(Received for publication, November 6, 1995; and in revised form, January 26, 1996)
From the
The allosteric transition of hemoglobin involves an extensive
reorganization of the interface, in
which two contact regions have been identified. This paper concerns the
effect of two mutations located in the ``switch'' (
C3
Thr
Trp) and the ``flexible joint'' (
C3 Trp
Thr). We have expressed and characterized one double and two
single mutants: Hb
T38W/
W37T, Hb
W37T, and Hb
T38W,
whose structure has been determined by crystallography.
We present data on: (i) the interface structure in the two contact regions, (ii) oxygen and CO binding kinetics and cooperativity, (iii) dissociation rates of deoxy tetramers and association rates of deoxy dimers, and (iv) the effect of NaI on deoxy tetramer dissociation rate constant.
All the mutants are tetrameric and T-state in the
deoxygenated derivative. Reassociation of deoxygenated dimers is not
modified by interface mutations. DeoxyHb T38W dimerizes 30% slower
than HbA; Hb
W37T and Hb
T38W/
W37T dissociate much
faster. We propose a binding site for I
at the switch
region.
The single mutants bind O cooperatively; the
double one is almost non-cooperative, a feature confirmed by CO
binding. The functional data, analyzed with the two-state model,
indicate that these mutations reduce the value of the allosteric
constant L
.
The three-dimensional structure of liganded and deoxyhemoglobin
(Baldwin and Chothia, 1979; Shaanan, 1983; Perutz et al.,
1987) indicates that the allosteric transition can be described
topographically as a change in the relative orientation of the two
dimers identified as and
, which rotate with respect to each
other and slide along the
and
interfaces. Therefore, the amino
acid residues that contribute to the
(and
) interface (Fig. 1) play a major role in controlling the relative stability
of the allosteric states and, as a consequence, cooperativity. This
interface contains residues of helix C and the FG corner of both
chains. In total 17 residues establish interactions across the
FG-
C and the
C-
FG contacts with a pseudo-symmetric
arrangement (Fig. 1). Other interactions of minor significance
are present across the
FG-
FG and
C-
C contacts
(Baldwin and Chothia, 1979).
Figure 1:
Schematic structure
of the interface of HbA. Ribbon
representation of
dimer. The regions
involved in the contact are framed by the black square, and
the residues represented in ball and stick mode are Thr
38 (C3)
and Trp
37 (C3).
The FG-
C contact is extensive
and forms a network of weak bonds, which is largely maintained in the
allosteric transition, in spite of some changes in the orientation of
specific amino acid side chains.
37 (C3) Trp in particular makes
contacts with
92(FG4) Arg,
94(G1) Asp, and
95(G2) Pro in
both oxy- and deoxyhemoglobin; nevertheless, it is a good probe of the
allosteric transition because its optical spectrum is perturbed upon
ligand binding (Briehl and Hobbs, 1970; Perutz et al., 1974).
The amino acid residues at the
C-
FG contact region of the
interface undergo an important
reorganization in the course of the T-R transition. In deoxyhemoglobin
the side chain of
97(FG4) His lies between
44(CD2) Pro and
41(C6) Thr, while in oxyhemoglobin this same His settles between
41(C6) Thr and
38(C3) Thr. Thus, this region of the
interface, which seems to be
compatible with only two states, plays a key role in determining the
allosteric transition (see Fermi and Perutz(1981)).
Baldwin and
Chothia(1979) summarize the function of the
interface with the definition of the
FG-
C contact region as a ``flexible joint'' and the
C-
FG as a ``switch.'' Hence the amino acid residue
at position C3 has a different role in the
and
chains;
37(C3) Trp is involved in a series of contacts, which stabilize
the tetramer in both the oxy and deoxy derivatives, while
38(C3)
Thr participates in the reorganization of the
interface associated with the
allosteric transition.
