Site-directed mutagenesis in hemoglobin: test of functional homology of the F9 amino acid residues of hemoglobin {alpha} and ß chains

Abdul Hassan Mohammed Mawjood, Gentaro Miyazaki1, Rina Kaneko2, Yoshinao Wada2 and Kiyohiro Imai3

Department of Physiology and Biosignaling, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, 1 Department of Biophysical Engineering, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka 560-8531 and 2 Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi, Osaka 594-1101, Japan


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The cysteine residue at F9(93) of the human hemoglobin (Hb A) ß chain, conserved in mammalian and avian hemoglobins, is located near the functionally important {alpha}1–ß2 interface and C-terminal region of the ß chain and is reactive to sulfhydryl reagents. The functional roles of this residue are still unclear, although regulation of local blood flow through allosteric S-nitrosylation of this residue is proposed. To clarify the role of this residue and its functional homology to F9(88) of the {alpha} chain, we measured oxygen equilibrium curves, UV-region derivative spectra, Soret-band absorption spectra, the number of titratable -SH groups with p-mercuribenzoate and the rate of reaction of these groups with 4,4'-dipyridine disulfide for three recombinant mutant Hbs with single amino acid substitutions: Ala->Cys at 88{alpha} (rHb A88{alpha}C), Cys->Ala at 93ß (rHb C93ßA) and Cys->Thr at 93ß (rHb C93ßT). These Hbs showed increased oxygen affinities and impaired allosteric effects. The spectral data indicated that the R to T transition upon deoxygenation was partially restricted in these Hbs. The number of titratable -SH groups of liganded form was 3.2–3.5 for rHb A88{alpha}C compared with 2.2 for Hb A, whereas those for rHb C93ßA and rHb C93ßT were negligibly small. The reduction of rate of reaction with 4,4'-dipyridine disulfide upon deoxygenation in rHb A88{alpha}C was smaller than that in Hb A. Our experimental data have shown that the residues at 88{alpha} and 93ß have definite roles but they have no functional homology. Structure–function relationships in our mutant Hbs are discussed.

Keywords: amino acid substitution/functional role/oxygen equilibrium/recombinant mutant hemoglobin/site-directed mutagenesis


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Until recently naturally occurring human hemoglobin (Hb A) mutants had been a unique source for studying the functional consequences of amino acid substitutions in normal Hb (Perutz and Lehmann, 1968Go; Imai, 1982Go; Bunn and Forget, 1986Go; Perutz, 1987Go). At present, site-directed mutagenesis provides a powerful means of carrying out protein engineering as it enables the substitution of any constituent single residue or several residues at will.

Since the success of site-directed mutagenesis of Hb (Nagai et al., 1985Go), various recombinant Hb mutants have been synthesized to mimic the function of certain animal Hbs (Nagai et al., 1985Go; Imai et al., 1989aGo; Jessen et al., 1991Go; Komiyama et al., 1995Go) as well as to investigate the structure–function relationships in Hb A (Nagai et al., 1987Go; Imai et al., 1991Go; Komiyama et al., 1991Go; Tame et al., 1991Go; Doyle et al., 1992Go; Ishimori et al., 1992Go, 1994Go; Hashimoto et al., 1993Go; Martin de Llano et al., 1993Go; Shih et al., 1993Go; Kim et al., 1994Go, 1995Go; Wakasugi et al., 1994Go; Yanase et al., 1994; Nagai et al., 1996Go; Inaba et al., 1998Go; Kono et al., 1998Go; Nakatsukasa et al., 1998Go).

Hb A contains three cysteine residues per {alpha}ß dimer, 104(G11){alpha}, 93(F9)ß and 112(G14)ß, of which only Cys93ß is partially exposed to the solvent and reactive toward reagents such as mercury compounds in the liganded form, whereas the other two are internal and unreactive both in the liganded and unliganded (deoxy) forms.

