©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Cathodic Hemoglobin of Anguilla anguilla
AMINO ACID SEQUENCE AND OXYGEN EQUILIBRIA OF A REVERSE BOHR EFFECT HEMOGLOBIN WITH HIGH OXYGEN AFFINITY AND HIGH PHOSPHATE SENSITIVITY (*)

(Received for publication, February 15, 1995; and in revised form, May 25, 1995)

Angela Fago (1) Vito Carratore (2) Guido di Prisco (2) Rene J. Feuerlein (1) Lars Sottrup-Jensen (3) Roy E. Weber (1)(§)

From the (1)Department of Zoophysiology, Institute of Biological Sciences, University of Aarhus, 8000 Aarhus C, Denmark, (2)Institute of Protein Biochemistry and Enzymology, C.N.R., 80125 Napoli, Italy, and (3)Department of Molecular Biology, University of Aarhus, 8000 Aarhus C, Denmark

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

As in other fish, the cathodic hemoglobin of the eel Anguilla anguilla is considered to play an important role in oxygen transport under hypoxic and acidotic conditions. In the absence of phosphates this hemoglobin shows a reverse Bohr effect and high oxygen affinity, which is strongly modulated over a wide pH range by GTP (whose concentration in the red blood cells varies with ambient oxygen availability). GTP obliterates the reverse Bohr effect in the cathodic hemoglobin. The molecular basis for the reverse Bohr effect in fish hemoglobins has remained obscure due to the lack of structural data. We have determined the complete amino acid sequence of the alpha and beta chains of the cathodic hemoglobin of A. anguilla and relate it to the oxygen equilibrium characteristics. Several substitutions in crucial positions are observed compared with other hemoglobins, such as the replacement of the C-terminal His of the beta chain by Phe (that suppresses the alkaline Bohr effect) and of residues at the switch region between alpha and beta subunits (that may alter the allosteric equilibrium, thus causing the high intrinsic oxygen affinity and low cooperativity). The residues binding organic phosphate in the beta cleft of fish hemoglobins are conserved, which explains the strong effect of GTP on oxygen affinity and suggests that these residues contribute to the reverse Bohr effect in the absence of alkaline Bohr groups. Moreover, His that is considered to be responsible for the reverse Bohr effect in human and tadpole Hbs is replaced by Lys.


INTRODUCTION

Fish hemoglobin (Hb) systems commonly exhibit functional heterogeneity, which appears to be adaptive to oxygen (O(2)) transport under varying environmental and physiological conditions (Weber, 1990). In eel such differentiation between individual Hb components may favor an efficient O(2) transport during exercise stress and migration between fresh and sea water or overland, when severe respiratory and metabolic acidosis occurs (Powers and Edmundson, 1972; Hyde et al., 1987). Eel Hb thus is well suited for studying molecular and functional adaptations of fish Hbs to environmental conditions, physiological requirements, and mode of life.

The hemolysate from the European eel Anguilla anguilla resolves into two major types of Hb components, an electrophoretically ``cathodic'' Hb (HbC) with high isoelectric point and an ``anodic'' fraction with low isoelectric point. As in the trout Hb system that has been intensively characterized in terms of molecular structure and oxygenation properties (e.g. Binotti et al. (1971) and Weber et al. (1976b)), the cathodic Hb fraction of eels displays high O(2) affinity and low pH sensitivity (a small Bohr effect), which appear to safeguard O(2) loading in the gills under hypoxic and acidotic conditions, whereas the anodic fraction displays a low affinity and a pronounced Bohr effect (Weber et al., 1976a). In contrast to the cathodic Hb of trout (HbI), which is insensitive to organic phosphates, that of the eel shows greater sensitivity to the endogenous red cell phosphates GTP and ATP than does the anodic Hb (Weber et al., 1976a).

