residues. However, we can not rule out the possibility that the tertiary structure changes of the isolated chains can be different from those of the chains incorporated into the Hb tetramer. In order to confirm this, we need to compare the CD spectra for the isolated chains with those for some non-cooperative Hb (chemically modified or mutant) which undergoes no quaternary structure change, and further compare the CD spectra for the non-cooperative Hb with those for normal Hb. Perutz et al. [15,16] presented absolute CD spectra of non-cooperative mutant Hb, Hb Kempsey (b99Asp→Asn), and modified Hb, Nes-des-ArgHb. Their deoxy forms exhibited the CD band with negative ellipticity at 287 nm like deoxyHb A but the ellipticity was nearly half [15]. When added inositol hexaphosphate (IHP) forced these non-cooperative Hbs into the T form, the negative CD band at 287 nm became more negative [15]. These facts indicate that about the half of the CD band at 287 nm is due to quaternary structure. Being not yet assigned, the other half is compatible with our finding that environmental alterations of Tyr and Trp residues with the tertiary structure change brought about by the oxygen binding contribute about half to the CD band at 287 nm. The present paper describes the changes in CD of aromatic side chains produced by tertiary and quaternary structural changes accompanying oxygen binding in hemo-
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globin and its separated subunits. This discrimination was accomplished by calculating the difference between the CD of the Hb tetramer and the sum of the spectra for the isolated chains. The spectra of the isolated chains contain information about the effects of tertiary structural changes, whereas the spectral difference between the tetramer and the subunits reveals the contribution of quaternary structural changes. It was found that about half of negative ellipticity of deoxyHb A at 287 nm came from the tertiary structural change and the other half was ascribed to the quaternary structure transition. Perutz et al. [15] have suggested that the aromatic residues at the a1b2 subunit interface (b37Trp and a42Tyr) are responsible for the negative CD band in the T form. Recently, using two abnormal Hbs, Hb Hirose (b37Trp→Ser) and Hb Rouen (a140Tyr→His), we have demonstrated that the b37Trp and a140Tyr contributions to the negative CD band were 26% and 30%, respectively [17]. These results indicate that the other aromatic residues, a42Tyr and / or b145Tyr, at the a1b2 subunit interface, are also responsible for changes of the CD band upon the R→T transition of Hb A. Recent time-resolved CD (TRCD) study on photodissociated HbCO has revealed the prompt appearance of a negative ellipticity in the aromatic bands near 285 nm with a time constant of approximately 500 ns [24]. This is evidence for a rapid shift of the a1b2 subunit interface to a more T-like conformation. Although the relative contributions of tertiary and quaternary structural changes were estimated to be equally 50% at 287 nm, this estimation varies with wavelengths (Fig. 5). Contribution of the tertiary structural changes become larger at longer wavelength than 287 nm, but that of the quaternary structural changes increase inversely at shorter wavelength than 287 nm as shown in Fig. 5B. Considering the spectral characteristic of the model compounds in Fig. 2, these results suggest that the environmental alterations of Tyr residues are mainly responsible for the quaternary structural changes of Hb A.
4.2. Far-UV CD spectra In far-UV region, the main CD bands originate from the peptide n–p* and p–p* transitions [25]. In the present study, distinct CD signals due to aromatic residues was also detected in the region upon the quaternary structure transition of recombined Hb (Fig. 7A). Although the difference in CD spectra between a and b chains were observed, the CD spectra of recombined Hb were greatly different from the arithmetic mean of a and b chains in both oxy and deoxy forms. The difference CD spectrum between the recombined Hb and the arithmetic mean of a and b chains in oxy form gave small negative peaks at 209 and 222 nm reflecting the a-helix content (Fig. 7A), indicating that the a-helix content increased slightly (2%) by recombination. On the other hand, the difference CD
spectrum in deoxy form exhibited a distinct negative band at 225 nm (Fig. 7A). This CD band might result from coupling with the far-UV transitions of neighboring aromatic side chains, the peptide p–p* transition and perhaps the 1 La tryptophanyl transition [7] or the 1 Lb CD band of Tyr [26]. Both Tyr and Trp showed large changes of ellipticity at 225 nm in different solvent (water and dioxane) (Fig. 8). Hence, it is likely that changes of environment of both Tyr and Trp residues to the hydrophobic conditions are responsible for the increased negative ellipticity at 225 nm upon the R→T transition.
