The Anodic Hemoglobin of Anguilla anguilla
MOLECULAR BASIS FOR ALLOSTERIC EFFECTS IN A ROOT-EFFECT HEMOGLOBIN*

(Received for publication, February 25, 1997, and in revised form, April 8, 1997)

Angela Fago Dagger §, Emøke Bendixen §par , Hans Malte Dagger and Roy E. Weber Dagger

From the Dagger  Department of Zoophysiology, Institute of Biological Sciences, Building 131 and the par  Department of Molecular Biology, University of Aarhus, 8000 Aarhus C, Denmark

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The functional and structural basis for the Root effect has been investigated in the anodic hemoglobin of the European eel, Anguilla anguilla. This hemoglobin exhibits a large Bohr effect, which is accounted for by oxygen-linked binding of seven to eight protons in the presence of GTP at pH 7.5. Oxygen equilibrium curves show nonlinear lower asymptote of Hill plots, indicating the occurrence of heme-heme interactions within the T state. Analysis of the curves according to the co-operon model (Brunori, M., Coletta, M., and Di Cera, E. (1986) Biophys. Chem. 23, 215-222) reveals that T state cooperativity is positive at high pH and in the stripped hemoglobin (where the T right-arrow R allosteric transition is operative) and negative at low pH and in the presence of organic phosphate (where the molecule is locked in the low affinity structure), indicating site heterogeneity. The complete amino acid sequence of eel anodic hemoglobin has been established and compared with that of other fish hemoglobins. The presence of the Root effect correlates with a specific configuration of the alpha 1beta 2 switch interface, which at low pH would stabilize subunit ligation in the T state without changing the quaternary structure. We propose that the major groups involved in the binding of oxygen-linked protons in eel anodic hemoglobin are located on the beta  chain and comprise His-HC3 at the C terminus, His-FG4 at the switch interface, and Lys-EF6 and the N terminus at the phosphate-binding site.


INTRODUCTION

In contrast to mammals, fish show a large variation in the number of hemoglobin (Hb) components and in the mechanisms of oxygen binding modulation, which relates to their ability to adapt to widely different environmental conditions (1, 2). Conventionally, fish hemoglobins are divided into electrophoretically "cathodic" components (with high isoelectric points, pI >=  8.0) and "anodic" ones (with low isoelectric points, pI <=  8.0) that differ markedly in their functional properties (3). The European eel (Anguilla anguilla) may be considered as the simplest model with two functionally distinct major hemoglobins (4): a cathodic Hb with high oxygen affinity that is weakly affected by pH (with a small Bohr effect) (5) and an anodic Hb showing low oxygen affinity and large Bohr and Root effects similar to many anodic fish hemoglobins.

The Root effect of teleost fish is a large decrease in the oxygen affinity at low pH whereby the hemoglobin molecule cannot be fully saturated with oxygen even at very high oxygen tensions (6). The physiological role of Root-effect hemoglobins is to secrete oxygen into the swim bladder and the eye against high oxygen pressures following local acidification of the blood in a countercurrent capillary system (7). During respiratory or metabolic acidosis, oxygen binding to anodic hemoglobins may be hampered by their strong Bohr and Root effects, whereby oxygen transport may increasingly depend on the pH-independent and highly cooperative cathodic components that are commonly found in active fish species.

The Root effect originates from a strong, proton-dependent stabilization of the low affinity T (tense) quaternary structure relative to the high affinity R (relaxed) state (8). This inhibits the T right-arrow R allosteric transition and causes a drastic reduction in the Hill coefficient n50 (the degree of cooperativity) to values close to unity or below. The T right-arrow R quaternary transition involves a rotation of the two alpha 1beta 1 and alpha 2beta 2 dimers relative to each other so that large conformational changes occur at the interdimer alpha 1beta 2 and alpha 2beta 1 interfaces, whereas the intradimer alpha 1beta 1 and alpha 2beta 2 interfaces remain virtually unaffected (9). Salt bridges and hydrogen bonds are broken in the transition from the T to the R state, resulting in the release of Bohr protons. These noncovalent interactions stabilize the T relative to the R state and act as constraints that determine the low affinity of the T state (10).

Although several mechanisms have been proposed, the molecular basis for the extraordinarily high stability of the T state in Root-effect hemoglobins is not yet fully understood. In the stereochemical model of Perutz and Brunori (11), the substitution of Cys-F9beta (in human HbA) with Ser (in Root-effect fish hemoglobins) was indicated as a crucial factor. However, site-directed mutagenesis experiments on human HbA showed that this substitution is not sufficient to induce the Root effect (12). More recent studies on the crystal structure of the carbonmonoxy form of spot Hb (Leiostomus xanthurus) have suggested that electrostatic repulsions between the positively charged residues (including the N terminus of the beta  chain Lys-EF6beta , Arg-H21, and His-HC3beta ) protruding into the central cavity between the beta  chains would destabilize the R state at low pH and induce the R right-arrow T transition that characterizes Root-effect hemoglobins (13). In spot Hb in the R state, the central cavity is narrower than in human HbA due to the presence of bulky Trp-NA3beta and Met-EF2beta residues (replacing Leu and Val in human HbA, respectively) and to a 3° larger rotation of the alpha 1beta 1 relative to the alpha 2beta 2 dimer (a similar rotation is found also in the Hb1 of the antarctic teleost Pagothenia bernacchii (14)). However, this mechanism alone does not satisfactorily explain the absence of the Root effect in Hb1 of another antarctic nototheniid, Trematomus newnesi (15), which has all the necessary key residues except that Lys-EF6beta is replaced by Ala (the same substitution is present in Notothenia angustata Hb1, which shows the Root effect (16)). A bulky Met residue at position E19beta in T. newnesi Hb1 has been suggested to interfere with the cluster formation (13), although a bulky Ile residue is present at this position in other Root-effect hemoglobins like those of goldfish or carp.