Another peculiarity of eel HbC is a reverse Bohr effect. A reverse Bohr effect has been demonstrated in few other cases, namely in cathodic Hbs from the the American eel, Anguilla rostrata (Gillen and Riggs, 1973) and the Amazonian catfishes Pterygoplichthys pardalis (Brunori et al., 1979; Weber and Wood, 1979), Hoplosternum littorale (Garlick et al., 1979) and Mylossoma sp. (Martin et al., 1979), where the first mentioned two species are facultative air breathers and H. littorale is an obligate air breather. A reverse Bohr effect has also been observed in some amphibians, as in Hbs from bullfrog (Rana catesbeiana) tadpoles (Atha et al., 1979), the primitive salamander, Amphiuma means (Bonaventura et al., 1977), and the newts Triton crisatus (Morpurgo et al., 1970) and Triturus crisatus carnifex (Condòet al., 1981). The occurrence of reverse Bohr effect Hbs in air-breathing fishes and in amphibians that still possess gills, such as tadpoles and salamanders, suggests an implication of these Hbs in the transition from water to air breathing, given that air breathing in fishes is induced by water hypoxia and associated with episodes of internal acidosis.

An increase in the Hb O(2) affinity at low pH (reverse Bohr effect) indicates that oxygenation is associated with proton binding rather than proton release as occurs in Hbs with a normal alkaline Bohr effect. This suggests that reverse Bohr effect Hbs have alternative ionizable groups or that their alkaline Bohr groups are radically modified. In deoxy human HbA, proton binding responsible for the alkaline Bohr effect occurs primarily at the N terminus of the alpha chain (Val(NA1)) and at the C-terminal histidine of the beta chain (His(HC3)) (Perutz, 1970; Riggs, 1988; Shih et al., 1993), whereas the endogenous phosphate (DPG) binds at the N terminus of the beta chain (Val(NA1)), Lys(EF6), and His(H21) that face the central cavity of the Hb molecule (Perutz, 1970; Arnone, 1972). The last mentioned residue is considered to be responsible for the reverse, ``acid'' Bohr effect, which in human HbA occurs at pH lower than 6.5 (Perutz et al., 1980). The basic molecular mechanism of the reverse Bohr effect in fish and amphibian Hbs is still obscure because of the lack of structural data on these Hbs. Apart from the amino acid sequences of the alpha and beta chains of the Hb from the tadpole of the bullfrog R. catesbeiana (Maruyama et al., 1980; Watt et al., 1980), sequences for Hbs with reverse Bohr effects are not available, and only the C-terminal residues in A. rostrata (Gillen and Riggs, 1973) and A. means (Bonaventura et al., 1977) Hbs have been identified.

In this paper we report the complete amino acid sequence of the alpha and beta chains of A. anguilla cathodic Hb and new information on oxygenation properties of this Hb. The elucidation of its primary structure provides a structural basis for comprehending the special functional properties of this Hb and provides insight into the mechanism of the reverse Bohr effect.


MATERIALS AND METHODS

HbC Purification

Specimens of A. anguilla were received from a local pisciculturist and kept at 15 °C in running freshwater in aquaria at the Zoophysiology Department, Aarhus University, for at least 2 weeks before blood sampling. Blood was drawn from the caudal vessels into heparinized syringes. The red blood cells were washed three times in 0.9% NaCl and hemolyzed by addition of 3 volumes of 20 mM Tris buffer, pH 8.1. After centrifugation (10 min at 14,000 rpm), the cathodic Hb (HbC) was stripped from organic phosphates and separated from the anodic Hb fraction by FPLC (^1)anion exchange chromatography on a HiLoad 16/10 Q Sepharose Hi Performance column (Pharmacia Biotech Inc.) as described previously (Feuerlein and Weber, 1994). The separated HbC was then dialyzed against three changes of CO-equilibrated 10 mM HEPES buffer, pH 7.7, containing 0.5 mM EDTA. No oxidation was evident from the visible spectrum. The Hb solution was divided into small aliquots that were stored at -80 °C until use (within 14 days). Control experiments showed no effect of freezing on O(2) affinity and cooperativity (see ``Results'').