Human Hb has been expressed in Escherichia coli and yeast, and several interesting mutants
have been prepared to test specific hypotheses (Nagai et al.,
1985; Martin de Llano et al., 1993; Komiyama et al.,
1995). In view of the considerations reported above, we have
investigated the role of the residue at C3 in both chains of human
hemoglobin by synthesizing the site-directed mutants bearing in the
chains the residue found in the
chains and vice versa. The
type of substitution to be inserted at C3 was chosen to probe the
effect of changes in the number of atomic contacts (Schaad et
al., 1993), avoiding the introduction of charged residues. Three
hemoglobins were therefore expressed in E. coli: the single
mutants
38 Thr
Trp (
T38W) (
)and
37 Trp
Thr (
W37T) and the double mutant
38 Thr
Trp/
37 Trp
Thr (
T38W/
W37T, called herein the
double mutant). It may be recalled that there are no known natural
mutants of
38 Thr, while the two natural mutants of
37 Trp,
Hb Hirose and Hb Rothschild, have Ser and Arg, respectively (Yamaoka,
1971; Gacon et al., 1977; Sasaki et al., 1978) (see
also Huisman(1992)).
Our three mutants were compared to wild type
HbA with respect to the reaction with oxygen and carbon monoxide, the
stability of liganded and unliganded tetramers, the kinetics of
association of deoxy dimers and the dissociation of deoxy tetramers,
the effect of sodium iodide, and the changes in the molecular contacts
at the interface by x-ray crystallography of deoxy-Hb 38 Thr
Trp or by modeling.
Structural analysis leads to simple
predictions on the role of the interface, suggesting that (a) semi-conservative
mutations at topological position C3 affect tetramer stability and
cooperativity in a simple manner, without loss of allosteric behavior; (b) substitutions at equivalent positions of the
and
chains should yield different functional effects, in spite of the
pseudo-symmetry of that interface. By-and-large these predictions are
fulfilled by our results.
The time course of dissociation of deoxyhemoglobin into dimers was monitored by recording the optical spectrum (in the Soret region) after mixing deoxyhemoglobin with a stoichiometric amount of haptoglobin (isoform 1.1 purchased from Sigma), as described by Ip et al.(1976). Since haptoglobin binds rapidly and tightly to the free dimers, the equilibrium is driven toward the dissociated state (Nagel and Gibson, 1972).
The time course of association of deoxyhemoglobin dimers was followed by mixing oxyhemoglobin (which at micromolar concentration contains a substantial amount of oxygenated dimers) with 50 mM sodium dithionite in the rapid scanning stopped flow spectrometer described by Bellelli et al.(1990). After rapid dissociation of oxygen, the dimers recombine in a slow, concentration-dependent process (Kellett and Gutfreund, 1970).
Transient spectra were collected into a matrix (A), each column being a difference spectrum and each row a time
course, and analyzed with the singular value deconvolution algorithm
(SVD; Golub and Reinsch, 1980; Henry and Hofrichter, 1992). This
deconvolution yields the three matrices U, S, and V, whose product U S
V
approximates A. The product U
S may be envisaged as a matrix of extinction
coefficients of the spectroscopic components detected by the algorithm,
while V columns represent their time evolution. The SVD analysis
allowed to magnify the small absorption changes coupled to the
allosteric transition and to increase the signal to noise ratio.
Molecular modeling was carried out on a Silicon Graphics workstation
(Silicon Graphics Inc., Mountain View, CA) by use of the
Discover/Insight package. Two structures were from the Brookhaven
Protein Data Bank: deoxygenated recombinant hemoglobin (Kavanaugh et al., 1992a) and wild type oxyhemoglobin (Shaanan, 1983).
For mutant T38W we used the coordinates of the deoxy derivative.
For the other mutants (i.e. the two proteins containing the
substitution
W37T and the oxy form of Hb
W38T) simulation of
the mutation in the corresponding wild type structure was performed by
substituting the side chain in the starting position leading to no
unfavorable contacts with neighboring side chains, followed by energy
minimization allowing only amino acids in a sphere of 6 Å from
the mutated amino acid to move.