4.3. Difference in CD spectra between a and b chains Three Tyr residues and one Trp residue exist in the a chain and three Tyr residues and two Trp residues are in the b chain. The a chain exhibited markedly different CD spectra from that of the b chain in both near-UV and far-UV regions (Figs. 1 and 6). The oxy-a chain gave a negative CD band at 290 nm and became deeper upon deoxygenation. In contrast, oxy-b chain showed positive peaks at 280|300 nm that changed to negative CD bands in the deoxy form (Fig. 1). There are totally three Trp residues in Hb A, that is, a14Trp (A12), b15Trp (A12) and b37Trp (C3). Of these, a14Trp and b15Trp are outside of the subunit interface, but b37Trp can probably make intraand / or inter-chain contact to many amino acid residues even in the isolated b chain. Two peaks in the 280|300nm region of the CD of b chain might be due to b37Trp, the environment of which must be altered by the tertiary structural change. The CD spectrum of Tyr is influenced by solvent and by modification at carboxyl group of Tyr. NAc-Tyr-amide in dioxane showed negative ellipticities in near-UV region (Fig. 2A), but NAc-Tyr-EE (ethyl ester) in dioxane exhibited positive peaks at 277 and 283 nm [27,28]. Although the N- and C-terminal-blocked amino acids are useful models for examining band positions, solvent effects, and (to some extent) approximate intensities in CD, it should be noted that aromatic residues in the protein might be somewhat different from those of model compounds.
Acknowledgements This work was supported in part by Grants-in Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan to M.N. (11116209 and 10670115).
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Changes of tyrosine and tryptophan residues in human hemoglobin by oxygen binding: near- and far-UV circular dichroism of isolated chains and recombined hemoglobin R. Li, Y. Nagai, M. Nagai* School of Health Sciences, Kanazawa University Faculty of Medicine, Kodatsuno 5 -11 -80, Kanazawa 920 -0942, Japan Received 7 March 2000; received in revised form 17 July 2000; accepted 19 July 2000
Abstract In order to assign the circular dichroism (CD) spectral change in the region between 280 and 300 nm of human adult hemoglobin (Hb A) upon the quaternary structure transition induced by oxygen binding, the near- and far-UV CD spectra of the isolated chains and the recombined hemoglobin were examined. Deoxygenation made the negative CD band at 290 nm of oxy-a chain deeper. On the other hand, positive CD bands of oxy-b chain at the 280|300 nm became negative upon deoxygenation. These changes were interpreted as being due to environmental alterations of tyrosine (Tyr) and / or tryptophan (Trp) perturbed by tertiary structural changes from the oxy to deoxy form in isolated chains, referring to the CD spectra of model compounds. From the difference between CD bands of the arithmetic mean of deoxy isolated chains and the CD band of deoxyHb tetramer, the contribution of tertiary structural change to the negative CD band of deoxyHb A at 287 nm was estimated to be 50%. This finding has revealed that the net contribution of quaternary structure transition to the negative band is 50%. In far-UV CD spectra, the environmental changes of aromatic residues upon the quaternary structure transition were also detected as a negative band at 225 nm. 2000 Elsevier Science B.V. All rights reserved. Keywords: Hemoglobin; Near-UV CD; Far-UV CD; Isolated chains; Recombined Hb
1. Introduction Human adult hemoglobin (Hb A), a well known allosteric protein, is a tetramer composed of two a and two b subunits. With an increase in the number of bound oxygens, the quaternary structure transition of Hb A occurs from the low-affinity deoxy, or T (tense) to the highaffinity oxy, or R (relaxed) form. This allosteric effect in Hb A has made it one of the most important proteins for studying the relation between structure and function. The a1b2 subunit interface is considered to play a pivotal role in the quaternary structure transition [1,2]. Aromatic residues such as a42Tyr, a140Tyr, b37Trp, and b145Tyr are located at the a1b2 subunit interface and undergo changes of environment with the quaternary structure transition from T to R [1,2]. All these aromatic residues remain invariant throughout the evolution of a and b chains [1], implying their indispensability for the mainte*Corresponding author. Tel.: 181-76-265-2581; fax: 181-76-2344360. E-mail address: [email protected] (M. Nagai).