Gel Filtration Experiments

The molecular weight of HbC was analyzed on a FPLC Sephacryl 16/10 (Parmacia) column equilibrated with 50 mM HEPES buffer, pH 8.1, at a flow rate of 0.5 ml/min. The HbC sample was injected at 0.1 mM heme concentration. The elution time was compared with that of human HbA.

Amino Acid Sequence Analysis

Globin chains of the cathodic Hb were precipitated according to the acid/acetone method (Rossi Fanelli et al., 1958) but using 20 mM HCl (instead of 5 mM) in acetone and incubating the acid/acetone-Hb mixture at -80 °C for 5 min before centrifugation. The alpha and beta chains were separated by reverse-phase high performance liquid chromatography (RP-HPLC) on a Waters µBondapak C(18) column (0.39 30 cm), using a multistep linear gradient of 70% acetonitrile (solvent B) in 45% acetonitrile containing 0.3% trifluoroacetic acid (solvent A), at a flow rate of 1 ml/min. The eluate was monitored at 280 nm. Alkylation at the SH residues by 4-vinylpyridine was performed as described (Caruso et al., 1991) at 30 °C. S-Pyridylethylated alpha and beta chains were digested with trypsin (D'Avino et al., 1989). The alpha chain was also cleaved by CNBr (Gross and Witkop, 1961). The resulting peptides were separated by RP-HPLC as described (D'Avino et al., 1992). Cleavage at the Asp-Pro bond was performed at 40 °C in 70% formic acid for 24 h (Landon, 1977). After cleavage, in sequencing the N-terminal Pro fragment, non-Pro N-terminal residues were blocked by reaction with o-phthalaldehyde (Brauer et al., 1984). RP-HPLC purifications of the fraction not dissolved by 0.1% trifluoroacetic acid after trypsin digestion (``core'') and of the fragments generated after formic acid treatment were achieved by a linear gradient of 80% acetonitrile (solvent B) in 30% acetonitrile containing 0.3% trifluoroacetic acid (solvent A). Separation on RP-HPLC of the CNBr-generated fragments was obtained using a multistep linear gradient as described (D'Avino et al., 1992). Amino acid composition analyses and automated Edman degradation were performed as described previously (Fago et al., 1992).

Mass Spectrometry Measurements

The molecular mass of the S-pyridylethylated alpha and beta chains was measured on a Hewlett Packard 5989B quadrupole mass spectrometer, equipped with a Hewlett Packard 59987A electrospray source.

Electrophoretic Analysis

Isoelectrofocusing on ultrathin polyacrylamide gel (0.3-mm thickness) in the pH range 3.5-10.0 was performed on a Multiphor II apparatus (Pharmacia) at 15 °C, according to the instructions contained in the manual. For the determination of the isoelectric point of HbC, proteins from an isoelectric focusing calibration kit (pH 5.0-10.5; Pharmacia) were run in parallel with the Hb sample. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-polyacrylamide gel electrophoresis) was performed according to Laemmli(1970) using linear gradients of 12-16% acrylamide and 6-8 M urea in the lower gel.

Oxygen Binding Studies

Oxygen equilibria of the cathodic Hb were measured in 0.1 M HEPES buffer, in the pH range 6.5-8.5, at 20 and 10 °C at a final Hb concentration of 100 µM on a heme basis. To obtain stepwise O(2) saturation, a modified gas diffusion chamber was used, coupled to cascaded Wosthoff pumps for mixing pure (>99.998%) N(2) and air or pure O(2) (Weber, 1981; Weber et al., 1987). pH values were measured by a Radiometer BMS Mk2 thermostatted electrode (Copenhagen). Sensitivity to Cl was tested by adding KCl to a final concentration of 0.1 M, which was assayed by coulometric titration (Radiometer CMT 10). The effect of GTP was investigated by adding a large (approximately 50) molar excess compared with the tetrameric Hb concentration. O(2) affinity and cooperativity were interpolated from P (half-saturation O(2) tension) and n (slope of the linearized Hill plot (log S/(1 - S) versus log PO(2)) at half-saturation, where S is the fractional O(2) saturation).


