(Received for publication, February 25, 1997, and in revised form, April 8, 1997)
From the The functional and structural basis for the Root
effect has been investigated in the anodic hemoglobin of the European
eel, Anguilla anguilla. This hemoglobin exhibits a large
Bohr effect, which is accounted for by oxygen-linked binding of seven
to eight protons in the presence of GTP at pH 7.5. Oxygen equilibrium
curves show nonlinear lower asymptote of Hill plots, indicating the
occurrence of heme-heme interactions within the T state. Analysis of
the curves according to the co-operon model (Brunori, M., Coletta, M.,
and Di Cera, E. (1986) Biophys. Chem. 23, 215-222) reveals that T state cooperativity is positive at high pH and in the stripped hemoglobin (where the T 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 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 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 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
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 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 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
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 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,
Department of Zoophysiology,
Department of
Molecular Biology, University of Aarhus, 8000 Aarhus C, Denmark
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
R allosteric transition is operative) and
negative at low pH and in the presence of organic phosphate (where the
molecule is locked in the low affinity structure), indicating site
heterogeneity. The complete amino acid sequence of eel anodic
hemoglobin has been established and compared with that of other fish
hemoglobins. The presence of the Root effect correlates with a specific
configuration of the
1
2 switch interface, which at low pH would stabilize subunit ligation in the T state without
changing the quaternary structure. We propose that the major groups
involved in the binding of oxygen-linked protons in eel anodic
hemoglobin are located on the
chain and comprise His-HC3 at the C
terminus, His-FG4 at the switch interface, and Lys-EF6 and the N
terminus at the phosphate-binding site.
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.
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
1
1 and
2
2
dimers relative to each other so that large conformational changes
occur at the interdimer
1
2 and
2
1 interfaces, whereas the intradimer
1
1 and
2
2
interfaces remain virtually unaffected (9). Salt bridges and hydrogen bonds are broken in the transition from the T to the R state, resulting
in the release of Bohr protons. These noncovalent interactions stabilize the T relative to the R state and act as constraints that
determine the low affinity of the T state (10).
(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
1
1 relative to the
2
2 dimer (a similar rotation is found
also in the Hb1 of the antarctic teleost Pagothenia
bernacchii (14)). However, this mechanism alone does not
satisfactorily explain the absence of the Root effect in Hb1 of another
antarctic nototheniid, Trematomus newnesi (15), which has
all the necessary key residues except that Lys-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.
1
2 interface acts as a constraint. The
quaternary transition to the R state occurs when the two dimers have at
least one ligated subunit, since the
1
2
interface is then no longer stable in the T conformation. Accordingly,
the release of the Bohr protons upon oxygenation is stepwise and
comprises a tertiary Bohr effect, which reflects ligation within the T
state and a quaternary Bohr effect, following the T
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.
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
1
2 interface remains stable in the T
state upon oxygen binding. This extends the basic stereochemical interpretation proposed by Perutz and Brunori (11) and, more recently,
by Mylvaganam et al. (13).
Purification of Anodic Hb
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.
-cyano-4-hydroxycinnamic acid, and 0.9 µl were applied to the
target. 30-100 calibrated mass spectra were averaged.
concentration was assayed by coulometric titration
(Radiometer CMT 10).
where KT is the intrinsic ligand affinity
and i describes the cooperative interactions within the T
quaternary structure. Values of i above or below unity
indicate positive and negative cooperativity, respectively. When
i = 1, the T state binds oxygen with no cooperativity,
and the model becomes identical to the classical two-state MWC model.
The binding polynomial for a noncooperative R state with ligand
affinity KR is therefore that of the MWC
model,
(Eq. 1)
According to these relations, the equation describing the
fractional saturation in tetrameric hemoglobins (36) is,
(Eq. 2)
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
(Eq. 3)
) were calculated as follows,
where A0
(Eq. 4)
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,
or
(Eq. 5)
where Y0 = (A0
(Eq. 6)
A0
)/(A100
A0
) and
Y100
= (A100
A0
)/(A100
A0
) were the parameters
fitted along with L, KT,
KR, and i, and Y is the
expression of the fractional saturation according to Equation 3.