nance of structure and function of Hb A. In fact, all mutant Hbs at these aromatic residues, recombinant Hb (rHb) (a42Tyr→His) [3], Hb Rouen (a140Tyr→His) [4], Hb Hirose (b37Trp→Ser) [5] and Hb Fort Gordon (b145Tyr→Asp) [6], have an increased oxygen affinity and decreased or no cooperativity. Circular dichroism (CD) is a sensitive physical technique for determining structures and monitoring structural changes of biomolecules. The far-UV CD spectra of proteins are highly sensitive toward protein structure, especially useful for determining the changes of protein secondary structure. On the other hand, the near-UV CD spectra of proteins reflect contributions of aromatic side chains and disulfide bonds [7]. The studies on CD spectra of Hb A have shown that both the tertiary structural change and the quaternary structure transition contribute to the spectral change upon oxygen binding [8–10] and that the CD band at the 280|290-nm region is ascribable to the aromatic residues [11–13]. It has been shown that the far-UV CD spectra in the 200|250-nm region also give information on aromatic residues [14]. Hb A shows a pronounced change of the CD spectra in
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the 280|300-nm region upon the T→R transition. The change of CD band in the 280|290-nm region from small positive peaks in oxyHb A to a distinct negative trough in deoxyHb A was suggested to be due to environmental alterations of Tyr or Trp residues located at the a1b2 subunit interface [11–13]. This spectral change has been thought to be due to b37Trp [15,16]. However, assignment of this spectral change to specific aromatic residues remains incomplete. Our recent CD study on Hb Hirose (b37Trp→Ser) has demonstrated that the contribution of b37Trp to the negative CD band in deoxyHb A is only 26%, indicating possible involvement of other aromatic residues at the a1b2 subunit interface [17]. We suspected that the environment of aromatic residues might be influenced by the tertiary structure change upon oxygen binding, resulting in the CD spectral changes in the 280| 300-nm region. Isolated a chains are monomeric with high oxygen affinity like myoglobin [18]. In contrast, isolated b chains aggregate to form a tetramer (b 4 ) without cooperativity [18]. Isolated chains enable us to distinguish the contribution of tertiary structural changes to the CD spectra on oxygen binding in the far- and near-UV region. In the present study, the near- and far-UV CD spectra of isolated chains and recombined Hb were examined to estimate the contribution of aromatic residues to the spectra due to the tertiary structural change, referring to the spectra of model compounds such as N-acetyltyrosineamide (NAc-Tyr-amide) and N-acetyltryptophanamide (NAc-Trp-amide).