Figure 2:
Oxygen binding isotherms of HbA and mutant
hemoglobins. Open squares, HbA; closed squares, Hb
T38W; open circles, Hb
W37T; closed
circles, Hb
T38W/
W37T. Continuous lines represent the best fit to a two-state model with the parameters
reported in Table 1. Conditions: 0.1 M Bis-Tris/HCl
buffer, pH 7.0, T = 20 °C. The magnitude of the symbols
corresponds to approximately ± 1 standard deviation (±
0.017 on the ordinate scale).
In spite of its
limitations (see, for example, Ackers et al. (1992)), the
analysis of the oxygen binding isotherms was carried out according to
the two-state allosteric model (Monod et al., 1965). Fit of
data is often beset by uncertainties in K and L
. Nonetheless, since kinetic data on O
and CO have provided independent support that K
is identical or very close to that of the isolated
and
chains and of the
dimer (Edelstein and Gibson, 1987;
Szabo and Karplus, 1972), we fitted the data in Fig. 2assuming K
to be the same for HbA and all the mutants, and
thus similar to the oxygen affinity of the isolated chains. This
assumption proved compatible with a good fit, with the parameters
presented in Table 1. By contrast, very poor fits were obtained
if K
of the mutants was imposed to be the same as
that of HbA, which is not surprising and agrees with the theory of
Szabo and Karplus(1972). Within the limitations of the two-state model,
the numerical values of the allosteric parameters indicate that
mutations at C3 (i) affect the equilibrium constant between the two
quaternary states, reducing the difference in stability between T
and R
in unliganded Hb (see
values of L
), and (ii) reduce to some extent the
constraints imposed on the subunits by the quaternary assembly as
deduced from the values of K
.
Figure 3:
Time course of CO combination by stopped
flow. Open squares, HbA; closed squares, Hb
T38W; open circles, Hb
W37T; closed
circles, Hb
T38W/
W37T. Lines are drawn according to a
sequential scheme (see text). CO was 50 µM after mixing;
other experimental conditions were as described for Fig. 2.
The data reported in Fig. 3were fitted to four consecutive and irreversible
pseudo-first order processes, as described by Hopfield et
al.(1971). The values of L and c (K
/K
) used to calculate
the relative amount of T- and R-state Hb at each
ligation state were taken from Table 1; the values of
k` were obtained by partial flash photolysis,
which is known to populate partially liganded species that recombine as
fast as the isolated chains (Sawicki and Gibson, 1976; Vandegriff et al., 1991; Jones et al., 1992). The CO combination
time courses in Fig. 3are satisfactorily fitted with this
model, regardless of the presence of evident kinetic cooperativity, and
the rate constants are given in Table 2. Regarding Hb
W37T,
an autocatalytic time course may have been expected. However, it is
known that speeding up of the CO combination is very sensitive to the
value of c, which may not be identical for O
and
CO, even though this difference is sometimes very difficult to
demonstrate. Under these conditions the estimate of
k` is subject to some uncertainties, and the overall time course is
essentially exponential.
Even complete photolysis of dilute
solutions of HbCO usually follows a biphasic rebinding time course
(Antonini and Brunori, 1971). This has been attributed to the presence
of rapidly reacting dimers in equilibrium with the slowly reacting
tetramers (Gibson and Antonini, 1967; Edelstein et al., 1970).
Thus flash photolysis has also been used to probe the extent of
dissociation of HbCO into dimers. Employing this technique, Vallone et al. (1993) have shown that Hb T38W CO is a more stable
tetramer than HbA CO, by approximately 0.6 kcal/mol. Similar
experiments carried out as a function of Hb concentration (data not
shown) have demonstrated that the CO derivative of the two
W37T
mutants is almost completely dissociated into dimers at [Hb]
= 15 µM, and thus a lower limit of 40 µM can be set to the value of the tetramer-dimer dissociation
constant of these mutants.
Since O and CO exhibit nearly
parallel equilibrium curves, the data in Table 1and Table 2may be considered together even though most of the
cooperativity is expressed in the dissociation rate constants in the
case of O
and in the association rate constants in the case
of CO (Antonini and Brunori, 1971; Szabo, 1978).