Fitting was performed on Hill-transformed data, log[Y
/(1
Y
)] versus log
PO2,, with equal weighting of data points. The
curve fitter employed the method of Levenberg-Marquardt, and the
standard errors on the parameters were estimated from the diagonal
elements of the curvature matrix associated with the fit.
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.
Functional Characterization
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
pKa values of the Bohr groups. The decrease in
saturation at low pH in the presence of GTP and 1 atm of oxygen
indicates the presence of the Root effect (Fig. 4, inset).
Accordingly, the Hill coefficient n50 falls to
approximately 0.5 at pH 6.6 (Fig. 4).
Good agreement is found between the P50 values obtained by the diffusion chamber and the tonometrical methods, provided that the incomplete oxygen saturation, which occurs at low pH because of the Root effect, is taken into account. By assuming 100% saturation in the presence of pure oxygen in diffusion chamber experiments, a lower P50 and higher n50 are found at low pH with GTP (Fig. 4). The agreement between the data obtained with the two techniques, moreover, indicates that small concentrations of the enzymatic reducing system (present only in tonometrical studies) does not influence the oxygen binding properties of hemoglobin. A weak effect of KCl on the oxygen affinity is illustrated in both tonometrical and diffusion chamber experiments (Fig. 4).
Extended Hill plots for oxygen equilibrium experiments at different pH
values and in the absence and presence of GTP are shown in Fig.
5, where deviations from linearity of the lower
asymptote reflecting cooperative interactions in the T state are
evident. A unitary slope in the upper asymptote indicates
noncooperative binding in the R state. The equilibrium data are
satisfactorily described by the co-operon model, as indicated by the
curves obtained by fitting Equation 5 to the data (Fig. 5). The
allosteric parameters obtained in the fitting procedures are reported
in Table I. The parameter i describes the
overall or the apparent cooperative interactions within the T state as
positive (i > 1) or negative (i < 1).
Reliable estimates for KR are in general
difficult to obtain (in particular, in a Root-effect hemoglobin) as
they require several data points at saturations 99%. The
KR value reported in Table I is thus the mean
value (±10%) calculated for the stripped hemoglobin at pH 7.935 and
held fixed to fit the allosteric constants in the other data sets as
described (37). No significant variations were observed in, and
regardless of, the values of the other parameters. Partly because of
the high number of parameters fitted, a large uncertainty was found in
the determination of L and i, but not in
KT. Compensating variations of L and
i are presumably implied in nonconvergence in three data
sets (Table I).
|
As illustrated in Table I, increasing proton concentration stabilizes the T relative to the R state (L increases) and decreases KT (as generally is observed in vertebrate hemoglobins) in the stripped hemoglobin. In the presence of GTP, KT does not decrease further below pH 7.48 where the apparent cooperativity in the T state becomes negative as i decreases to values significantly below unity at low pH. Under these conditions, the hemoglobin molecule can be considered as locked in the low affinity conformation as indicated by the high values of L. In the absence of cofactors, i remains above unity in the pH range investigated.