It appears that the search for the structural basis of the Root effect should not only concern the structural differences between the two end states, i.e. the fully deoxygenated T and the fully ligated R state, but should also focus on the molecular mechanisms that allow the hemoglobin molecule to remain in the T state even when it is in the ligated form.

According to the two-state MWC model1 (17), cooperativity of oxygen binding arises from a concerted transition between the T and R states, both with noncooperative oxygen binding. The MWC model satisfactorily describes highly cooperative systems with a major allosteric equilibrium, which is the reason for its widespread utility in the analysis of ligand interactions. Nevertheless, T state crystals of human HbA bind oxygen cooperatively (18-20). The presence of complex positive (intradimer) and negative (interdimer) cooperative interactions has been revealed in the T state of human HbA (21, 22). Based on analysis of tetramer-dimer dissociation equilibria at different ligation stages, Ackers and co-workers (22) identified a third and intermediate allosteric structure between the T and R states of human HbA where ligand-induced tertiary conformational changes are accommodated within the T quaternary structure while the alpha 1beta 2 interface acts as a constraint. The quaternary transition to the R state occurs when the two dimers have at least one ligated subunit, since the alpha 1beta 2 interface is then no longer stable in the T conformation. Accordingly, the release of the Bohr protons upon oxygenation is stepwise and comprises a tertiary Bohr effect, which reflects ligation within the T state and a quaternary Bohr effect, following the T right-arrow R transition (23). The presence of cooperative interactions nested within a quaternary structure (24) becomes apparent when the cooperativity originating from the major T right-left-harpoons  R allosteric equilibrium is inhibited as it is for Root-effect hemoglobins. As they may remain in the T state, even in the presence of oxygen, Root-effect hemoglobins represent ideal candidates for the study of the cooperative interactions occurring within the T state of tetrameric vertebrate hemoglobins.

We have analyzed the allosteric properties of the Root-effect anodic eel Hb following the co-operon model (25, 26) by assuming cooperative interactions within the T quaternary state along with the T right-left-harpoons  R cooperative transition. The co-operon model has been successfully applied to many cooperative systems, including those of the giant oxygen-carrying molecules from invertebrates, where a concerted allosteric reaction of >100 subunits (according to the MWC model) appears unlikely (25). To our knowledge, this is the first report of such analysis of the allosteric properties of a Root-effect hemoglobin. Moreover, this study reports the amino acid sequence of the anodic eel Hb, which reveals a possible structural mechanism for the Root effect and allows identification of the potential binding sites for Bohr protons. It emerges from this study that an essential condition for the occurrence of the Root effect is that at low pH the alpha 1beta 2 interface remains stable in the T state upon oxygen binding. This extends the basic stereochemical interpretation proposed by Perutz and Brunori (11) and, more recently, by Mylvaganam et al. (13).


EXPERIMENTAL PROCEDURES

Purification of Anodic Hb

Preparation of the hemolysate, separation from the cathodic Hb, and simultaneous stripping from organic phosphates by fast protein liquid anion-exchange chromatography were performed as described previously (5).

Amino Acid Sequence Analysis

Heme removal and precipitation of the globin chains were performed by the acid/acetone method (27). The alpha  and beta  chains were separated by RP-HPLC on a Waters µBondapak C18 column (0.39 × 30 cm) by a linear gradient of 70% acetonitrile (solvent B) in 50% acetonitrile, 0.3% trifluoroacetic acid (solvent A) as described (5). Reduction and carboxymethylation of SH groups were performed by incubating 2 mg of globin chain with 5 mM dithiothreitol in M guanidine hydrochloride, 0.25 M Tris-HCl buffer, pH 9.0, for 50 min at room temperature followed by the addition of solid iodoacetic acid to a final concentration of 80 mM and adjustment of the pH with 1 M KOH. The reaction was stopped after 20 min by RP-HPLC. Alternatively, the carboxymethylation reaction was performed on the globin mixture containing both alpha  and beta  chains and terminated by dialysis against double distilled water before RP-HPLC separation of the globin chains. The change in retention time on RP-HPLC of the globin chains after carboxymethylation indicated full alkylation at the SH groups. The alkylated chains were dissolved in 1% ammonium bicarbonate and digested overnight by L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin at room temperature at an enzyme:protein weight ratio of 1:100 (w/w). Digestion of the alpha chain was performed in 2 M urea. Tryptic peptides were separated by RP-HPLC on a Waters µBondapak C18 column by a linear gradient of 70% acetonitrile, 0.08% trifluoroacetic acid (solvent B) in 2% acetonitrile, 0.1% trifluoroacetic acid (solvent A) at a flow rate of 1 ml/min. Cleavage by CNBr was performed in 70% formic acid (28). CNBr-generated fragments of the beta  chain were separated by RP-HPLC on a Nucleosil C18 column eluted by a linear gradient of 90% acetonitrile, 0.075% trifluoroacetic acid (solvent B) in 0.1% trifluoroacetic acid (solvent A). Selective cleavage at glutamic acid residues by Staphylococcus aureus protease V8 was performed in 0.1 M ammonium bicarbonate at 37 °C for 3 h (28). Deacylation at the N terminus of the alpha  chain was obtained by incubating the N-terminal blocked peptide with 30% trifluoroacetic acid at 55 °C for 3 h. Amino acid composition analyses were performed as described (29). The amino acid sequence was determined by automated Edman degradation using a pulsed liquid sequencer model 477A from Applied Biosystems equipped with a 120A analyzer for the detection of the phenylthiohydantoin-derivatives.

Mass Spectrometry Measurements

MALDI-TOF-MS was carried out on a Bruker Biflex instrument (Bruker-Franzen, Bremen, Germany) equipped with an ultraviolet laser at 337 nm. The samples dissolved in 0.1% trifluoroacetic acid were mixed with 2 µl of alpha -cyano-4-hydroxycinnamic acid, and 0.9 µl were applied to the target. 30-100 calibrated mass spectra were averaged.