Cys93ß of Hb A is one of the important residues whose role has been extensively studied. It is located between the heme-linked proximal His92(F8)ß and Asp94(FG1)ß. This Asp residue stabilizes the deoxy conformation of the ß chain by forming an intrasubunit salt bridge with the imidazole group of His146(HC3)ß, which is responsible for 40% of the alkaline Bohr effect (Perutz et al., 1969Go; Perutz, 1970Go). The reactivity of Cys93ß was utilized for the heavy atom isomorphous replacement method (Green et al., 1954Go), which led to elucidation of the three-dimensional structure of Hb by X-ray crystallography, and for cleaving the Hb tetramer into single chains with mercuribenzoate derivatives (Bucci and Fronticelli, 1965Go). Cys93ß served as a probe for detecting ligation-induced conformational changes in the protein moiety. Its form-dependent reactivity toward various sulfhydryl reagents indicated that it underwent environmental changes during ligation of the heme (Riggs, 1961Go; Antonini and Brunori, 1971Go; Baldwin, 1975Go; Shulman et al., 1975Go; Imai, 1982Go). Spin-labels attached to Cys93ß exhibited oxygenation-induced changes in the electron spin resonance spectrum which reflected both tertiary and quaternary structure states (Ogawa and McConnell, 1967Go; Ogawa et al., 1968Go). The chemical shift in the 19F nuclear magnetic resonance spectrum of Hb trifluoroacetonylated at Cys93ß showed dependencies not only on the ligation state, but also on the conformational state of the ß subunits (Huestis and Raftery, 1972Go). Although CysF9ß is conserved in avian and mammalian Hbs, it is replaced by Ser in Hbs of teleosts, such as carp and trout, and of amphibia, such as the aquatic frog Xenopus. This Ser was predicted to be a key residue responsible for the Root effect (Perutz, 1984Go) but site-directed mutagenic studies showed that the Root effect is not mimicked only by introducing a Ser into the F9 site of Hb A (Nagai et al., 1985Go; Imai et al., 1989aGo).

As briefly reviewed above, the functional role of Cys93ß is still unknown in spite of a tremendous number of experimental observations concerning this residue. Recently, Stamler and co-workers (Jia et al., 1996Go; Stamler et al., 1997Go; Gow and Stamler, 1998Go) proposed a new possible function for Hb; that is, Hb contributes to the regulation of local blood flow through allosteric S-nitrosylation at Cys93ß. Its physiological significance is still under debate (Yonetani et al., 1998Go).

To get an insight into the functional role of Cys93ß we have carried out mutagenesis studies. We considered that it was useful, as well as being of interest, to test the functional homology between site 93(F9)ß and its structurally analogous site in the {alpha} chain of Hb A, 88(F9){alpha}. Since the amino acid residue at 88{alpha} of Hb A is Ala and no natural mutant with a replacement of Cys93ß by Ala has been reported, we prepared a recombinant mutant Hb with such a replacement (designated rHb C93ßA). We also prepared a mutant Hb with a replacement of Ala88{alpha} by Cys (designated rHb A88{alpha}C) as the reverse mutation. Two other 93ß-site mutant Hbs are already known: one is a natural mutant, Hb Okazaki (Cys->Arg) (Harano et al., 1984Go) and the other is a recombinant Hb named `Nympheas' (Cys->Ser) (Nagai et al., 1985Go). To investigate further the functional consequences of the 93ß-site mutation, we also prepared a recombinant Hb Cys93ß->Thr (designated rHb C93ßT). Thr is polar like Cys and Ser but bulkier than them. Three other naturally occurring 88{alpha}-site mutant Hbs are known: Hb Loire (Ala->Ser) (Baklouti et al., 1988Go), Hb Columbia Missouri (Ala->Val) (Perry et al., 1991Go) and Hb Valparaiso (Ala->Gly) (Wajcman et al., 1994Go). In the present study, we measured oxygen equilibrium, UV-range derivative spectra, Soret-band absorption spectra, the number of titratable -SH groups and their reactivity toward sulfhydryl reagents for our three recombinant Hbs. Then by comparison of these data with those from other 93ß-site and 88{alpha}-site mutant Hbs, we discuss the functional consequences of the Cys93ß substitutions with special reference to the functional homology of the F9 sites in the {alpha} and ß chains.


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 Materials and methods
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Preparation of mutant hemoglobins

The mutant Hbs (rHb A88{alpha}C, rHb C93ßA and rHb C93ßT) were synthesized basically as described by Nagai and co-workers (Nagai et al., 1985Go; Nagai and Thøgersen, 1987Go; Jessen et al., 1994Go). The intended mutations were introduced into the respective human globin gene ({alpha}-globin or ß-globin gene) using the M13 phage vectors, M13mp18cIIFX{alpha}-globin and M13mp18cIIFXß-globin, and Escherichia coli cells (CJ236 and TG1). The mutated {alpha}-globin and ß-globin genes were then cut out with BamHI from each M13 vector and cloned into a BamHI-cleaved two-cistron version (Schoner et al., 1987Go) of the T7 RNA polymerase-dependent expression vector (Studier et al., 1990Go), yielding plasmids pT7cIIFX{alpha}-globin and pT7cIIFXß-globin, respectively. It was confirmed by DNA sequencing, using a DNA sequencer (model 373A, Applied Biosystems), that these plasmids contained the correct base substitutions. Escherichia coli strain BL21 (DE3) was transformed with these plasmids and the mutant globin genes were expressed. The resultant fusion proteins CIIFX{alpha}-globin and CIIFXß-globin were extracted, purified and then digested with activated blood coagulation factor Xa to liberate their respective authentic mutant globins. The mutant {alpha}- and ß-globins were combined with CO-saturated native ß- and {alpha}-globins, respectively, which had been isolated from native Hb beforehand, in the presence of hemin chloride to reconstitute the recombinant Hb tetramers. Ferric heme was reduced with sodium dithionite under a CO atmosphere and in the presence of catalase and superoxide dismutase. The CO-saturated mutant Hbs were purified by passing through ion-exchange columns of CM-52 and then DE-23 (Whatman).

