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(Received for publication, February 15, 1995; and in revised form, May 25, 1995) From the
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
Fish hemoglobin (Hb) systems commonly exhibit functional
heterogeneity, which appears to be adaptive to oxygen (O 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 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 In this paper we report
the complete amino acid sequence of the
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,
Figure 2:
RP-HPLC of the
Tryptic peptides of S-pyridylethylated HbC
Figure 3:
Amino acid sequence of the
In the The sequence-deduced molecular weights of the S-pyridylethylated
Figure 4:
Transformed electrospray mass spectra of S-pyridylethylated
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.
O
The inhibition of the alkaline Bohr effect that is
due to the C-terminal histidine of the His
Another element which could affect O
The presence of both Cl In conclusion, the elucidation of
the primary structure of the cathodic Hb of eel provides new insight
into the the molecular basis for O 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
Volume 270,
Number 32,
Issue of August 11, pp. 18897-18902, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
AMINO ACID SEQUENCE AND OXYGEN EQUILIBRIA OF A REVERSE BOHR
EFFECT HEMOGLOBIN WITH HIGH OXYGEN AFFINITY AND HIGH PHOSPHATE
SENSITIVITY (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and 
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
chain by Phe (that suppresses the alkaline Bohr effect) and of residues
at the switch region between and subunits (that may alter
the allosteric equilibrium, thus causing the high intrinsic oxygen
affinity and low cooperativity). The residues binding organic phosphate
in the 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.
)
transport under varying environmental and physiological conditions
(Weber, 1990). In eel such differentiation between individual Hb
components may favor an efficient O 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. affinity and low pH sensitivity (a small Bohr
effect), which appear to safeguard O 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). 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
chain (Val(NA1)) and at the C-terminal histidine
of the
chain (His(HC3)) (Perutz, 1970; Riggs,
1988; Shih et al., 1993), whereas the endogenous phosphate
(DPG) binds at the N terminus of the
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
and 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. and 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.
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 (
)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 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 and chains were separated by
reverse-phase high performance liquid chromatography (RP-HPLC) on a
Waters µBondapak C
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
and chains were digested with
trypsin (D'Avino et al., 1989). The 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 and 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 saturation, a modified gas diffusion chamber was used, coupled to
cascaded Wosthoff pumps for mixing pure (>99.998%) N and
air or pure O (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
affinity and
cooperativity were interpolated from P (half-saturation
O
tension) and n (slope of the
linearized Hill plot (log S/(1 - S) versus log PO
) at half-saturation, where S is the
fractional O saturation).
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 and
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.
-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.
and chains of
HbC. Absorbance was monitored at 280 nm. Details are given under
``Materials and Methods.''
and 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 and 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 chain was blocked and thus unavailable to Edman
degradation.
and
chains of the cathodic Hb of A. anguilla. T1 to T17 indicate the tryptic peptides; in the chain, CB indicates the peptide obtained after subdigestion of T13 with cyanogen bromide; in the chain, C indicates
fragment recovered in the ``core.'' The arrows denote the sequenced fragments of the intact 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 chain. Helix notation as established for human HbA is
also reported.
chain an incomplete trypsin cleavage after
Lys
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
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
chain and of the corresponding region of the chain. In the
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
chain was not
recovered at all after RP-HPLC purification. After trypsin digestion of
the 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
chain the
last two residues of the T16 peptide were identified by cleavage of the
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
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. and 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
chain and 15,997 Da for the chain (Fig.4), which is
consistent with the presence of an acetyl group at the N terminus of
the chain. Molecular weights of 15,100 and 15,300 obtained for
the and chains, in SDS-polyacrylamide gel electrophoresis
experiments are in accordance with these measurements.
(A) and 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=
log P/
pH = +0.2 at pH 7.5), a high
O 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
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 affinity at low pH values and had virtually no effect at pH 8.3.
In the presence of chloride O affinity was not decreased as
pH fell below 7.3. Cooperativity was slightly enhanced by the presence
of KCl. The O 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
affinity less than GTP alone.
O 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).
affinity of eel blood adapts rapidly to ambient
O 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 transport function to environmental hypoxia. The
associated increase in blood O affinity improves O loading and the arteriovenous O 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 tensions, O 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 affinity and low pH sensitivity
cathodic Hbs will predictably secure O 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 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
chain is available as a Bohr group, since that
of the chain is acetylated. The five histidine residues of the
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 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
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.
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
chain, which cannot form the salt bridge with the
C-terminal His in the T state. His(HC3) 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.(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
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 affinity and low cooperativity of O binding
of HbC (Fig.5) could be due to a destabilized T state relative
to the R state. A high O affinity may also result, at least
in part, from an altered intrinsic affinity in the and
subunits. In eel HbC some of the residues in contact with the heme
groups of the and subunits are replaced in comparison with
human HbA and other fish Hbs (Kleinschmidt and Sgouros, 1987; di Prisco et al., 1991). In the 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 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 subunit, as verified in human HbA mutants
(Mathews et al., 1989; Tame et al., 1991). In the
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 heme propionic groups that may alter the
orientation of the heme plan and the subunit affinity, whereas in
human HbA the side chain of Ser(CD3) is too short to allow such a bond
(Fermi and Perutz, 1981). binding at the 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
None of the side chains forming the
Switch
Region
``dovetailed'' switch
interface in human HbA (Pro(CD2), Thr
(C6),
and Thr
(C3) of the
subunit with
His(FG4) of the
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
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 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 ( FG corner
with 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
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). and GTP causes a
lesser decrease in the O
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
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 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
chain
(Fronticelli et al., 1994). On the basis of our results it is
not possible to discriminate between these two mechanisms because the
chain N terminus also participates in the binding of phosphates.
However, the far smaller effect of Cl than of GTP on
the O
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 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
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 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). binding regulation. Some
amino acid substitutions are identified that could affect the and
particularly the heme ligand affinities and the side chain
packing at the 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 affinity. and subunits by reaction
with p-mercuribenzoate for O binding studies,
chemical modification of the N terminus of the 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.
The technical assistance of Anny Bang, Annie Wetter,
and Lene Kristensen (Aarhus) is gratefully acknowledged.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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