Oxygen Binding Studies

Oxygen equilibria were measured in the pH range of 6.5-8.3 in 0.1 M HEPES buffer in the absence and presence of 0.1 M KCl or 1 mM GTP at 20 °C and at a hemoglobin concentration of 200 µM heme. The pH values of the hemoglobin solutions were measured by a thermostated Radiometer BMS Mk2 capillary microelectrode. Cl- concentration was assayed by coulometric titration (Radiometer CMT 10).

In tonometrical oxygen equilibrium experiments (30), hemoglobin solutions were equilibrated to different oxygen tensions in thermostated tonometers (Eschweiler, Kiel, Germany) coupled to cascaded Wösthoff pumps for mixing humidified pure N2 (99.998%) with air or O2. To obtain the spectrum of the fully oxygenated hemoglobin at the end of the measurement, the pH of the sample (equilibrated with pure O2) was raised to pH 8.0-8.5 by the addition of solid HEPES salt. To minimize autooxidation during oxygen binding experiments, the methemoglobin-reducing system (31) was added to the hemoglobin solution (1 ml) as described by Imai (32) but using 1 µl of each reagent instead of 20 µl to minimize the possible effects of phosphates (as NADP or glucose 6-phosphate) on oxygen binding. No absorbance peak at 630 nm could be detected during the experiments, indicating that methemoglobin formation was negligible. To evaluate the Root effect, oxygen saturation was measured at different pH values in Eschweiler tonometers equilibrated with pure oxygen under the same experimental conditions used to measure the oxygen equilibria. The spectrum of the deoxygenated hemoglobin was obtained after equilibration with pure N2. The value of 100% saturation was assigned to stripped hemoglobin at pH > 8.2.

Oxygen binding equilibria were also measured using a thin layer equilibration technique (modified gas diffusion chamber) utilizing cascaded Wösthoff pumps for mixing humidified pure N2 (99.998%) with air or O2 to obtain stepwise increases in oxygen tension (33, 34). The fractional saturation values were corrected for the incomplete oxygen saturation in the presence of 1 atm of oxygen due to the Root effect. The saturation achieved in the presence of oxygen (Y) was interpolated from the plot Y versus pH obtained in tonometrical experiments. Because of the rapidity of this method and the possibility to correct graphically for the small amount (<5%) of methemoglobin formed during the experiment (35), no reducing system was needed.

For detailed analysis of the allosteric interactions, diffusion chamber measurements at very low and high fractional saturation were carried out in the presence of the reducing system described above. Nonlinear least squares fitting was performed according to the co-operon model (25, 26). The binding polynomial for hemoglobin in the T state is as follows,
P<SUB><UP>T</UP></SUB>=(1+2K<SUB><UP>T</UP></SUB>P<UP>O</UP><SUB>2</SUB>+iK<SUP>2</SUP><SUB><UP>T</UP></SUB>P<UP>O</UP><SUP>2</SUP><SUB>2</SUB>)<SUP>2</SUP> (Eq. 1)
where KT is the intrinsic ligand affinity and i describes the cooperative interactions within the T quaternary structure. Values of i above or below unity indicate positive and negative cooperativity, respectively. When i = 1, the T state binds oxygen with no cooperativity, and the model becomes identical to the classical two-state MWC model. The binding polynomial for a noncooperative R state with ligand affinity KR is therefore that of the MWC model,
P<SUB><UP>R</UP></SUB>=(1+K<SUB><UP>R</UP></SUB>P<UP>O</UP><SUB>2</SUB>)<SUP>4</SUP> (Eq. 2)
According to these relations, the equation describing the fractional saturation in tetrameric hemoglobins (36) is,
Y=<FR><NU>L K<SUB><UP>T</UP></SUB>P<UP>O</UP><SUB>2</SUB>[1+(2+i)K<SUB><UP>T</UP></SUB>P<UP>O</UP><SUB>2</SUB>+3iK<SUP>2</SUP><SUB><UP>T</UP></SUB>+i<SUP>2</SUP>K<SUP>3</SUP><SUB><UP>T</UP></SUB>P<UP>O</UP><SUP>3</SUP><SUB>2</SUB>]+K<SUB><UP>R</UP></SUB>P<UP>O</UP><SUB>2</SUB>(1+K<SUB><UP>R</UP></SUB>P<UP>O</UP><SUB>2</SUB>)<SUP>3</SUP></NU><DE>L(1+2K<SUB><UP>T</UP></SUB>P<UP>O</UP><SUB>2</SUB>+iK<SUP>2</SUP><SUB><UP>T</UP></SUB> P<UP>O</UP><SUP>2</SUP><SUB>2</SUB>)<SUP>2</SUP>+(1+K<SUB><UP>R</UP></SUB>P<UP>O</UP><SUB>2</SUB>)<SUP>4</SUP></DE></FR> (Eq. 3)
where L is the allosteric constant in the absence of ligand (17). The parameters of the equation were estimated from nonlinear least squares curve fitting. In addition, to minimize errors introduced by incomplete saturation or desaturation when equilibrating with pure oxygen or pure nitrogen, respectively, the absorbance values at zero (A0) and full saturation (A100) were extrapolated from the data in the fitting procedure. In practice, the apparent saturation values (Y') were calculated as follows,
Y′=(A−A′<SUB>0</SUB>)/(A′<SUB>100</SUB>−A′<SUB>0</SUB>) (Eq. 4)
where A0' and A100' are the absorbance values measured in the presence of pure nitrogen and pure oxygen, respectively. The relationship between the true (Y) and the apparent (Y') saturations is given by,
Y=(Y′−Y′<SUB>0</SUB>)/(Y′<SUB>100</SUB>−Y′<SUB>0</SUB>) (Eq. 5)
or
Y′=Y′<SUB>0</SUB>+(Y′<SUB>100</SUB>−Y′<SUB>0</SUB>)Y (Eq. 6)
where Y0 = (A0 - A0')/(A100' - A0') and Y100' = (A100 - A0')/(A100' - A0') were the parameters fitted along with L, KT, KR, and i, and Y is the expression of the fractional saturation according to Equation 3. Fitting was performed on Hill-transformed data, log[Y'/(1 - Y')] versus log PO2,, with equal weighting of data points. The curve fitter employed the method of Levenberg-Marquardt, and the standard errors on the parameters were estimated from the diagonal elements of the curvature matrix associated with the fit.