Oxygen equilibrium experiments and analysis

Oxygen equilibrium curves (OECs) were measured with an on-line system (Imai, 1994Go) of an improved version (Imai, 1981aGo) of the automatic recording apparatus (Imai et al., 1970Go) and analyzed according to the Adair equation (Imai, 1981bGo, 1994Go). The spectrophotometer used for the apparatus was a double-beam spectrophotometer (model U-4000, Hitachi, Tokyo). The wavelength of detection light was 560 nm. The temperature of the sample in the oxygenation cell was held constant at 25 ± 0.05°C. Immediately before measurement, the CO-form Hb was converted to the oxy-form by intense illumination under a constant stream of pure oxygen. To reduce metHb pre-existing in the sample and to minimize its formation during OEC measurement, an enzymic metHb reducing system (Hayashi et al., 1973Go) was added to each Hb sample. The metHb content of each sample determined immediately after the OEC measurement by the method of Evelyn and Malloy (1938) were from 2 to 10% of the total Hb.

The oxygen equilibrium data were analyzed by fitting the Adair equation to each OEC by a nonlinear least-squares method (Imai, 1981bGo, 1994Go). Overall oxygen affinity was expressed by P50 (partial pressure of oxygen at half saturation) and cooperativity in oxygen binding was expressed by the maximal slope, nmax, of the Hill plot, log [Y/(1 – Y)] versus log P, where Y is fractional oxygen saturation and P is partial pressure of oxygen. These parameter values were calculated from the best-fit values of the Adair constants. Magnitude of the alkaline Bohr effect was expressed by the Bohr coefficient, .

Oxygen equilibrium data for native Hb A were used as a control since it has been established that a wild-type recombinant Hb prepared by the same method shows oxygen binding properties identical with those for Hb A (Nagai et al., 1987Go; Tame et al., 1991Go).

Spectrophotometric measurements

Visible- and UV-range absorption spectra of Hb were recorded with a double-beam spectrophotometer (model 320L, Hitachi, Tokyo). The concentration of Hb samples was calculated on the basis of millimolar absorption coefficient values at 576 and 540 nm for oxy and cyanmet forms, respectively (van Assendelft and Zijlstra, 1975Go).

UV-region derivative spectra (the first derivative of absorption spectra) were recorded with a first-derivative mode of the spectrophotometer as described by Imai (1973). The Hb sample was placed in a Benesch-type versatile tonometer with a cuvette of 10 mm light-path length (Benesch et al., 1965Go) and its deoxygenation was gently performed by repeated evacuation and flushing with pure nitrogen (99.999%).

Titration of sulfhydryl groups

Spectrophotometric titration of reactive -SH groups of Hb was performed basically as described by Benesch and Benesch (1962). The concentration of the p-mercuribenzoate (PMB) solution was determined spectrophotometrically using the absorption coefficient at 232 nm, 16.9 mM–1cm–1 (Boyer, 1954Go) immediately before each titration experiment. The PMB solution of known concentration, as a titrant, was added both to the reference cell containing the buffer and to the sample cell containing the Hb solution and the absorbance change at 250 nm was recorded on each addition.

Reactivity of sulfhydryl groups

Rate of reaction of 4,4'-dipyridinedisufide (4-PDS) with titratable -SH groups of Hb was determined by recording time courses of the reaction according to Ampulski et al. (1969) with minor modifications. The spectrophotometer used was equipped with a thermostated cell holder. The reaction was monitored at 324 nm. The Benesch-type tonometer was used for deoxyHb samples.

Mass spectrometry

Globin was prepared from mutant Hbs and Hb A using the acid–acetone method (Rossi-Fanelli et al., 1958); that is, the Hb samples were dissolved in a solvent of 75 vol/24.8 vol/0.2 vol of methanol/H2O/acetic acid to make a protein concentration of 10 µM. An aliquot (20 µl) of the globin sample was injected into the electrospray ionization ion source of a mass spectrometer (model JMS SX 102A, JEOL, Akishima, Japan) at a flow rate of 0.8 µl/min (Wada et al., 1992Go). Mass spectrometry was also performed with mutant Hb samples treated with PMB.