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
(
1
1 and
2
2)
cannot be considered as functionally unrelated but that in the T state,
ligation at one subunit enhances ligation at the other subunit of the
same dimer and inhibits ligation at the opposite dimer (22). In our
study, the parameter i therefore includes not only the
intradimer cooperativity as in the original model but also any
functional interaction between dimers across the
1
2 interface. Moreover, the situation in
Root-effect hemoglobins is complicated by a large functional
heterogeneity of the chains in the T state (40, 41), which is
responsible for biphasic oxygen equilibrium curves at low pH and with
organic phosphates and would contribute negatively to the T state
cooperativity and decrease the value of i. This would
explain both the low values of i (Table I) and
n50 (Fig. 4) found under these conditions, where
the tetrameric molecule is essentially in the T state. It is important
to note that the effect of organic phosphates is to shift the
allosteric equilibrium toward the T state and to increase the
pKa of Bohr groups (Fig. 4) rather than to enhance
functional subunit heterogeneity in itself (41), in agreement with the
conclusion that organic phosphates and protons have a similar
allosteric effect (42). The apparent negative cooperativity between the
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 KT (32) so that the decrease in
oxygen saturation is larger at low oxygen tension. These two different
mechanisms may relate to the different physiological roles of the two
pH effects. The Bohr effect enhances the amount of oxygen released in
the metabolizing tissues (at low oxygen tension), and the Root effect
allows release of a large amount of oxygen in the eye and swim bladder
(at high oxygen tensions (38)).
The inverse relationship between L and i suggests
that in the T state of eel anodic Hb, positive cooperative interactions prevail in the presence of the T 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
1
2
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
1
2 interface remains stable in the T
state upon oxygenation, whereas human HbA switches to the R
conformation. The comparison of the primary structure of the anodic
hemoglobin of the eel with that of the cathodic Hb and other fish
hemoglobins reveals that several concomitant factors may contribute to
the expression of the Root effect in fish hemoglobins: a particular
configuration of the
1
2 and
1
2 interfaces and the presence of proton
binding groups.
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 1
2 interface
in fish hemoglobins is consistent with the lower tendency to split into
dimers than human HbA (43).
In human HbA, at the dovetailed 1
2 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.
|
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
1
2 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 pKa 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
pKa and participating in the Bohr effect (45).
We propose that another major Bohr group in eel anodic Hb and other
Root-effect hemoglobin may be His-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 pKa increase in the T state (by interaction
with polar or negatively charged groups, including the C-terminal
end of the C helix of the
chain (50)) or by a
pKa decrease in the R state (e.g. by a
more hydrophobic environment of this His residue in the R than in the T
state). An Ile residue at position 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).
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 pKa of Bohr
groups, the remaining oxygen-linked proton-binding sites in anodic eel
hemoglobin thus appear to be localized in the central cavity, and the
most likely candidates appear to be the N terminus of the
chain and Lys-EF6
, given the high pKa of the Arg side
chain. The excess of positive charges at the
1
2 interface represents a destabilizing
factor for the T state of human HbA, which, in the absence of anions,
results in an increased oxygen affinity due to the shift toward the
high affinity state (54). The pKa of the
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 pKa of these
groups (13). By this mechanism, the groups in the central cavity attain
an increased proton affinity in the T state so that they can contribute
to the alkaline Bohr effect. Replacement of Lys-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 pKa.
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
1
2 are replaced, as proposed for the
cathodic eel Hb (5).
This site is of primary importance for phosphate modulation of blood oxygen affinity. In the eel, under hypoxic conditions, oxygen affinity increases rapidly through a decrease in the intra-erythrocytic concentration of organic phosphates (56), particularly GTP, whereas the relative amount of the two hemoglobin components remains unaffected (4).
ConclusionThe present data on eel anodic Hb indicate that
several regions of the tetrameric hemoglobin molecule contribute to the
Root effect: 1) the 1
2 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
1
2
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
1
2 interface in the T quaternary state
(even in the presence of oxygen) and destabilize the
1
2 interface in the R state, as proposed
for spot Hb (13), thereby shifting the allosteric equilibrium toward the low affinity conformation. As all the proton-binding sites appear
to be on the
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
1
2 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.
The amino acid sequences reported in this paper have been deposited in the SWISS-PROT database under the accession numbers [GenBank] and [GenBank].
We thank Drs. Lone K. Rasmussen and Esben S. Sørensen for mass spectrometry measurements, Dr. Lars Sottrup-Jensen for the use of sequencing equipment, and Dr. Mogens Kruhøffer for helpful criticism. The technical assistance of Anny Bang, Faith Post, Annie Wetter, and Lene Kristensen is gratefully acknowledged.