RESULTS

Structural Characterization

The anodic Hb of eel is a single component with an isoelectric point of 6.35, as indicated by isoelectrofocusing on polyacrylamide gel (5). Consistently, MALDI-TOF-MS measurements and RP-HPLC separation of the globin chains showed only two peaks corresponding to the alpha  and beta  chains that differ from those of the cathodic Hb (Fig. 1). Tryptic peptides of the S-carboxymethylated alpha  and beta chains were purified by RP-HPLC as shown in Fig. 2. All peptides were sequenced and aligned by homology with the sequences of other fish globin chains. The complete amino acid sequence of the alpha and beta  chains of the anodic Hb of eel is reported in Fig. 3 where the sequence portions elucidated by peptide sequencing are indicated. The intact beta  chain was sequenced up to Ile-20, whereas the alpha  chain was blocked and thus not accessible to Edman degradation. The sequence of the N-terminal peptide T1 of the alpha  chain was obtained after unblocking the N terminus as described under "Experimental Procedures." The difference between the molecular mass of the alpha  chain measured by mass spectrometry (15,970 Da) and that deduced from the amino acid sequence (15,904 Da) is consistent with the presence of an acetyl group at the N terminus, as found in the alpha  chains of other fish hemoglobins. The molecular mass of the beta  chain measured by mass spectrometry (16,783 Da) is in agreement with that deduced from the sequence (16,764 Da) within experimental error. In the alpha  chain, trypsin failed to cleave at Lys-128, and the tryptic peptide from Phe-129 to Arg-140 was not recovered after RP-HPLC purification. The corresponding sequence was obtained after subfragmentation of the peptide T15 (extending from Ile-101 to Arg-140, sequenced up to Phe-130). T15 was subjected to CNBr cleavage, followed by S. aureus V8 digestion. In this way, small fragments were generated (Ile-101-Met-107, Val-108-Met-112, and Thr-113-Glu-121) together with the larger V1 (Val-122-Arg-140). The peptide mixture was then subjected to automated Edman degradation, where the amino acid sequence of V1 could be unequivocally established by taking advantage of the early termination of the shorter peptides. Incomplete trypsin digestion was found at Lys-47, Lys-58, and Arg-93 in the alpha  chain and at Arg-29 and Lys-59 in the beta  chain. No tryptic cleavage occurred after Arg-8 in the beta  chain probably because it follows three acid residues in the sequence. The fragments Thr-9-Lys-17 and Val-60-Lys-62 of the beta  chain were not recovered after RP-HPLC purification, and the corresponding sequences were obtained from N-terminal sequencing of the intact chain and from peptide T5, respectively. The beta  chain was also cleaved by CNBr, and the fragments were separated by RP-HPLC. The presence of two adjacent Arg residues (Arg-29-Arg-30) in the sequence of the beta chain was confirmed by sequencing the first three steps of the CNBr-generated fragment CB1 (Fig. 3). The sequences of peptides T3 and T12 in the beta  chain (eluting in the same peak in RP-HPLC in Fig. 2) were obtained after their separation in a second purification step on RP-HPLC.


Fig. 1. RP-HPLC separation of the alpha  and beta  chains of the anodic (alpha a and beta a) eel Hb compared with that of the cathodic Hb globin chains (alpha c and beta c). Details are given under "Experimental Procedures." %B, % buffer B.
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Fig. 2. RP-HPLC separation of the tryptic peptides of the S-carboxymethylated alpha  (upper panel) and beta  (lower panel) chains of anodic eel Hb. Details are given under "Experimental Procedures." %B, % buffer B.
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Fig. 3. Amino acid sequence of the alpha  and beta  chains of the anodic Hb of the eel. The segments indicate the sequence portions elucidated by automated Edman degradation of peptides obtained after cleavage with trypsin (T1-T16), V8 protease (V1 in the alpha chain), and cyanogen bromide (CB1 in the beta  chain). The arrow indicates the N-terminal sequence obtained from the intact beta  chain.
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Functional Characterization

A large Bohr effect is observed in eel anodic Hb (Fig. 4). The Bohr factor (phi  = delta  log P50/delta pH, which represents the average number of protons bound upon heme oxygenation) at pH 7.5 is -0.27 in the stripped hemoglobin and -1.85 in the presence of GTP, the major erythrocytic phosphate in eel (4). This indicates that at pH 7.5 the number of oxygen-linked protons bound by tetrameric hemoglobin in the deoxygenated state can be increased by GTP from 1.08 (in the stripped hemoglobin) to 7.4. The Bohr effects measured in the absence and in the presence of GTP can be superimposed by a simple shift of the pH scale, indicating that the effect of GTP is essentially to increase the pKa values of the Bohr groups. The decrease in saturation at low pH in the presence of GTP and 1 atm of oxygen indicates the presence of the Root effect (Fig. 4, inset). Accordingly, the Hill coefficient n50 falls to approximately 0.5 at pH 6.6 (Fig. 4).