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Oxygen equilibrium

Oxygen equilibrium curves were recorded at physiological pH, pH 7.4, and at two other pHs, pH 7.9 and 8.4, to measure the alkaline Bohr effect. At pH 7.4, an OEC was also recorded in the presence of 2 mM inositol hexaphosphate (IHP) to measure the effect of this allosteric effector. The OECs of mutant Hbs and Hb A at pH 7.4 are presented by Hill plots in Figure 1Go. In the absence of IHP the main part of the OECs for the mutant Hbs are shifted toward the left relative to the OEC for Hb A (Figure 1AGo), indicating that the oxygen affinities of the mutants are higher than that of Hb A. In the presence of 2 mM IHP the leftward shifts of the OECs for the mutant Hbs become larger (Figure 1BGo).



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Fig. 1. Oxygen equilibrium curves for recombinant mutant Hbs and HbA as presented by Hill plots. Y, fractional saturation of Hb with oxygen; P, partial pressure of oxygen in mmHg. In the absence (A) and presence (B) of 2 mM inositol hexaphosphate (IHP). Other experimental conditions: Hb concentration, 60 µM on a heme basis; in 0.05 M bis–Tris (pH 7.4) containing 0.1 M Cl ; 25°C. {circ}, rHb A88{alpha}C; {square}, rHb C93ßA; {lozenge}, rHb C93ßT; line without symbols, HbA.

 
The values of oxygenation parameters for mutant Hbs and Hb A obtained from their OECs are listed in Table IGo. At pH 7.4, in the absence of IHP, the oxygen affinity of rHb C93ßA, rHb A88{alpha}C and rHb C93ßT was 1.3-, 5.9- and 9.8-fold, respectively, higher than that of Hb A. The effect of IHP on P50 was somewhat decreased for rHb C93ßA and rHb C93ßT, whereas it was completely diminished in rHb A88{alpha}C. The Bohr effect and cooperativity for the mutant Hbs were smaller than those for Hb A and the degree of these functional impairments was in the order rHb C93ßA < rHb C93ßT < rHb A88{alpha}C. On the addition of 2 mM IHP, the cooperativity of rHb C93ßA and rHb C93ßT was restored to some extent, whereas that of rHb A88{alpha}C remained unchanged. In rHb A88{alpha}C, cooperativity was nearly absent and the Bohr effect was very small. Thus, both homotropic and heterotropic effects were nearly lost in this Hb.


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Table I. Oxygen equilibrium parameters for recombinant mutant Hbs and Hb Aa
 
Spectroscopic properties

The UV-range derivative spectra for oxy- and deoxy-forms of mutant Hbs and Hb A are shown in Figure 2Go. All three mutant Hbs and Hb A showed a characteristic fine structure composed of one maximum at 289.5 nm and two minima at 286.5 and 293.5 nm, in agreement with previous observations (Imai, 1973Go). Upon deoxygenation of Hb A the magnitude of the fine structure became 0.59-fold that of oxy-Hb A. The magnitudes of the fine structure for the oxy-form of mutant Hbs were nearly identical to that of oxy-Hb A, whereas the magnitudes of the fine structure for the deoxy-form of the mutant Hbs were somewhat larger than that of deoxy-Hb A. The change in the magnitude of fine structure upon deoxygenation was expressed by its ratio between the deoxy- and oxy-forms (Deoxy {Delta}/Oxy {Delta}) and its values are listed in Table IIGo, which also includes the data for recombinant mutant Hbs previously studied by our group. The ratio values for the present mutant Hbs ranged from 0.63 to 0.91 and were larger than that for Hb A (0.59). The ratio values for the previous mutant Hbs, which exhibited high oxygen affinities and reduced cooperativity, were also larger than that for Hb A. The value of 0.57 for rHb T38{alpha}S was the only one exception.



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Fig. 2. UV-range derivative spectra for recombinant mutant Hbs and Hb A. The ordinate is the first derivative of absorbance. Solid lines, oxy-form; broken lines, deoxy-form. Experimental conditions are as in Figure 1AGo. Light-path length, 10 mm. The short horizontal lines attached to the left end of spectra indicate their respective base lines.

