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  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  R quaternary transition involves a rotation of the two ₁₁ and ₂₂ dimers relative to each other so that large conformational changes occur at the interdimer ₁₂ and ₂₁ interfaces, whereas the intradimer ₁₁ and ₂₂ 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-F9 (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  chain Lys-EF6, Arg-H21, and His-HC3) protruding into the central cavity between the  chains would destabilize the R state at low pH and induce the R  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-NA3 and Met-EF2 residues (replacing Leu and Val in human HbA, respectively) and to a 3° larger rotation of the ₁₁ relative to the ₂₂ 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-EF6 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 E19 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 ₁₂ 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 ₁₂ 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  R transition (23). The presence of cooperative interactions nested within a quaternary structure (24) becomes apparent when the cooperativity originating from the major T  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  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 ₁₂ 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

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).

Heme removal and precipitation of the globin chains were performed by the acid/acetone method (27). The  and  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 6 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  and  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 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  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  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.

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 -cyano-4-hydroxycinnamic acid, and 0.9 µl were applied to the target. 30-100 calibrated mass spectra were averaged.

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,

(Eq. 1)

where K_T 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 K_R is therefore that of the MWC model,

(Eq. 2)

According to these relations, the equation describing the fractional saturation in tetrameric hemoglobins (36) is,

(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,

(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,

(Eq. 5)

or

(Eq. 6)

where Y₀ = (A₀  A₀)/(A₁₀₀  A₀) and Y₁₀₀ = (A₁₀₀  A₀)/(A₁₀₀  A₀) were the parameters fitted along with L, K_T, K_R, 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 PO_2,, 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

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  and  chains that differ from those of the cathodic Hb (Fig. 1). Tryptic peptides of the S-carboxymethylated  and 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 and  chains of the anodic Hb of eel is reported in Fig. 3 where the sequence portions elucidated by peptide sequencing are indicated. The intact  chain was sequenced up to Ile-20, whereas the  chain was blocked and thus not accessible to Edman degradation. The sequence of the N-terminal peptide T1 of the  chain was obtained after unblocking the N terminus as described under "Experimental Procedures." The difference between the molecular mass of the  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  chains of other fish hemoglobins. The molecular mass of the  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  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  chain and at Arg-29 and Lys-59 in the  chain. No tryptic cleavage occurred after Arg-8 in the  chain probably because it follows three acid residues in the sequence. The fragments Thr-9-Lys-17 and Val-60-Lys-62 of the  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  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 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  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.

RP-HPLC separation of the  and  chains of the anodic (_a and _a) eel Hb compared with that of the cathodic Hb globin chains (_c and _c).

RP-HPLC separation of the tryptic peptides of the S-carboxymethylated  (upper panel) and  (lower panel) chains of anodic eel Hb.

Amino acid sequence of the  and  chains of the anodic Hb of the eel.

chain

 chain

A large Bohr effect is observed in eel anodic Hb (Fig. 4). The Bohr factor ( =  log P₅₀/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 pK_a 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).

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 K_R are in general difficult to obtain (in particular, in a Root-effect hemoglobin) as they require several data points at saturations  99%. The K_R 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 K_T. Compensating variations of L and i are presumably implied in nonconvergence in three data sets (Table I).

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 Y100 Y0 torr1 torr1 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 × 109  ± 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 K_T (as generally is observed in vertebrate hemoglobins) in the stripped hemoglobin. In the presence of GTP, K_T 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 and  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 dimers, each representing a cooperative unit or co-operon. However, later studies have shown that the two dimers (₁₁ and ₂₂) 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 ₁₂ 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 pK_a 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  and  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  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 K_T (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  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 ₁₂ interface, cooperative interactions in the T state may either be negative (and produce the T  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 ₁₂ 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 ₁₂ and ₁₂ interfaces and the presence of proton binding groups.

The ₁₂ 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 ₁₂ interface in fish hemoglobins is consistent with the lower tendency to split into dimers than human HbA (43).