Fig. 4. Bohr effect (pH dependence of oxygen tensions and cooperativity at half-saturation, P50 and n50) of the anodic eel Hb measured at 20 °C in 0.1 M HEPES buffer in the absence and presence of 0.1 M KCl or 1 mM GTP. Oxygen binding experiments were performed with a modified diffusion chamber (open symbols) or tonometrically (closed symbols). Stars (star ) indicate the low P50 and high n50 values obtained in the presence of 1 mM GTP with the diffusion chamber technique when 100% saturation in pure oxygen is assumed. The heme concentration is 200 µM. The inset represents the pH-dependent saturation decrease in the presence of pure oxygen (Root effect) measured tonometrically under the same experimental conditions.
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Good agreement is found between the P50 values obtained by the diffusion chamber and the tonometrical methods, provided that the incomplete oxygen saturation, which occurs at low pH because of the Root effect, is taken into account. By assuming 100% saturation in the presence of pure oxygen in diffusion chamber experiments, a lower P50 and higher n50 are found at low pH with GTP (Fig. 4). The agreement between the data obtained with the two techniques, moreover, indicates that small concentrations of the enzymatic reducing system (present only in tonometrical studies) does not influence the oxygen binding properties of hemoglobin. A weak effect of KCl on the oxygen affinity is illustrated in both tonometrical and diffusion chamber experiments (Fig. 4).

Extended Hill plots for oxygen equilibrium experiments at different pH values and in the absence and presence of GTP are shown in Fig. 5, where deviations from linearity of the lower asymptote reflecting cooperative interactions in the T state are evident. A unitary slope in the upper asymptote indicates noncooperative binding in the R state. The equilibrium data are satisfactorily described by the co-operon model, as indicated by the curves obtained by fitting Equation 5 to the data (Fig. 5). The allosteric parameters obtained in the fitting procedures are reported in Table I. The parameter i describes the overall or the apparent cooperative interactions within the T state as positive (i > 1) or negative (i < 1). Reliable estimates for KR are in general difficult to obtain (in particular, in a Root-effect hemoglobin) as they require several data points at saturations >=  99%. The KR value reported in Table I is thus the mean value (±10%) calculated for the stripped hemoglobin at pH 7.935 and held fixed to fit the allosteric constants in the other data sets as described (37). No significant variations were observed in, and regardless of, the values of the other parameters. Partly because of the high number of parameters fitted, a large uncertainty was found in the determination of L and i, but not in KT. Compensating variations of L and i are presumably implied in nonconvergence in three data sets (Table I).


Fig. 5. Extended Hill plots of stripped anodic eel Hb in the presence (right panel) and absence (left panel) of 1 mM GTP at different pH values. Oxygen binding equilibria were measured at 20 °C in 0.1 M HEPES buffer and at a heme concentration of 200 µM. The curves represent fitting of the experimental data (open and closed symbols) obtained according to the co-operon model (the parameters derived from fitting are reported in Table I). The data shown have been corrected for the incomplete oxygen saturation due to the Root effect as described under "Experimental Procedures."
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Table I. Parameters for oxygen binding in eel anodic hemoglobin derived from fitting the oxygen equilibrium data reported in Fig. 5 to the co-operon model, as described under "Experimental Procedures"

The experimental conditions were as described in Fig. 5.

pH GTP KT KR L i Y'100 Y'0

torr-1 torr-1
7.935  - 0.0856  ± 0.0238 0.4920 15.60  ± 16.86 4.0846  ± 7.2996 0.9973  ± 0.0021 0.0077  ± 0.0006
7.481a  - 0.1080  ± 0.0275 0.4920 27.29  ± 71.37 2.5970  ± 6.3769 0.9967  ± 0.0009 0.0048  ± 0.0005
7.106a  - 0.0891  ± 0.0124 0.4920 2.35 × 102  ± 1.04 × 103 2.7747  ± 3.7093 1.0010  ± 0.0027 1 × 10-9  ± 0.0036
6.630  - 0.0300  ± 0.0019 0.4920 1.03 × 104  ± 5.29 × 103 3.2821  ± 0.8328 1.0103  ± 0.0018 0.0047  ± 0.0003
7.882 + 0.0515  ± 0.0048 0.4920 247.30  ± 358.01 5.4527  ± 4.8637 0.9997  ± 0.0008 0.0089  ± 0.0008
7.482 + 0.0313  ± 0.0020 0.4920 11.91 × 105  ± 1.32 × 103 0.8797  ± 0.2456 1.0170  ± 0.0024 0.0050  ± 0.0008
7.093 + 0.0272  ± 0.0055 0.4920 3.13 × 107  ± 9.24 × 107 0.1066  ± 0.0779 1.1404  ± 0.1703 0.0011  ± 0.0010
6.630a + 0.0357  ± 0.0015 0.4920 1.08 × 109  ± 1.55 × 1010 0.0085  ± 0.0070 1.1351  ± 0.0302 0.0009  ± 0.0001

a Not converged.

As illustrated in Table I, increasing proton concentration stabilizes the T relative to the R state (L increases) and decreases KT (as generally is observed in vertebrate hemoglobins) in the stripped hemoglobin. In the presence of GTP, KT does not decrease further below pH 7.48 where the apparent cooperativity in the T state becomes negative as i decreases to values significantly below unity at low pH. Under these conditions, the hemoglobin molecule can be considered as locked in the low affinity conformation as indicated by the high values of L. In the absence of cofactors, i remains above unity in the pH range investigated.