 

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Table II. Comparison of spectral and oxygen equilibrium properties of mutant Hbs and Hb Aa
 
Figure 3Go shows the Soret-band absorption spectra for the oxy- and deoxy-forms of the mutant Hbs and Hb A. The peak height values are listed in Table IIGo (see {varepsilon}430 values). The Soret peak of oxy-form was the same height for all the mutant Hbs and Hb A. On the other hand, the peak height values for all the deoxy mutant Hbs were lower than that for deoxy-Hb A; that for deoxy-rHb A88{alpha}C was the lowest of all the deoxy-Hbs, even lower than that for the oxy-form.



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Fig. 3. Soret-band absorption spectra for recombinant mutant Hbs and Hb A. Experimental conditions are as in Figure 1AGo. Light-path length, 2 mm. The thin line stands for oxy-form (common to all the Hbs). The bold lines stand for deoxy-form: ––, rHb A88{alpha}C; – – –, rHb C93ßA; ···, rHb C93ßT; –··, Hb A.

 
To examine the relationships between spectral properties and oxygen binding properties, the oxygen affinity relative to Hb A and cooperativity for mutant Hbs are listed in Table IIGo.

Titration of sulfhydryl groups

Figure 4Go shows spectrophotometric titration of reactive (titratable) -SH groups with PMB for the CO-form. The number of titratable -SH groups obtained from the refraction point of these titration plots was 2.2 per tetramer for oxy- and CO-Hb A (Table IIIGo) in good agreement with Benesch and Benesch (1962). For rHb A88{alpha}C, the number was 3.2 and 3.5 per tetramer for the oxy- and CO-forms, respectively. Those for rHb C93ßA and rHb C93ßT were very small, less than one-tenth that for Hb A (the titration plots are not shown).



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Fig. 4. Titration of carbonmonoxy rHb A88{alpha}C and Hb A with p-mercuribenzoate (PMB). Experimental conditions: Hb concentration, ~10 µM on a tetramer basis; in 0.05 M phosphate buffer (pH 7.0); light-path length, 10 mm; wavelength, 250 nm. The refraction points give the number of titratable -SH groups. •, CO-rHb A88{alpha}C; {blacktriangleup}, CO-HbA.

 

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Table III. Number of titratable -SH groups in recombinant mutant Hbs and Hb A
 
Absorbance in the titration plots for Hb A, rHb C93ßA and rHb C93ßT became constant after the refraction point was reached, showing that the reaction was complete. On the other hand, that for oxy- and CO-rHb A88{alpha}C showed gradual increases on further additions of PMB even after the refraction point was reached (Figure 4Go). These absorbance changes were not apparently time-dependent. When the titration was performed quickly with fewer data points, the total absorbance change indicated that the total number of the reactive -SH groups was still approximately 3.5. Conversely, when PMB was added to the Hb sample at long time intervals beyond the refraction point, the absorbance values were stable and showed no significant change even for several hours after the last addition of PMB.

The unusual behavior of rHb A88{alpha}C in -SH titration led us to the consideration that rHb A88{alpha}C had four titratable -SH groups (presumably two at 93ß and the other two at 88{alpha}) as an original form but those groups underwent a modification (e.g. oxidation) in part during preparation procedures and the modified groups were gradually reacting with PMB during titration. To test this possibility we treated this mutant Hb and Hb A with dithiothreitol to regenerate the modified -SH groups if present and performed titration experiments. However, the numbers of titratable -SH groups per tetramer were the same: 3.5 for the mutant and 2.2 for Hb A.

Reactivity of sulfhydryl groups

The reactivity of the titratable -SH groups of rHb A88{alpha}C and Hb A was studied by binding kinetics of 4-PDS. The other two mutant Hbs were not studied since they showed very small numbers of titratable -SH groups. The reaction of CO-rHb A88{alpha}C and CO- and deoxy-Hb A was completed within 60 min. The number of reactive -SH groups per tetramer, which was estimated from the total absorbance change at 324 nm, was 2.2 for CO-Hb A and 3.6 for CO-rHb A88{alpha}C, in good agreement with those determined by titration with PMB. In the case of deoxy-Hb A, the number was 2.3 per tetramer. As some side-reactions (e.g. turbidity caused by protein denaturation) occurred before the completion of the reaction of deoxy-rHb A88{alpha}C, the total absorbance change of reaction was assumed to be equal to that for the CO-form. The time courses are presented in Figure 5Go by means of a first-order reaction plot. These plots were not completely linear. The pseudo-first-order rate constant was evaluated from the initial slope and the results are summarized in Table IVGo. In the CO-form, rHb A88{alpha}C showed a reactivity very close to that of Hb A. In the deoxy-form, on the other hand, the rate constant for rHb A88{alpha}C was 2.3-fold larger than that for Hb A. The enhancement of 4-PDS reaction upon CO binding was 11-fold for rHb A88{alpha}C, while that of Hb A was 22-fold.