2. Materials and methods
2.1. Isolation of a and b chains After fresh human blood cells were washed with 0.9% NaCl solution and hemolyzed, a five-fold excess of phydroxymercuribenzoate ( p-HMB) was added to hemoglobin solution containing 0.1 M NaCl and adjusted to pH 5.8, which was allowed to stand overnight at 48C [19,20]. a-HMB and b-HMB chains were separated by isoelectric focusing electrophoresis on a Sephadex G-75 superfine gel bed containing 5% ampholine, pH 3.5–9.5. The p-HMB of a and b chains were removed by adding 3 mg dithiothreitol (DTT) / ml in 0.1 M Tris–HCl buffer, pH 8.2, and passage through a Sephadex G-25 column. Regeneration of sulfhydryl (SH) groups of a and b chains was confirmed by SH-titration with p-chloromercuribenzoate. The concentration of hemoglobin was determined after conversion to pyridine hemochrome using emM (557 nm)534. The recombined Hb was prepared by mixing a chains and b chains at equimolar concentration just before the CD measurements.
2.2. CD measurements CD measurements were performed with a Jasco J-725
spectropolarimeter at 258C. Spectra were measured several times for each sample using different preparations, recorded as an average of 40 (far-UV region) or 20 scans (near-UV region). The solvent spectrum obtained under the same conditions was subtracted to obtain the actual sample spectrum. Hbs used were 100 mM (in heme) for the 240|340-nm region and 10 mM for the 200|250-nm region in 0.1 M phosphate buffer, pH 7.0, respectively. The path length of the cell used was 2 mm. The optical density of the Hb samples was maintained below 2.0 for CD measurements in all regions. DeoxyHb was prepared by adding small quantities of sodium dithionite powder to the oxyHb. As excess amounts of sodium dithionite used for deoxygenation interfered with the measurement of CD in the UV region by increasing the noise, we usually measured the CD spectra of the samples with OD less than 0.5 at 310 nm. (1)-10-Camphorsulfonic acid was used for the calibration of the instrument. CD spectra were expressed as molar CD, De (M 21 cm 21 ).
3. Results
3.1. Near-UV CD spectra of isolated chains and recombined Hb CD spectra of the isolated a and b chains in the near-UV region are shown in Fig. 1. As shown by Ueda et al. [8], a distinctive difference in positive band near 260 nm can be seen between the isolated chains. The ellipticity associated with the 260-nm band is strongly influenced by the attached ligand and, thereby, the spin state of the iron atom [21]. A large decrease of the band near 260 nm from oxy to deoxy form was attributed to changes of optical activity of the heme moiety in both a and b chains [15,16]. The CD spectrum in the 280|300-nm region is useful for monitoring the environmental changes of Tyr and Trp residues in the globin [7]. Upon deoxygenation of the a chain, the CD band at 290 nm became more negative and the positive ellipticity at 280 nm increased. On the other hand, the positive CD spectrum at the 280|300-nm region of the b chain became negative upon deoxygenation. These results suggest that the tertiary structural changes upon oxygen binding also induce change of local environment of Tyr and Trp residues in both a and b chains. In order to assess, upon oxygen binding, the above changes of CD spectra of isolated chains to Tyr and Trp residues, we determined CD spectra of NAc-Tyr-amide and NAc-Trp-amide as model compounds in water and in dioxane (Fig. 2). In water, NAc-Tyr-amide showed a negative CD band in the 270|290-nm region that remained negative but red-shifted in dioxane (less polar solvent). NAc-Trp-amide is distinctive in exhibiting two positive bands at 282 and 288 nm and a negative band at 298 nm in water, and changing into negative troughs at 287 and 295 nm in dioxane. Since NAc-Tyr-amide does not exhibit the CD band in the 290|300-nm region, changes of CD bands
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Fig. 1. Near-UV CD spectra of isolated a chains (A) and b chains (B). The dotted line refers to the oxy form and the solid one to deoxy form. The spectra shown here are the mean of two spectra (each spectrum was an average of 20 scans). The Hb solution was 100 mM (in heme) in 0.1 M phosphate buffer, pH 7.0.
Fig. 2. Near-UV CD spectra of N-acetyl-tyrosineamide (A) and N-acetyl-tryptophanamide (B). The dotted line refers to the spectra in water and the solid one to those in dioxane. The spectra are an average of 40 scans with 1 mM NAc-Tyr-amide or 0.33 mM NAc-Trp-amide in 1 cm cell.