DISCUSSION

A remarkable feature of the oxygen equilibrium curves for the anodic Hb of eel is the deviation from linearity of the lower asymptote, which reflects the presence of cooperative interactions within the T state. Such behavior has not been reported before in other fish hemoglobins where such low oxygen saturation levels were not analyzed. In T state crystals of human HbA, a small amount of cooperativity compensates for inequivalent binding to the alpha and beta  subunits, resulting in perfectly noncooperative (n50 = 1) oxygen binding (18, 19). This fits neatly with the unitary slope of the lower asymptote observed in an extended Hill plot of human HbA in solution, which allows the use of the two-state MWC model for analysis of the allosteric properties. In the anodic eel Hb as well as in other Root-effect hemoglobins (38, 39), highly biphasic oxygen binding curves with apparent negative cooperativity are observed at low pH and in the presence of organic phosphate (Fig. 5). These particular oxygen binding properties together with the nonlinear lower asymptote cannot be described by the MWC model. We show that the functional properties of eel anodic Hb can be described by assuming the presence of cooperative interactions in the T state, as included in the co-operon model (25, 26). In its original formulation, the model assumes that the hemoglobin tetramer consists of two independent alpha beta dimers, each representing a cooperative unit or co-operon. However, later studies have shown that the two alpha beta dimers (alpha 1beta 1 and alpha 2beta 2) cannot be considered as functionally unrelated but that in the T state, ligation at one subunit enhances ligation at the other subunit of the same dimer and inhibits ligation at the opposite dimer (22). In our study, the parameter i therefore includes not only the intradimer cooperativity as in the original model but also any functional interaction between dimers across the alpha 1beta 2 interface. Moreover, the situation in Root-effect hemoglobins is complicated by a large functional heterogeneity of the chains in the T state (40, 41), which is responsible for biphasic oxygen equilibrium curves at low pH and with organic phosphates and would contribute negatively to the T state cooperativity and decrease the value of i. This would explain both the low values of i (Table I) and n50 (Fig. 4) found under these conditions, where the tetrameric molecule is essentially in the T state. It is important to note that the effect of organic phosphates is to shift the allosteric equilibrium toward the T state and to increase the pKa of Bohr groups (Fig. 4) rather than to enhance functional subunit heterogeneity in itself (41), in agreement with the conclusion that organic phosphates and protons have a similar allosteric effect (42). The apparent negative cooperativity between the alpha  and beta  chains found at low pH with GTP therefore reflects a larger pH dependence (or a larger tertiary Bohr effect) of one of the two chains in the T state. By this mechanism, eel anodic Hb may release oxygen even in the absence of the T right-arrow R quaternary transition, which is the basis for the large Bohr effect observed in Root-effect hemoglobins. The increase in L and in the functional subunit heterogeneity at low pH is the basis for the larger decrease in oxygen saturation at high oxygen pressures found in Root-effect hemoglobins. In human HbA, a pH decrease produces an increase in L and a decrease in KT (32) so that the decrease in oxygen saturation is larger at low oxygen tension. These two different mechanisms may relate to the different physiological roles of the two pH effects. The Bohr effect enhances the amount of oxygen released in the metabolizing tissues (at low oxygen tension), and the Root effect allows release of a large amount of oxygen in the eye and swim bladder (at high oxygen tensions (38)).

The inverse relationship between L and i suggests that in the T state of eel anodic Hb, positive cooperative interactions prevail in the presence of the T right-arrow R transition, whereas negative interactions become apparent in the absence of the quaternary transition. Although a detailed analysis of the opposing factors contributing to heme-heme interactions is impossible at this stage, it appears that at the level of the alpha 1beta 2 interface, cooperative interactions in the T state may either be negative (and produce the T right-arrow R transition, as in human HbA (22)) or positive, thereby allowing ligand binding to proceed through the T state. Thus, a fundamental difference between Root-effect hemoglobins and hemoglobins with a normal Bohr effect such as human HbA is that at low pH, Root-effect hemoglobins remain in the T state, indicating that the alpha 1beta 2 interface remains stable in the T state upon oxygenation, whereas human HbA switches to the R conformation. The comparison of the primary structure of the anodic hemoglobin of the eel with that of the cathodic Hb and other fish hemoglobins reveals that several concomitant factors may contribute to the expression of the Root effect in fish hemoglobins: a particular configuration of the alpha 1beta 2 and beta 1beta 2 interfaces and the presence of proton binding groups.

The alpha 1beta 2 Contact

Side-chain packing at this interface is likely to be the major reason for the larger rotation of the two dimers in the R state found in the hemoglobins of spot and P. bernacchii compared with human HbA. Moreover, a different alpha 1beta 2 interface in fish hemoglobins is consistent with the lower tendency to split into dimers than human HbA (43).

In human HbA, at the dovetailed alpha 1beta 2 switch contact (between the C helix and CD corner of the alpha  subunit with the FG corner of the beta  subunit), His-FG4beta packs between the side chains of Pro-CD2alpha and Thr-C6alpha in the T state, passes over one helix turn during the T right-arrow R transition, and packs between Thr-C6alpha and Thr-C3alpha in the R state. The ability of Root-effect hemoglobins to remain in the T state even when ligated may be related to a switch region different from that of human HbA and other fish hemoglobins (Table II). On the alpha  chain, Gln is present at position C3, Thr or Ala is present at C6, a small residue (Ser, Ala, Thr) replaces the bulky Pro at position CD2, and Trp is highly conserved at CD4 whereas His is conserved at position FG4 of the beta  chain. Moreover, fish hemoglobins possess an additional residue in position CD5 of the alpha  chain compared with human HbA (Table II). Ligation of beta  subunits within the T state of human HbA results in profound steric hindrance between the side chains of His-FG4beta and Pro-CD2alpha (44). The substitution of Pro-CD2alpha with a smaller and more flexible residue in Root-effect hemoglobins (Ser, Ala, or Thr; Table II) is likely to stabilize beta  chain ligations in the T state. Ligation of alpha  subunits in the quaternary T state may be favored by replacements at the flexible joint interface between the FG corner of the alpha chain and the C helix of the beta  chain. In deoxy human HbA, Arg-FG4alpha is bound to Glu CD2beta , which is replaced by a neutral amino acid residue in Root-effect hemoglobins (Ser, Ala or Gly; Table II), so that Arg-FG4alpha may instead form a hydrogen bond with Gln-C5beta (conserved in all fish hemoglobins) as found in deoxy trout (Oncorhynchus mykiss) HbI (45). The same interaction between Arg-FG4alpha and Gln-C5beta is present in oxygenated crystals of T state human HbA (20), which indicates that the replacement of Glu-CD2beta may stabilize intermediates in the oxygenation process of T state molecules (45). The location of the groups at the switch contact and at the flexible joint that may stabilize ligand binding in the T state is shown in Fig. 6.