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Fig. 5. The time courses of the reaction of rHb A88{alpha}C and Hb A with 4,4'-dipyridine disulfide (4-PDS). A0, A{infty} and At are absorbance at the starting time, the termination time and a given time, respectively. Hb concentration, 40 µM on a heme basis; 0.05 M Bis–Tris (pH 7.4) containing 0.1 M Cl; 25°C. Initial concentration of 4-PDS, 161 µM for the CO-form and 1.67 mM for the deoxy-form. {square}, deoxy-rHb A88{alpha}C; {blacksquare}, CO-rHb A88{alpha}C; {circ}, deoxy-Hb A; •, CO-Hb A.

 

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Table IV. Pseudo-first-order rate constant values (M–1S–1) for reaction with 4-PDS and their ratios
 
Mass spectrometry

The mass spectra showed that the present three recombinant mutant Hbs had the amino acid substitutions as expected. No unexpected substitutions or post-translational modifications occurred in those proteins.

To determine the positions at which PMB was attached we carried out mass spectroscopy for PMB-treated rHb A88{alpha}C and Hb A. However, we detected no signal peak of PMB-attached {alpha} and ß chains. This proved to be attributed to removal of the bound PMB during the acid–acetone treatment for globin preparation.


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Functional characteristics of mutant Hbs

The present study showed that the oxygen equilibrium properties of the three recombinant mutant Hbs were impaired by different degrees, those of rHb A88{alpha}C most extensively. Table VGo compares the oxygen equilibrium properties of these recombinant Hbs with those of one recombinant mutant Hb and four natural mutant Hbs which have single amino acid substitutions at either F9(88){alpha} or F9(93)ß. In all of these mutant Hbs oxygen affinity was increased while allosteric effects were normal or partially impaired. Hb Loire (Ala88->Ser) showed a very high oxygen affinity (7-fold) whereas the effect of 2,3-dihposphoglycerate (DPG) and the Bohr effect were normal with cooperativity slightly decreased. The smaller increase in oxygen affinity (5.9-fold) and the intensive impairments of allosteric effects of rHb A88{alpha}C were in striking contrast with those of Hb Loire.


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Table V. Oxygen equilibrium properties of recombinant and natural mutant Hbsa
 
Meaning of spectral data

The magnitude of the fine structure of UV derivative spectrum around 290 nm is closely related to the quaternary structure, which is detected by aromatic amino acid residues at the {alpha}1–ß2 interface undergoing environmental changes during heme ligation (Imai, 1973Go). Recent UV resonance Raman studies using mutant Hbs (Nagai et al., 1995Go, 1996Go, 1999Go) suggest that the major aromatic residue acting as the quaternary state probe in UV derivative spectrum is Trp37ß, with smaller contributions from tyrosines at 42{alpha}, 140{alpha} and 145ß. In Hb A, the magnitude of the fine structure for the oxy-form (R state) was 0.056 nm–1 whereas that for the deoxy-form (T-state) was 0.033 nm–1 (Figure 2Go). It is well documented, through extensive studies using many mutant Hbs (Table IIGo), that the Hb which shows the same magnitude of fine structure in the oxy-form as that of oxy-Hb A assumes a normal R-state quaternary structure and exhibits a normal affinity for the fourth oxygen molecule and that the Hb which shows a larger magnitude in the deoxy-form than that of deoxy-Hb A assumes a T-state quaternary structure more favored to the R-state and exhibits an increased affinity for the first oxygen molecule and diminished cooperativity. It is also well documented that a reduced peak height of the Soret band of the deoxy-form is a diagnostic indicator for a `deoxygenated R structure' as detected by heme strain [Hopfield, 1973; see Imai et al. (1989b) for examples]. The increased deoxy {Delta}/oxy {Delta} values (Table IIGo), together with the reduced peak height of the Soret band for our three mutant Hbs, indicate that these Hbs assume a normal R structure in the oxy-form but the transition toward the T-state upon deoxygenation is partially restricted, most intensively in rHb A88{alpha}C. These structural features essentially agree with the functional properties of the present three mutant Hbs.