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at the 290|300-nm region are ascribable to local environment alteration of Trp residues. On the other hand, two negative CD bands in the 270|290-nm region of NAc-Tyramide are characteristic of Tyr residues but obscured by those of Trp residues. In order to extract, upon oxygen binding, the change in CD bands due to local environment alterations of Tyr and Trp residues induced by tertiary structure changes in a and b chains, we examined the difference spectra (deoxy-minus-oxy) of a and b chains (Fig. 3). The difference spectrum of the a chain displayed a positive peak at 278 nm and a broad negative bands in the 285|310-nm region. In contrast, the b chain exhibited broad negative bands in the 280|300-nm region without distinct peak at 278 nm as in the a chain. These results indicate that oxygen binding in the a chain brings about local environment alteration of both Tyr and Trp residues due to tertiary structure change but in the b chains induces distinctively that of Trp. Fig. 4 shows the CD spectra of recombined Hb from equimolar a and b chains (solid lines) in comparison with that of the arithmetic mean of a and b chains (dotted lines). The CD spectra of the recombined Hb in the 250|270-nm region were very similar to the arithmetic mean of the isolated chains in both the oxy and deoxy form, indicating that the optical activity of the heme in the isolated chains was scarcely influenced by recombination to tetramer. In the 270|300-nm region, the difference between the arithmetic mean and recombined Hb was very Fig. 4. Comparison of CD spectra of the recombined Hb with the arithmetic mean of the spectra of a and b chains in the near-UV region. Recombined Hb was prepared by mixing a chain and b chain at equimolar concentration. The arithmetic mean was calculated from 1 / 2 (CD spectrum of a1CD spectrum of b). The dotted line refers to the arithmetic mean of a and b chains and the solid one to recombined Hb. The spectra shown here are the mean of two spectra (each spectrum was an average of 20 scans). The Hb solution was 100 mM (in heme) in 0.1 M phosphate buffer, pH 7.0.
Fig. 3. Deoxy-minus-oxy difference spectra of the isolated a and b chains in the near-UV region. The dotted line refers to the difference CD spectrum between deoxy-a chain minus oxy-a chain, and the solid one to that between deoxy-b and oxy-b chain.
small in the oxy form, but distinct in the deoxy form. Fig. 5 shows the same CD spectra in the 260|310-nm region as Fig. 4 and the difference spectra between the recombined Hb and the arithmetic mean of the isolated chains in an expanded scale. The spectra of the arithmetic mean of the a and b chains reflect only environmental conditions of the aromatic side chains of Tyr and Trp residues in the tertiary structure. But the effects of further changes of environment induced by tetramerization (quaternary structure) are added to the above spectra in recombined Hb. Therefore, difference spectra in both oxy (Fig. 5A) and deoxy (Fig. 5B) forms show the net contribution of quaternary structure to both CD spectra in the 270|300-nm region. The difference spectrum of the oxy form exhibited no apparent CD band. In contrast, the arithmetic mean of deoxy chains gave a negative CD band with a peak at 292 and a shoulder at 287 nm, but recombined Hb gave a single negative CD band at 287 nm and the depth was twice of the arithmetic mean.
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Fig. 5. Expanded CD spectra of recombined Hb and the arithmetic mean of a and b chains and difference between them in the near-UV region in oxy (A) and deoxy form (B). The dotted line refers to the arithmetic mean of the a and b chains and the thin solid line to recombined Hb. The bold solid line designates the difference spectrum (recombined Hb — the arithmetic mean of a and b chains). Experimental conditions were the same as in Fig. 4.
These results reveal that about half of negative CD band of deoxyHb A at 287 nm reflects the tertiary structure change and the other half is due to the quaternary structure transition upon the R→T transition. In other words, the contribution of the quaternary structure transition to the negative CD band at 287 nm is estimated to be 50%. The difference spectrum showed a broad band with peaks at 281 and 285 nm in addition to 287 nm. Judging from their band positions, Tyr residues seem to contribute to the former two peaks.