Table II. Conservation of functionally important residues in Root-effect hemoglobins in comparison with non-Root-effect hemoglobins

Amino acid sequences are from the Swiss protein data bank except for P. antarcticum (46), T. newnesi (15), and A. mitopteryx (52).

 alpha Chain
 beta Chain
C3 C6 CD2 CD4 CD5 NA2 NA3 CD2 EF6 F9 FG1 FG4 H21 HC3

Root-effect Hb
  A. anguilla anodic Hb Gln Ala Ala Trp Lys Glu Trp Ala Lys Ser Glu His Arg His
  O. mykiss HbIV Gln Ala Ser Trp Ala Glu Trp Ser Lys Ser Glu His Arg His
  Cyprinus carpio Gln Thr Ala Trp Ala Glu Trp Ala Lys Ser Glu His Arg His
  N. angustata Hb1 Gln Thr Ser Trp Pro Lys Trp Ser Ala Ser Glu His Lys His
  Chelodonichtys kumu Gln Thr Thr Trp Thr Glu Trp Ala Lys Ser Glu His Arg His
  P. bernacchii Hb1 Gln Thr Ser Trp Pro Glu Trp Ser Ala Ser Glu His Lys His
  P. antarcticum Hb1 Gln Thr Ser Trp Pro Glu Trp Gly Ala Ser Gln His Lys His
Non-Root-effect Hb
  Human Thr Thr Pro Phe His Leu Glu Lys Cys Asp His His His
  Lepidosiren paradoxus Gly Ser Pro Phe Gly His Trp Asn Lys Ser Glu His Arg His
  Latimeria chalumnae Gln Val Asp Phe Thr His Trp Lys Lys Phe His His Arg His
  Electrophorus electricus Glu Thr Ala Trp Ser Glu Leu Ala Lys Ser Glu His Lys His
  G. acuticeps Gln Ile Ser Trp Pro Asn Trp Ser Glu Ser Glu His Lys His
  T. newnesi Hb1 Gln Ile Ser Trp Pro Glu Trp Ser Ala Ser Glu His Lys His
  A. mitopteryx Gln Ile Asn Trp Pro Glu Trp Gly Ala Ser Glu His Lys Val
  O. mykiss HbI Gln Thr Ser Trp Ala Glu Trp Gly Leu Ala Asn Phe Ser Phe
  A. anguilla cathodic Hb Ala Val Ser Trp Pro Glu Trp Gly Lys Asn Glu Asn Lys Phe


Fig. 6. Schematic representation of the deoxygenated structure of a Root-effect hemoglobin (P. bernacchii Hb1 is from the Brookhaven National Laboratory Protein Data Bank, file 1HBH), where the residues Gln-C3(38)alpha , Thr-C6(41)alpha , Ser-CD2(44)alpha , Ser-CD2(43)beta , and His-FG4(97)beta at the alpha 1beta 2 and alpha 2beta 1 interfaces are indicated. The notations A, B, C, and D at the residues refer to the subunits alpha 1, beta 1, alpha 2, and beta 2, respectively.
[View Larger Version of this Image (106K GIF file)]

Hemoglobin molecules that remain in the low affinity T state at low pH even when ligated will also have a larger number of oxygen-linked protons or a larger Bohr factor than hemoglobins that switch to the R state upon ligation. This means that the number of proton-binding sites in Root-effect hemoglobins does not need to be higher than in human HbA. The stabilization of the T state in Root-effect hemoglobins may be achieved not by an increased number of salt bridges but by a different allosteric mechanism where the stabilization of the alpha 1beta 2 interface upon oxygenation is an essential condition.

Proton-binding Sites

Anodic eel Hb can bind seven to eight Bohr protons per tetramer in the presence of GTP at physiological pH. A major candidate as a proton-binding site is His-HC3beta , which is conserved in all Root-effect hemoglobins (Table II). Glu-FG1beta is also generally conserved in Root-effect hemoglobins (except for one of the three Root-effect hemoglobins of the antarctic Pleuragramma antarcticum where it is substituted by Gln (46)), indicating that a salt bridge between these two residues may be formed in the T state, as known for human HbA (where Asp is in position FG1beta ). Quite unexpectedly, in the crystal structure of the deoxygenated P. bernacchii Hb1 (47), His-HC3beta was found free in solution and not bound to Glu-FG1beta , which leaves a major part of the Bohr and Root effects difficult to explain. Asp-G1alpha and Asp-G3beta were indicated as possible Bohr groups in this hemoglobin, as they move closer to each other upon deoxygenation and could thereby increase their pKa and share a proton. By analogy with P. bernacchii, Hb1, Asp-G1alpha , and Asp-G3beta have been proposed as binding sites for two of the four oxygen-linked protons in spot Hb, where the remaining two protons would bind between the N terminus and His-HC3 of each beta  subunit (13). However, the contribution to the Bohr effect of these two Asp residues appears questionable since they are conserved in all fish hemoglobins including trout HbI, which exhibits pH-independent oxygen binding. Moreover, in the deoxygenated form of trout HbI, Asp-G3beta makes a salt bridge with Arg-G6beta (conserved in fish hemoglobins or replaced by Lys in antarctic teleosts), thus preventing the two Asp residues from increasing their pKa and participating in the Bohr effect (45).

We propose that another major Bohr group in eel anodic Hb and other Root-effect hemoglobin may be His-FG4beta , located at the switch interface. Histidines in both positions HC3beta and FG4beta are replaced in the cathodic hemoglobins of eel (5), trout (48), and moray (49), all showing pH-independent oxygen binding or a weak reverse Bohr effect. In human HbA where the C-terminal His of the beta  chain has been cleaved, His-FG4beta contributes to the Bohr effect under specific conditions of ionic strength and pH (50). This residue packs against different side chains of the C helix and CD corner of the alpha  chain in the T and the R state. Proton uptake by His-FG4beta upon deoxygenation would be favored by a pKa increase in the T state (by interaction with polar or negatively charged groups, including the C-terminal end of the C helix of the alpha  chain (50)) or by a pKa decrease in the R state (e.g. by a more hydrophobic environment of this His residue in the R than in the T state). An Ile residue at position C6alpha in the hemoglobins of T. newnesi (15), Gymnodraco acuticeps (51), and Aethotaxis mitopteryx (Ref. 52 and Table II) may not be able to provide the favorable environment for oxygen-linked protonation of His-FG4beta , which is consistent with the lack of a Root effect in these hemoglobins. The highly cooperative oxygen binding of these hemoglobins, even at low pH values (n50 > 2), indicates that the allosteric T right-arrow R transition is fully operative or, in other words, that the switch interface in the T state is not stable upon oxygenation. Other replacements at the C helix of the alpha  chain in the presence of His-FG4beta agree well with the absence of the Root effect (Table II).