RESULTS

Structural Characterization

The cathodic Hb fraction of A. anguilla separated from the anodic one and from the organic phosphates in FPLC anion exchange chromatography, where it eluted unretarded (Fig.1). In isoelectrofocusing on polyacrylamide gel in the pH range 3.5-10.0 (see Fig.1, inset) the CO derivative of HbC appeared as a single band with an isoelectric point of 9.05. Globin chain separation on RP-HPLC (Fig.2) revealed only two peaks corresponding to the alpha and beta chains, consistent with the presence of a single cathodic Hb. Gel filtration experiments on a Sephacryl column revealed the Hb to be tetrameric with no evidence for dissociation. SDS-polyacrylamide gel electrophoresis and RP-HPLC experiments indicate that the cathodic and anodic Hbs have no chains in common.


Figure 1: FPLC separation and simultaneous stripping of the cathodic (HbC) and anodic (HbA) Hb fractions on a HiLoad 16/10 Q Sepharose Hi Performance anion exchange column. Absorbance was monitored at 254 nm (continuousline) and at 540 nm (discontinuousline). The inset shows isoelectrofocusing in the pH range 3.5-10.0 on ultrathin polyacrylamide gel of the separated HbC (C), HbA (A), unfractionated hemolysate (T), and isoelectric point standard proteins (S), including, from top to bottom, beta-lactoglobulin A, bovine carbonic anhydrase B, myoglobin acidic band, acidic, middle and basic bands of lentil lectin (with pI values of 5.2, 5.85, 6.85, 8.15, 8.45 and 8.65, respectively) and marker protein near the cathode wick. The cathode is at the bottom.




Figure 2: RP-HPLC of the alpha and beta chains of HbC. Absorbance was monitored at 280 nm. Details are given under ``Materials and Methods.''



Tryptic peptides of S-pyridylethylated HbC alpha and beta chains were purified by RP-HPLC. All peptides were sequenced and aligned by homology with the corresponding sequences of other fish globin chains. The amino acid compositions deduced from the sequence of the individual tryptic peptides matched those found by amino acid analyses. The primary structure of the alpha and beta chains of the cathodic Hb from A. anguilla is reported in Fig.3, where the sequence portions elucidated by automated Edman degradation of the intact chain, of the Asp-Pro cleaved chain and of the peptides (obtained after CNBr and trypsin cleavage) are indicated. As in other fish Hbs, the N terminus of the alpha chain was blocked and thus unavailable to Edman degradation.


Figure 3: Amino acid sequence of the alpha and beta chains of the cathodic Hb of A. anguilla. T1 to T17 indicate the tryptic peptides; in the alpha chain, CB indicates the peptide obtained after subdigestion of T13 with cyanogen bromide; in the beta chain, C indicates fragment recovered in the ``core.'' The arrows denote the sequenced fragments of the intact beta chain and of the Asp-Pro cleaved chains. Dottedlines indicate the sequence portion of the tryptic peptides not resolved by automated Edman degradation. Discontinuouslines indicate the sequence portions elucidated after trypsin digestion of the Asp-Pro cleaved beta chain. Helix notation as established for human HbA is also reported.