Structure–function relationships

In T-state Hb A, the side chain of Cys93ß is external but screened by the imidazole group of His146(HC3)ß, which forms a salt bridge with the {gamma}-carboxyl of Asp94(FG1) of the same ß chain (Perutz, 1970Go). In R-state Hb A, the side chain of Cys93ß becomes internal but is partially exposed to the solvent since the salt bridge between His146ß and Asp94ß is broken (Perutz, 1970Go). In the internal position, this side chain is in contact with the phenyl ring of Tyr145ß, sharing a pocket between helices F and H (Baldwin, 1980Go; Shaanan, 1983Go). The movement of the Cys93ß side chain between these two conformations can take place only in conjunction with a complementary movement of the Tyr145ß side chain. The -OH group of Tyr145ß forms a hydrogen bond with the main chain carbonyl of Val98(FG5) of the same ß chain, but its orientation depends on the quaternary state (Baldwin, 1980Go; Shaanan, 1983Go). An X-ray crystallographic study of rHb C93ßS (Luisi et al., 1987Go) showed that in this mutant the side chain of Asp94ß moved closer to Ser93ß and formed a new hydrogen bond with it, being in equilibrium between this hydrogen bond and the normal salt bridge with His146ß. This partial disruption of the normal salt bridge caused destabilization of the T state, consequent increase in oxygen affinity and reduction of the Bohr effect [the imidazole group of His146ß is responsible for 40% of the alkaline Bohr effect (Perutz, 1970Go)]. The same mechanism may apply to rHb C93ßT. Its more drastic change in oxygen equilibrium properties (Table VGo) requires an additional mechanism: in the T state the bulky side chain of Thr causes displacement of the imidazole group of His146ß and pushes the HC region of the ß chain away from the normal position, resulting in more complete disruption of the salt bridge between His146ß and Asp94ß and even the disruption of the intersubunit salt bridge between the ß chain {alpha} carboxyl group and the {varepsilon}-amino group of Lys40(C5){alpha} (Perutz, 1970Go). A similar mechanism for increase in oxygen affinity was previously proposed for N-ethylsuccinimide Hb in which the bulky chain attached to Cys93ß caused displacement of the imidazole group of His146ß in the T state (Perutz et al., 1969Go). Although the mechanism for the mild change in oxygen equilibrium properties of rHb C93ßA was not clear, it may be pointed out that the loss of the sulfur atom upon mutation caused a conformational change of the side chain of Tyr145ß that was in contact with Cys93ß of Hb A. This conformational change could destabilize the salt bridge that normally exists between His146ß and Asp94ß, causing a mild increase in oxygen affinity and reduction of the Bohr effect.

Ala88(F9){alpha} in Hb A is located near the {alpha}1–ß2 interface and the C-terminal region of the {alpha} chain. In the T-state this residue is involved in the intersubunit network of van der Waal's contacts and hydrogen bonds among Tyr140(HC2){alpha}, Pro36(C2)ß and Trp37(C3)ß, whereas it is excluded from this network in the R-state (Fermi and Perutz, 1981Go). Therefore, any perturbation of this network in the T-state by replacement of Ala88{alpha} by Cys could cause an increase in oxygen affinity and a decrease in cooperativity as actually observed in rHb A88{alpha}C. Another plausible mechanism for the altered properties of rHb A88{alpha}C may be formation of a new hydrogen bond between Cys88{alpha} and His89(FG1) of the same {alpha} chain in a way analogous to the hydrogen bond between Ser93ß and Asp94(FG1)ß found in rHb C93ßS (Luisi et al., 1987Go). His89{alpha} is a functionally important residue, contributing to the alkaline Bohr effect (Ohe and Kajita, 1980Go; Imai et al., 1989aGo). Although this new hydrogen bond may cause some secondary effect on function via the interference with this histidine, it is not easy to estimate its magnitude. One more possibility, as proposed in Hb Loire (Baklouti et al., 1988Go), may be formation of a hydrogen bond between Cys88{alpha} and the carboxyl group of Lys139(HC1) of the same {alpha} chain. This hydrogen bond could maintain Tyr140{alpha} in its position in the R-state, thus relatively destabilizing the T-state and causing an increase in oxygen affinity and a decrease in cooperativity. The extensive changes in oxygen binding properties of rHb A88{alpha}C may be explained by the combination of some or all of these mechanisms.

The partial or complete impairment of the IHP effect observed in the present recombinant Hbs was not attributed to direct effects by amino acid substitutions, because none of the residues substituted were included in the binding site of IHP (Arnone and Perutz, 1974Go). Since IHP is preferentially bound to the T quaternary structure (Benesch et al., 1968Go), destabilization of the T state generally leads to reduction of the IHP effect on oxygen equilibrium, as demonstrated in many mutant Hbs (Imai et al., 1989bGo, 1991Go; Ishimori et al., 1992Go, 1994Go; Hashimoto et al., 1993Go). The degree of reduction in the IHP effect for the present three recombinant Hbs accord with the degree of impairment of cooperativity (Table VGo).