Proton Binding at the beta 1beta 2 Interface

GTP binds in the central cavity between the two beta  chains in the T state. The binding site for organic phosphates in fish hemoglobins involves the N terminus, Asp- or Glu-NA2, Lys-EF6, and Arg-H21 (53). Since GTP increases the pKa of Bohr groups, the remaining oxygen-linked proton-binding sites in anodic eel hemoglobin thus appear to be localized in the central cavity, and the most likely candidates appear to be the N terminus of the beta  chain and Lys-EF6beta , given the high pKa of the Arg side chain. The excess of positive charges at the beta 1beta 2 interface represents a destabilizing factor for the T state of human HbA, which, in the absence of anions, results in an increased oxygen affinity due to the shift toward the high affinity state (54). The pKa of the beta  N terminus and Lys-EF6beta in the T state may therefore be lowered by adjacent positive charges (e.g. Arg-H21beta ) to values between 6.0 and 8.0, where the Bohr effect is observed. Moreover, in Root-effect hemoglobins, a narrower central cavity in the R state than in human HbA would further reduce the pKa of these groups (13). By this mechanism, the groups in the central cavity attain an increased proton affinity in the T state so that they can contribute to the alkaline Bohr effect. Replacement of Lys-EF6beta by Ala in the hemoglobins of P. bernacchii, P. antarcticum, and N. angustata may be compensated by the substitution of Arg-H21beta with Lys (Table II) that has a lower pKa. Moreover, Lys replaces Glu-NA2beta in N. angustata Hb1, in agreement with a Bohr effect larger than in P. bernacchii Hb1 (55). In the absence of organic phosphates, the positively charged residues in the central cavity appear to act as reverse Bohr groups when the alkaline Bohr groups and the residues at the alpha 1beta 2 are replaced, as proposed for the cathodic eel Hb (5).

This site is of primary importance for phosphate modulation of blood oxygen affinity. In the eel, under hypoxic conditions, oxygen affinity increases rapidly through a decrease in the intra-erythrocytic concentration of organic phosphates (56), particularly GTP, whereas the relative amount of the two hemoglobin components remains unaffected (4).

Conclusion

The present data on eel anodic Hb indicate that several regions of the tetrameric hemoglobin molecule contribute to the Root effect: 1) the alpha 1beta 2 interface with Gln-C3alpha , Thr- (or Ala)-C6alpha , a small apolar or weakly polar residue (Ala, Ser, Thr) at CD2alpha , Trp-CD4alpha and His-FG4beta at the switch contact, and a nonnegatively charged residue at CD2beta (Ala, Gly, or Ser) at the flexible joint; 2) the beta 1beta 2 interface (as indicated by Mylvaganam et al. (13)), including the N terminus of the beta  chain, Lys-EF6beta , Trp-NA3beta , and Arg-H21beta ; and 3) His-HC3beta . Substitutions in at least one of these regions correlate with the absence of the Root effect (Table II).

In the anodic eel Hb, the Bohr effect may be accounted for by proton binding at the N terminus, His-HC3, His-FG4, and Lys-EF6 of the beta  chains. Protons appear to stabilize the alpha 1beta 2 interface in the T quaternary state (even in the presence of oxygen) and destabilize the beta 1beta 2 interface in the R state, as proposed for spot Hb (13), thereby shifting the allosteric equilibrium toward the low affinity conformation. As all the proton-binding sites appear to be on the beta  chain, the increase in the functional heterogeneity observed at low pH may be consistent with a larger pH dependence of oxygen binding in the beta  than in the alpha  subunit. The role of the alpha  subunit would be to provide a favorable structure of the alpha 1beta 2 interface, which stabilizes protonation at His-FG4beta and allows the residues in the central cavity to orientate differently from human HbA. This is consistent with the observation that hybrid hemoglobin tetramers consisting of human alpha  chain and carp beta  chain do not show the Root effect (57). Another residue that may be important to the Root effect is Ser-F9beta , which is replaced in the cathodic hemoglobins of trout and eel. Although the role of Ser-F9beta in the Root effect was ruled out by site-directed mutagenesis in human HbA (12), the possibility that it may have a different environment in fish hemoglobins cannot be excluded. Site-directed mutagenesis experiments on recombinant anodic and cathodic eel hemoglobins are in progress to further investigate the molecular basis of the Root effect.


FOOTNOTES

*   This work was supported by the Danish Center for Respiratory Adaptation and the Danish Center for Molecular Gerontology (to Ole Westergaard).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The amino acid sequences reported in this paper have been deposited in the SWISS-PROT database under the accession numbers [GenBank] and [GenBank].


§   The first two authors contributed equally to this work.
   To whom correspondence should be addressed. Tel.: 45 8942 2594; Fax: 45 8619 4186; E-mail: angela{at}bio.aau.dk.
1   The abbreviations used are: MWC, Monod, Wyman, and Changeaux; RP-HPLC, reverse-phase high performance liquid chromatography; MALDI-TOF-MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

ACKNOWLEDGEMENTS

We thank Drs. Lone K. Rasmussen and Esben S. Sørensen for mass spectrometry measurements, Dr. Lars Sottrup-Jensen for the use of sequencing equipment, and Dr. Mogens Kruhøffer for helpful criticism. The technical assistance of Anny Bang, Faith Post, Annie Wetter, and Lene Kristensen is gratefully acknowledged.


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