In the alpha chain an incomplete trypsin cleavage after Lys^7 and Arg yielded also the uncleaved T2-T3 and T5-T6 fragments. Again, the peptide bond Lys-Lys was only partially digested, and therefore the peptides T7 and T8 were eluted as double peaks, i.e. one containing and the other lacking an additional Lys residue. The sequences of the tryptic peptides T13 and T6 of the beta chain that coeluted in the RP-HPLC purification step were unequivocally established, taking advantage of their different yields (77 and 23%, respectively). Some difficulties were encountered in the elucidation of the amino acid sequence of the penultimate tryptic peptide T16 of the beta chain and of the corresponding region of the alpha chain. In the beta chain the peptide T16 was not recovered in sufficient amounts to allow its complete sequencing, and the expected tryptic peptide from Phe to Lys of the alpha chain was not recovered at all after RP-HPLC purification. After trypsin digestion of the alpha chain the peptide from Thr to Arg was obtained as an uncleaved fragment, despite the presence of Lys and Lys. Its sequencing proceeded up to Lys. To obtain the sequence of the final segment this fragment was subjected to further subdigestion with CNBr, and the resulting peptide from Asp to the C-terminal Arg was sequenced, after a purification step on RP-HPLC. In the beta chain the last two residues of the T16 peptide were identified by cleavage of the beta chain at the Asp-Pro bond with formic acid, purification of the C-terminal fragment (from Pro to Phe) by RP-HPLC and its subsequent digestion with trypsin. The peptide mixture was then subjected directly to automated Edman degradation without purification, and the complete amino acid sequence of the T16 peptide was elucidated by difference. In another preparation the sequence of the T16 peptide was determined after RP-HPLC purification of the core, where the fragment Leu-Lys coeluted with the (presumably) undigested beta globin. The sequence of this fragment (containing the T16 peptide) was determined after reaction of the mixture with OPA at the 6th step of the Edman degradation to eliminate undesired (i.e. non-Pro N-terminal) sequences.

The sequence-deduced molecular weights of the S-pyridylethylated alpha and beta chains were 15,338 (including acetylation at the N terminus) and 15,999, respectively. These values are in agreement with the mass of the two globins determined by electrospray mass spectrometry of 15,341 Da for the alpha chain and 15,997 Da for the beta chain (Fig.4), which is consistent with the presence of an acetyl group at the N terminus of the alpha chain. Molecular weights of 15,100 and 15,300 obtained for the alpha and beta chains, in SDS-polyacrylamide gel electrophoresis experiments are in accordance with these measurements.


Figure 4: Transformed electrospray mass spectra of S-pyridylethylated alpha (A) and beta chains (B).



Oxygen Binding Properties

The effects of pH, Cl, and organic phosphates on the oxygen affinity and cooperativity are shown in Fig.5. In the absence of Cl the stripped HbC showed a reverse Bohr effect throughout the pH range investigated (Ø= Deltalog P/DeltapH = +0.2 at pH 7.5), a high O(2) affinity (P = 1.8 and 0.8 mm Hg at 20 °C at pH 8.3 and 6.6, respectively), and a low cooperativity (n varying from approximately 2.0 at pH 8.3 to 1.3 at pH 6.6). Chloride ions, whose effect on O(2) binding can be more accurately estimated in HEPES (Weber, 1992) than in Tris or bis-Tris buffers used in an earlier study (Weber et al., 1976a), produced a small decrease in the O(2) affinity at low pH values and had virtually no effect at pH 8.3. In the presence of chloride O(2) affinity was not decreased as pH fell below 7.3. Cooperativity was slightly enhanced by the presence of KCl. The O(2) affinity was drastically reduced by GTP (P = 11.4 mm Hg at pH 8.3), which is the major allosteric effector in this species and is a more potent effector than ATP (Weber et al., 1976a). Addition of GTP, in the absence and presence of Cl, raised cooperativity to 2.8. At equimolar ratio to Hb tetramers, GTP virtually obliterated the reverse Bohr effect, whereas a high GTP:Hb ratio induced a slight, normal Bohr effect. Interestingly, the presence of both Cl and GTP decreased O(2) affinity less than GTP alone. O(2) affinity and cooperativity values of eel HbC samples that had been frozen at -80 °C were the same as in freshly prepared Hb solutions (Fig.5).


Figure 5: Bohr effect of HbC measured in 0.1 M HEPES buffer at 20 °C, in the presence and absence of 0.1 M KCl and at different GTP:Hb tetramer ratios. Heme concentration was 0.1 mM. Circles, freshly prepared Hb; othersymbols, samples that had been frozen at -80 °C.