In human HbA, at the dovetailed ₁₂ switch contact (between the C helix and CD corner of the  subunit with the FG corner of the  subunit), His-FG4 packs between the side chains of Pro-CD2 and Thr-C6 in the T state, passes over one helix turn during the T  R transition, and packs between Thr-C6 and Thr-C3 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  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  chain. Moreover, fish hemoglobins possess an additional residue in position CD5 of the  chain compared with human HbA (Table II). Ligation of  subunits within the T state of human HbA results in profound steric hindrance between the side chains of His-FG4 and Pro-CD2 (44). The substitution of Pro-CD2 with a smaller and more flexible residue in Root-effect hemoglobins (Ser, Ala, or Thr; Table II) is likely to stabilize  chain ligations in the T state. Ligation of  subunits in the quaternary T state may be favored by replacements at the flexible joint interface between the FG corner of the chain and the C helix of the  chain. In deoxy human HbA, Arg-FG4 is bound to Glu CD2, which is replaced by a neutral amino acid residue in Root-effect hemoglobins (Ser, Ala or Gly; Table II), so that Arg-FG4 may instead form a hydrogen bond with Gln-C5 (conserved in all fish hemoglobins) as found in deoxy trout (Oncorhynchus mykiss) HbI (45). The same interaction between Arg-FG4 and Gln-C5 is present in oxygenated crystals of T state human HbA (20), which indicates that the replacement of Glu-CD2 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).   Chain   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

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), Thr-C6(41), Ser-CD2(44), Ser-CD2(43), and His-FG4(97) at the ₁₂ and ₂₁ interfaces are indicated.

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 ₁₂ interface upon oxygenation is an essential condition.

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-HC3, which is conserved in all Root-effect hemoglobins (Table II). Glu-FG1 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 FG1). Quite unexpectedly, in the crystal structure of the deoxygenated P. bernacchii Hb1 (47), His-HC3 was found free in solution and not bound to Glu-FG1, which leaves a major part of the Bohr and Root effects difficult to explain. Asp-G1 and Asp-G3 were indicated as possible Bohr groups in this hemoglobin, as they move closer to each other upon deoxygenation and could thereby increase their pK_a and share a proton. By analogy with P. bernacchii, Hb1, Asp-G1, and Asp-G3 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  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-G3 makes a salt bridge with Arg-G6 (conserved in fish hemoglobins or replaced by Lys in antarctic teleosts), thus preventing the two Asp residues from increasing their pK_a 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-FG4, located at the switch interface. Histidines in both positions HC3 and FG4 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  chain has been cleaved, His-FG4 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  chain in the T and the R state. Proton uptake by His-FG4 upon deoxygenation would be favored by a pK_a increase in the T state (by interaction with polar or negatively charged groups, including the C-terminal end of the C helix of the  chain (50)) or by a pK_a 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 C6 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-FG4, 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  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  chain in the presence of His-FG4 agree well with the absence of the Root effect (Table II).

Proton Binding at the ₁₂ Interface

GTP binds in the central cavity between the two  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 pK_a 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  chain and Lys-EF6, given the high pK_a of the Arg side chain. The excess of positive charges at the ₁₂ 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 pK_a of the  N terminus and Lys-EF6 in the T state may therefore be lowered by adjacent positive charges (e.g. Arg-H21) 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 pK_a 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-EF6 by Ala in the hemoglobins of P. bernacchii, P. antarcticum, and N. angustata may be compensated by the substitution of Arg-H21 with Lys (Table II) that has a lower pK_a. Moreover, Lys replaces Glu-NA2 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 ₁₂ 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).

The present data on eel anodic Hb indicate that several regions of the tetrameric hemoglobin molecule contribute to the Root effect: 1) the ₁₂ interface with Gln-C3, Thr- (or Ala)-C6, a small apolar or weakly polar residue (Ala, Ser, Thr) at CD2, Trp-CD4 and His-FG4 at the switch contact, and a nonnegatively charged residue at CD2 (Ala, Gly, or Ser) at the flexible joint; 2) the ₁₂ interface (as indicated by Mylvaganam et al. (13)), including the N terminus of the  chain, Lys-EF6, Trp-NA3, and Arg-H21; and 3) His-HC3. 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  chains. Protons appear to stabilize the ₁₂ interface in the T quaternary state (even in the presence of oxygen) and destabilize the ₁₂ 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  chain, the increase in the functional heterogeneity observed at low pH may be consistent with a larger pH dependence of oxygen binding in the  than in the  subunit. The role of the  subunit would be to provide a favorable structure of the ₁₂ interface, which stabilizes protonation at His-FG4 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  chain and carp  chain do not show the Root effect (57). Another residue that may be important to the Root effect is Ser-F9, which is replaced in the cathodic hemoglobins of trout and eel. Although the role of Ser-F9 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.