3.2. Far-UV CD spectra of isolated chains and recombined Hb The far-UV CD spectra give a measure of helical content in the secondary structure of proteins. The in-
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fluence of secondary structure on the CD spectra of hemoglobin was well illustrated by the dramatic changes that occurred upon heme binding to the apoprotein [22]. However, it was also reported that no significant difference was observed between isolated chains and native Hb [8]. Fig. 6 shows the far-UV CD spectra of a chain, b chain and recombined Hb. Oxygen binding did slightly influence the spectra of b chain but increased the intensity of the two negative CD bands at 209 and 222 nm of a chain. On the other hand, deoxygenation brought about a very small change of the CD band at 209 nm but caused an obvious increase in negative intensity at 222 nm in the recombined Hb. The observed change in CD spectra of recombined Hb was the same as that in the CD spectra of native Hb (not shown). The difference between reconstituted Hb and the arithmetic mean of isolated chains in oxy (dotted line) and deoxy (solid line) forms are shown in Fig. 7A. The difference in oxy form exhibited small negative bands at 209 and 222 nm, suggesting that helical content underwent a slight change by tetramerization. In contrast, a distinct negative band at 225 nm was detected in the deoxy form. Since a negative CD band at 209 nm is not observed in the difference spectrum of the deoxy form, it is unlikely that an increase in helical content accompanying by deoxygenation contributes to this band. Fig. 7A shows that the deoxy-minus-oxy difference in the CD band at 225 nm is 77 / M / cm. This difference is attributable to the quaternary structure change upon deoxygenation, as the spectra in Fig. 7A represent ‘recombined-minus-arithmetic mean’. On the other hand, the deoxy-minus-oxy difference of recombined Hb in the CD band at 225 nm which includes contribution of both tertiary and quaternary structure change is estimated to be approximately 60 / M / cm from Fig. 6C. In order to examine this discrepancy, we compared the deoxy-minus-oxy difference spectra of the isolated chains and recombined Hb (Fig. 7B). As shown in the figure, the deoxy-minus-oxy difference spectrum in the CD band at 225 nm reflecting the quaternary structure change is estimated to be 79 / M / cm, in good accordance with 77 / M / cm in Fig. 7A. We used 10 mM Hb (in heme) for far-UV CD measurements. As the tetramer–dimer dissociation constant have shown to be 2 mM [23], the majority of the Hb molecule is assumed to stay as tetramer under the present experimental conditions. Fig. 8 shows the far-UV CD spectra of model compounds of Tyr and Trp residues dissolved in either water or dioxane. The CD spectra of aromatic side chains are strongly influenced by the hydrophobicity of solvent. In particular, the positive peak at around 225 nm of both aromatic amino acids in water was greatly diminished upon exposing to hydrophobic environment in dioxane. The negative CD band at 225 nm in the difference spectrum between the recombined Hb and the arithmetic mean of isolated chains in deoxy form is probably attributable to environmental alterations of aromatic residues upon the R→T transition. These results suggest that aromatic
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Fig. 6. Far-UV CD spectra of isolated a chain (A), b chain (B) and their recombination (C). The dotted line refers to the oxy form and the thin solid one to the deoxy form. The bold solid line designates the difference spectrum (deoxy-oxy). The spectra shown here are an average of 40 scans. The Hb solution was 10 mM (in heme) in 0.1 M phosphate buffer, pH 7.0.
Fig. 7. Difference CD spectra between recombined Hb and the arithmetic mean of isolated subunits (recombined — the arithmetic mean) in oxy- and deoxy-forms (A) and the deoxy-minus-oxy difference spectra of the isolated chains and recombined Hb (B) in the far-UV region. (A) The dotted line refers to the oxy form and the solid one to the deoxy form. (B) The dotted line refers to the isolated chains (a), the solid one to the recombined Hb (b) and the dashed line (c) to the difference spectrum between (b)-spectrum minus (a)-spectrum.