DISCUSSION

O(2) affinity of eel blood adapts rapidly to ambient O(2) depletion primarily through a decrease in the concentration of organic phosphates, particularly GTP, in the red cells, whereas Hb heterogeneity (number and relative concentration of the individual Hb components) remains the same after hypoxic and normoxic acclimation (Wood and Johansen, 1972; Weber et al., 1976a). Related to its higher sensitivity to nucleotide triphosphates, the cathodic Hb from eel plays a major role in the adaptation of the O(2) transport function to environmental hypoxia. The associated increase in blood O(2) affinity improves O(2) loading and the arteriovenous O(2) content difference under hypoxia (Wood and Johansen, 1973). As shown here, phosphate modulation occurs even at high pH, such as results from hyperventilation, which is a primary response in fish subjected to hypoxic conditions (cf. Weber, 1981). In response to variable O(2) tensions, O(2) affinity of HbC can thus be modulated over a wide pH range by modification of the GTP:Hb ratio. As a result of the high O(2) affinity and low pH sensitivity cathodic Hbs will predictably secure O(2) loading under internal acidosis induced by exercise stress. Although the physiological significance of eel HbC is clear, the molecular mechanisms governing the regulation of intrinsic O(2) affinity of this Hb are not yet understood. The knowledge of the primary structure reported here provides a basis for insight.

The Bohr Groups

The reverse Bohr effect observed in A. anguilla cathodic Hb reflects an increase in the affinity for oxygen as pH is lowered, showing that protons have a higher affinity for the oxygenated, relaxed (R) state than for the deoxygenated, tense (T) state. Such effects require that some ionizable amino acid residues increase their pK in the transition from the T to the R state. Likely candidates for such groups are the N termini of both chains and the histidine residues, since these normally have pK values in the pH range in which the Bohr effect is observed. Among the N termini only that of the beta chain is available as a Bohr group, since that of the alpha chain is acetylated. The five histidine residues of the alpha chain (including the ``distal'' and ``proximal'' histidines) are conserved among fish Hbs (as in human HbA). It seems, therefore, unlikely that they are responsible for the reverse Bohr effect, although this possibility cannot be excluded. The beta chain has only the two heme-linked histidines, His(E7) and His(F8). Remarkably, two important histidine residues found in human HbA are substituted in the beta chain of A. anguilla HbC; the C-terminal His(HC3), accounting for the major fraction of the alkaline Bohr effect in other Hbs (Perutz, 1970; Kilmartin and Wootton, 1970; Shih et al., 1984) is replaced by a Phe residue, and His(H21), which is involved in the reverse acid Bohr effect in human HbA below pH 6.5 (Perutz et al., 1980), is replaced by Lys.

The inhibition of the alkaline Bohr effect that is due to the C-terminal histidine of the beta chain seems to be a common feature of several reverse Bohr effect Hbs; His(HC3) is replaced by Phe also in the American eel A. rostrata (Gillen and Riggs, 1973) and is deleted in A. means (Bonaventura et al., 1977). Although the C-terminal His residue is conserved in R. catesbeiana tadpole Hb (Watt et al., 1980), the alkaline Bohr effect here is inhibited by the presence of an Asn (instead of Asp or Glu) residue at position 94(FG1) of the beta chain, which cannot form the salt bridge with the C-terminal His in the T state. His(HC3)beta is preserved in T. crisatus carnifex Hb (Condòet al., 1981), but no further sequence data are available for this Hb. Sequence data for Amazonian fish Hbs that show a reverse Bohr effect are similarly lacking.

His(H21) is considered to be largely responsible for the reverse Bohr effect in human and tadpole Hbs. In the deoxy human HbA this residue is located between Lys(EF6) and Lys(HC1), so that its imidazole group has a lower affinity for protons (i.e. a lower pK) than in the oxygenated form (Perutz et al., 1980). In tadpole Hb, position 144 of the beta chain is occupied by Ser, which is expected to raise the pK of His, explaining the occurrence of reverse Bohr effects in tadpole Hb in the pH range 8.5-6.0 and in human HbA below pH 6.5 (Watt et al., 1980). The replacement His Lys in eel cathodic Hb thus indicates a different allosteric mechanism in this Hb.