It may be argued that part of the functional impairments of our three mutant Hbs was accounted for by partial dissociation of the tetramer into {alpha}ß dimers via cleavage along the {alpha}1–ß2 interface in the oxy-form. However, such a dissociation does not seem extensive and probably has no significant contribution to the functional impairments for the following reasons. The Soret peak at 430 nm of the three mutant Hbs did not change during repeated wavelength scannings immediately after adding sodium dithionite. This means that no significant amount of dimers was present in the oxy-form of the Hb samples because, if present, the peak height should have increased due to slow reassociation of dimers following the instantaneous shift of the tetramer–dimer equilibrium toward tetramers. Amino acid substitutions at Ala88{alpha} and Cys93ß cause no loss of intersubunit bond in the oxy-form because they have no contacts at the {alpha}1–ß2 interface in the oxy-form of Hb A (Fermi and Perutz, 1981Go). The side chains of the newly introduced amino acid residues in the present mutant Hbs could cause enhancement of dissociation into dimers by possible steric hindrances. However, such a possibility is not likely to occur since the tertrameric nature is established for chemically modified Hbs, such as N-ethylsuccinimide Hb and iodoacetamide Hb, in which bulky chains are attached to Cys93ß (Guidotti, 1967Go). No evidence for the presence of dimers was noted in the kinetic studies of Hb Loire (Baklouti et al., 1988Go).

State of sulfhydryl groups

Of the 3.2 to 3.5 titratable -SH groups of liganded rHb A88{alpha}C (Table IIIGo), two were assigned to Cys93ß. Initially, we expected the number of titratable -SH groups (per tetramer) to be two or four for this mutant Hb, depending on the reactivity of the two groups of cysteins-88{alpha}. The extra 1.2–1.5 -SH groups may be assigned to Cys88{alpha}, but it is not straightforward to explain this fractional number. This number was not time-dependent, was independent of the speed of titration and agreed with the number determined by 4-PDS kinetics. The missing number, 0.5–0.8, was not ascribed to some modification of Cys88{alpha}, such as oxidation, as shown by dithiothreitol treatment. Mass spectrometry also detected no sign of modification of Cys88{alpha}. If the number of titratable -SH groups for the deoxy-form was obtained, a clearer picture could be given. Unfortunately, experiments with the deoxy-form was not successful due to its instability.

The pseudo-first-order rate constant values in Table IVGo express the reactivity of the -SH groups of Cys93ß in Hb A and, possibly, the same groups in rHb A88{alpha}C. The similar constant values for the CO-form of these two Hbs indicate that they are equally in the R-state and Cys93ß is not screened by the imidazole group of His146(HC3)ß. The lesser reduction of the rate constant upon deoxygenation for rHb A88{alpha}C than that for Hb A means that the R to T transition was partially restricted and the screening by His146(HC3)ß was also weakened in rHb A88{alpha}C.

Functional roles and homology of the F9{alpha}and F9ß residues

The structural features common to the sites 88(F9){alpha} and 93(F9)ß are that they are adjacent to the proximal His and located near the {alpha}1–ß2 interface and the C-terminal region of their own chain. The residues at these sites sense the ligand-induced conformational changes at the interface and the C-terminal region. In each {alpha} and ß chain, the -OH group of the penultimate Tyr at HC2 forms a hydrogen bond with the main chain carbonyl of Val-FG5, stabilizing its phenyl ring in the tyrosine pocket between helices F and H. The side chain of Cys93ß is in contact with the phenyl ring of the penultimate Tyr in the pocket. If the residues at 88{alpha} and 93ß in these similar environments were functionally homologous, the mutation Ala->Cys at 88{alpha} should have caused functional changes opposite to those caused by the mutation Cys->Ala at 93ß. Obviously, this is not the case according to our present experimental results.

Tolerance of substituting other residues for Ala88{alpha} and Cys93ß is very narrow (Table VGo). The present study has added further evidence that these residues play definite roles in Hb. Their roles still remain undefined. However, in addition to the new functional model proposed by Stamler's group (see Introduction), the recent finding by Balagopalakrishna et al. (1998) that Cys93ß contributes to inhibition of autooxidation of the heme iron seems interesting and promising.


    Acknowledgments
 
We thank Drs Y.Igarashi and K.Ichimura (Department of Biochemistry, Dokkyo University School of Medicine) for their kind cooperation in preparation of the recombinant Hbs and for discussions.


    Notes
 
3 To whom correspondence should be addressed Email: kimai{at}phys1.med.osaka-u.ac.jp Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received September 30, 1999; revised November 11, 1999; accepted November 25, 1999.





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