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Fig. 8. Far-UV CD spectra of N-acetyl-tyrosineamide (A) and N-acetyl- tryptophanamide (B). The dotted line refers to the spectra in water and the solid one to those in dioxane. The spectra are an average of 40 scans with 1 mM NAc-Tyr-amide or 0.33 mM NAc-Trp-amide in 0.2-cm cell.
residues at the a1b2 subunit interface also contribute to the CD band at the far-UV region upon the quaternary structure transition.
4. Discussion
4.1. Near-UV CD spectra The R and T forms of Hb A differ both in quaternary structure (the relative arrangements of the four subunits) and in tertiary structure (the conformation of the individual subunits) [1]. In particular, aromatic residues (b37Trp, a42Tyr, a140Tyr and b145Tyr) undergo large changes in their environment at the a1b2 subunit interface with the structural transition [1,2]. It is generally accepted that these changes of aromatic residues contribute dominantly to the near-UV CD spectral differences between the R and T forms [15,16,21]. Perutz et al. [15,16] stated that the sharp negative peak in the CD spectrum at 287 nm is characteristic of the change in quaternary structure of the globin, but independent of the ligation of the heme and the accompanying changes in tertiary structure of the subunits. From these results they also suggested that part of the intensity of the band at 287 nm reflected the contribution of the change of the heme from oxy to deoxy form [15]. In the present paper, we compared CD spectra of the arithmetic mean of deoxy-a and deoxy-b chains with that of recombined Hb in the deoxy form (Fig. 5B) to remove the
contribution of the heme. Since both spectra contain the contribution of the deoxy heme, the observed difference between them represents only the contribution of aromatic residues. However, we can not rule out the possibility that the tertiary structure changes of the isolated chains can be different from those of the chains incorporated into the Hb tetramer. In order to confirm this, we need to compare the CD spectra for the isolated chains with those for some non-cooperative Hb (chemically modified or mutant) which undergoes no quaternary structure change, and further compare the CD spectra for the non-cooperative Hb with those for normal Hb. Perutz et al. [15,16] presented absolute CD spectra of non-cooperative mutant Hb, Hb Kempsey (b99Asp→Asn), and modified Hb, Nes-des-ArgHb. Their deoxy forms exhibited the CD band with negative ellipticity at 287 nm like deoxyHb A but the ellipticity was nearly half [15]. When added inositol hexaphosphate (IHP) forced these non-cooperative Hbs into the T form, the negative CD band at 287 nm became more negative [15]. These facts indicate that about the half of the CD band at 287 nm is due to quaternary structure. Being not yet assigned, the other half is compatible with our finding that environmental alterations of Tyr and Trp residues with the tertiary structure change brought about by the oxygen binding contribute about half to the CD band at 287 nm. The present paper describes the changes in CD of aromatic side chains produced by tertiary and quaternary structural changes accompanying oxygen binding in hemo-
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globin and its separated subunits. This discrimination was accomplished by calculating the difference between the CD of the Hb tetramer and the sum of the spectra for the isolated chains. The spectra of the isolated chains contain information about the effects of tertiary structural changes, whereas the spectral difference between the tetramer and the subunits reveals the contribution of quaternary structural changes. It was found that about half of negative ellipticity of deoxyHb A at 287 nm came from the tertiary structural change and the other half was ascribed to the quaternary structure transition. Perutz et al. [15] have suggested that the aromatic residues at the a1b2 subunit interface (b37Trp and a42Tyr) are responsible for the negative CD band in the T form. Recently, using two abnormal Hbs, Hb Hirose (b37Trp→Ser) and Hb Rouen (a140Tyr→His), we have demonstrated that the b37Trp and a140Tyr contributions to the negative CD band were 26% and 30%, respectively [17]. These results indicate that the other aromatic residues, a42Tyr and / or b145Tyr, at the a1b2 subunit interface, are also responsible for changes of the CD band upon the R→T transition of Hb A. Recent time-resolved CD (TRCD) study on photodissociated HbCO has revealed the prompt appearance of a negative ellipticity in the aromatic bands near 285 nm with a time constant of approximately 500 ns [24]. This is evidence for a rapid shift of the a1b2 subunit interface to a more T-like conformation. Although the relative contributions of tertiary and quaternary structural changes were estimated to be equally 50% at 287 nm, this estimation varies with wavelengths (Fig. 5). Contribution of the tertiary structural changes become larger at longer wavelength than 287 nm, but that of the quaternary structural changes increase inversely at shorter wavelength than 287 nm as shown in Fig. 5B. Considering the spectral characteristic of the model compounds in Fig. 2, these results suggest that the environmental alterations of Tyr residues are mainly responsible for the quaternary structural changes of Hb A.