Oxygen Affinity and Subunit Structure

The high O(2) affinity and low cooperativity of O(2) binding of HbC (Fig.5) could be due to a destabilized T state relative to the R state. A high O(2) affinity may also result, at least in part, from an altered intrinsic affinity in the alpha and beta subunits. In eel HbC some of the residues in contact with the heme groups of the alpha and beta subunits are replaced in comparison with human HbA and other fish Hbs (Kleinschmidt and Sgouros, 1987; di Prisco et al., 1991). In the alpha chain, Leu(FG3) is substituted by Met (as in tuna Hb; Rodewald et al.(1987)), and Val(FG5) is replaced by Ile (as in goldfish Hb; Rodewald and Braunitzer, 1984). Val(E11), normally present at the distal side of the alpha heme, is replaced by Ile, as also found in other fish Hb (in HbI and HbIV of trout, in goldfish Hb, and in red gurnard Hb; Bossa et al.(1978), Petruzzelli et al.(1989), and Fago et al.(1993)), although this substitution is not likely to alter ligand affinity of the alpha subunit, as verified in human HbA mutants (Mathews et al., 1989; Tame et al., 1991). In the beta chain, Ser(CD3) and Lys(E10) present in human HbA are replaced, respectively, by Lys and Val (this latter is also found in HbI of trout; Barra et al.(1983)). The presence of Lys in position CD3 could allow the formation of a hydrogen bond (or even of a salt bridge) with one of the beta heme propionic groups that may alter the orientation of the heme plan and the beta subunit affinity, whereas in human HbA the side chain of Ser(CD3) is too short to allow such a bond (Fermi and Perutz, 1981).

Another element which could affect O(2) binding at the beta subunit is the presence of an Asn residue in position 93(F9), replacing Cys in human HbA or Ser in other fish Hbs. In human HbA in the R state the SH group of Cys(F9) can be either internal between the F and H helices, sharing the pocket with Tyr(HC2), or external, depending on the spin state of the iron, whereas in the T state it is external and in contact with the C terminus (Perutz and Brunori, 1982). The side chain of Asn in the cathodic eel Hb may therefore produce perturbations in the F and H helix packing and in the C-terminal region because of steric hindrance factors and the ability of Asn to form hydrogen bonds.

A Novel alpha(1)betaSwitch Region

None of the side chains forming the alpha(1)beta(2) ``dovetailed'' switch interface in human HbA (Pro(CD2), Thr(C6), and Thr(C3) of the alpha(1) subunit with His(FG4) of the beta(2) subunit) are conserved in HbC of A. anguilla (see Table1), although this region, which has a primary role in the cooperative, quaternary transition T-R, is highly conserved among vertebrate Hbs. Switching to the oxy quaternary conformation, His(FG4) in human HbA shifts over one helix residue of the C helix and CD corner of the alpha(1) subunit (Perutz, 1970; Baldwin and Chothia, 1979). In eel HbC a unique side chain packing is expected to be present at this interface, which may influence the functional properties of this Hb. The increase in the buried surface at this interface in the deoxy conformation indicates that the increase in hydrophobic interactions at this region in human HbA may contribute to the stability of the T state (Lesk et al., 1985). A less constrained T state produced by a looser side chain packing at this interface or the possibility of intermediates in the quaternary transition process may increase O(2) affinity and reduce cooperativity, as observed in HbC. It must be noted, however, that the residues forming the hydrogen bond network in the switch region, which also contribute to the stability of the deoxy state (LiCata et al., 1993) are conserved. The residues forming the ``joint'' region (alpha(1) FG corner with beta(2) C helix) that maintains the same structure in both quaternary states are also conserved. Moreover, a significant change in proton affinity of His(FG4) in human HbA arises when the C-terminal His has been cleaved, so that it becomes a Bohr group (Perutz et al., 1985). The replacement His Asn at this position in eel HbC (where the C-terminal His is replaced by Phe) eliminates another possible contributor to the normal Bohr effect.



The Anion Binding Site, a Role in the Reverse Bohr Effect?