4.2. Far-UV CD spectra In far-UV region, the main CD bands originate from the peptide n–p* and p–p* transitions [25]. In the present study, distinct CD signals due to aromatic residues was also detected in the region upon the quaternary structure transition of recombined Hb (Fig. 7A). Although the difference in CD spectra between a and b chains were observed, the CD spectra of recombined Hb were greatly different from the arithmetic mean of a and b chains in both oxy and deoxy forms. The difference CD spectrum between the recombined Hb and the arithmetic mean of a and b chains in oxy form gave small negative peaks at 209 and 222 nm reflecting the a-helix content (Fig. 7A), indicating that the a-helix content increased slightly (2%) by recombination. On the other hand, the difference CD
spectrum in deoxy form exhibited a distinct negative band at 225 nm (Fig. 7A). This CD band might result from coupling with the far-UV transitions of neighboring aromatic side chains, the peptide p–p* transition and perhaps the 1 La tryptophanyl transition [7] or the 1 Lb CD band of Tyr [26]. Both Tyr and Trp showed large changes of ellipticity at 225 nm in different solvent (water and dioxane) (Fig. 8). Hence, it is likely that changes of environment of both Tyr and Trp residues to the hydrophobic conditions are responsible for the increased negative ellipticity at 225 nm upon the R→T transition.
4.3. Difference in CD spectra between a and b chains Three Tyr residues and one Trp residue exist in the a chain and three Tyr residues and two Trp residues are in the b chain. The a chain exhibited markedly different CD spectra from that of the b chain in both near-UV and far-UV regions (Figs. 1 and 6). The oxy-a chain gave a negative CD band at 290 nm and became deeper upon deoxygenation. In contrast, oxy-b chain showed positive peaks at 280|300 nm that changed to negative CD bands in the deoxy form (Fig. 1). There are totally three Trp residues in Hb A, that is, a14Trp (A12), b15Trp (A12) and b37Trp (C3). Of these, a14Trp and b15Trp are outside of the subunit interface, but b37Trp can probably make intraand / or inter-chain contact to many amino acid residues even in the isolated b chain. Two peaks in the 280|300nm region of the CD of b chain might be due to b37Trp, the environment of which must be altered by the tertiary structural change. The CD spectrum of Tyr is influenced by solvent and by modification at carboxyl group of Tyr. NAc-Tyr-amide in dioxane showed negative ellipticities in near-UV region (Fig. 2A), but NAc-Tyr-EE (ethyl ester) in dioxane exhibited positive peaks at 277 and 283 nm [27,28]. Although the N- and C-terminal-blocked amino acids are useful models for examining band positions, solvent effects, and (to some extent) approximate intensities in CD, it should be noted that aromatic residues in the protein might be somewhat different from those of model compounds.
Acknowledgements This work was supported in part by Grants-in Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan to M.N. (11116209 and 10670115).
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