The phosphate binding site in fish Hb includes the N terminus (Val(NA1)), Glu(NA2), Lys(EF6), and Arg(H21) of the beta chains (Perutz and Brunori, 1982; Gronenborn et al., 1984). As shown (Fig.3), all these are conserved in the cathodic Hb of A. anguilla. The possibility to form an additional hydrogen bond (between Val(NA1) and the O2` of the ribose) explains why GTP is a stronger allosteric effector than ATP, in agreement with the stereochemical model proposed for carp Hb (Gronenborn et al., 1984).

The presence of both Cl and GTP causes a lesser decrease in the O(2) affinity than that induced by GTP alone. This indicates that the binding sites for the two anions overlap, at least in part, and suggests the absence of additional sites for allosteric Cl binding in this Hb. It has been proposed that the excess of positive charges in the beta cleft of human Hb can cause a destabilization of the T state relative to the R state in the absence of anions (Bonaventura and Bonaventura, 1978). The lowering of the O(2) affinity in the presence of Cl has recently been explained in terms of neutralization of the positive charges in the cavity without binding at any specific residue (Perutz et al., 1993, 1994) or, alternatively, to binding at the N-terminal group of the beta chain (Fronticelli et al., 1994). On the basis of our results it is not possible to discriminate between these two mechanisms because the beta chain N terminus also participates in the binding of phosphates. However, the far smaller effect of Cl than of GTP on the O(2) affinity of HbC indicates that in the low affinity conformation electrostatic repulsions in the central cavity are not a major factor destabilizing the T state and increasing O(2) affinity. However, the close packing of positive side chains at this site in the deoxy form could conceivably induce abnormally low pK values in the N-terminal group of the beta chain or possibly even in the Lys residues, thus making them acid Bohr groups. The residues Val(NA1), Glu(NA2), Lys(EF6), and Arg(H21) (or Lys) of the beta chain have so far only been found in Root effect fish Hbs, such as those from carp (Grujic-Injac et al., 1980), goldfish (Rodewald and Braunitzer, 1984), and red gurnard (Fago et al., 1993), where the contribution of these residues to the pH effect may be masked by the presence of strong alkaline Bohr groups. In this regard it is suggestive that a small reverse Bohr effect (+0.13 in the pH range 7.8-9.0, where the alkaline Bohr groups are inoperative), which was inhibited by ATP, also is seen in carp Hb (Gillen and Riggs, 1972).

In conclusion, the elucidation of the primary structure of the cathodic Hb of eel provides new insight into the the molecular basis for O(2) binding regulation. Some amino acid substitutions are identified that could affect the alpha and particularly the beta heme ligand affinities and the side chain packing at the alpha(1)beta(2) switch interface that could alter the T-R allosteric transition. The presence of the GTP binding residues in this Hb is in agreement with the stereochemical model proposed for carp Hb and explains the strong effect of organic phosphates on the O(2) affinity.

For further investigation into the structure-function relationships preparation of Hb crystals in the T and R state suitable for x-ray diffraction analysis is under way. Moreover preparation of native alpha and beta subunits by reaction with p-mercuribenzoate for O(2) binding studies, chemical modification of the N terminus of the beta chain, and cross-linking at the central cavity of the Hb molecule are also in progress to verify the hypothesis regarding the cause of the reverse Bohr effect.


FOOTNOTES

*
This work was supported by the Danish Research Academy, the Danish Natural Science Research Council, the Danish Biomembrane Centre, and the Carlsberg Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Zoophysiology, Inst. of Biological Sciences, Bldg. 131, Aarhus University, DK-8000 Aarhus C, Denmark. Tel.: 45-8942-2599; Fax: 45-86-194186.

^1
The abbreviations used are: FPLC, fast protein liquid chromatography; RP-HPLC, reverse phase high performance liquid chromatography; R state, relaxed state; T state, tense state.


ACKNOWLEDGEMENTS

The technical assistance of Anny Bang, Annie Wetter, and Lene Kristensen (Aarhus) is gratefully acknowledged.


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