A significant
conclusion is that all our mutants display a ligand affinity higher
than HbA in the T-state. However, this increase is smaller for
the two single mutants, and substantial for the double mutant, in
keeping with the reduced number of contacts at the
interface (see below). More
interestingly, the value of L
decreases
significantly in the mutants, being, in the double mutant,
15-fold
smaller than in HbA.
The
experiment is described in the following scheme ().()
An example of this type of experiment for HbA and the three
mutants is reported in Fig. 4. The plot shows the second column
of V (from the SVD analysis), which contains most of the slow
optical transition. The faster phase seen in this figure is the time
course of O dissociation (upon mixing with dithionite), and
is described by steps 1 and 3 of . The slower phase (step
2 in the same scheme) relates to the time course of association of
deoxy dimers. The relative amplitude of this phase is a measure of the
fraction of oxy dimers in equilibrium with oxy tetramers. Since
addition of 0.5 M NaI promotes complete dissociation of the
liganded tetramers but does not prevent the reassociation of unliganded
dimers (Kellett and Gutfreund, 1970), experiments carried out under
these experimental conditions yield the full amplitude of the slow
phase. Estimates for the rate constant of deoxy dimers association for
HbA and the three mutants (in the absence and in the presence of 0.5 M NaI) are reported in Table 3.
Figure 4:
Time course of the oxygen dissociation
(rapid upward phase) and dimers reassociation (slower downward phase)
for HbA (A), Hb T38W (B), Hb
W37T (C), and Hb
T38W/
W37T (D). Each panel
depicts the time evolution of the amplitude of column 2 of the V matrix
obtained form the SVD of an experiment carried out at two hemoglobin
concentrations, namely 2.5 (open symbols) and 1.25 (closed
symbols) µM after mixing (heme basis). Conditions
were as described for Fig. 2.
The static difference
spectra obtained upon binding to Hp are very similar to those obtained
by the kinetics of dimer recombination (data not shown). The total
absorbance change for complete dissociation of deoxy tetramers into
dimers was found to be 13% of the total absorbance of the sample at 430
nm. This is in good agreement with the value expected for the T-R
spectral change (for a
review, see Bellelli and Brunori(1994)).
Fig. 5shows the observed time courses of dissociation. This experimental approach suffers from the extremely slow time course, which, at lower temperatures, extends over several days; thus, for some proteins and under some conditions, the optical transition could not be followed to completion. Nonetheless, the time course was fitted in all cases to a single exponential following Ip et al.(1976). In addition, the asymptotic value was independently estimated from the total optical density change (see above).
Figure 5:
Time course of dissociation of the
deoxygenated tetramer of HbA and mutants induced by binding of
haptoglobin, in the presence and absence of NaI. The percentage of the
reaction, followed by the decrease of absorbance at 430 nm, is plotted versus time, and fitted using a monoexponential equation. Panel A, HbA; panel B, Hb T38W; panel
C, Hb
W37T; panel D, Hb
T38W/
W37T. Open symbols, buffer 0.1 M Bis-Tris pH 7.0; closed symbols, the same buffer as before plus 0.5 M NaI, temperature = 20 °C.
Table 4reports the first order
rate constants determined from the data in Fig. 5. HbA and Hb
T38W behave similarly, while both Hb
W37T and the double
mutant dissociate faster than HbA (in spite of being tetrameric in the
deoxygenated derivative, as judged by the recovery of the expected
optical transition). The effect of NaI on the dissociation of HbA and
Hb
W37T is the same (see the ratio k`/k in Table 4). However, quite unexpectedly, this effect is
considerably reduced in the two mutants in which
38 (C3) is
mutated to Trp; this point will be discussed below, with reference to
the structure of the interface.
Figure 6:
Symmetry averaged difference map F(Hb T38W) - F(HbA). The map is contoured
at +0.15 (green contours) and -0.15 (red
contours), or approximately 3 times the root-mean-square density
value of the unaveraged map. Panel a, the superimposed model
is that of wild type HbA with
38 Thr replaced by Trp with
1
angle -60°. Residues of the
chain are
labeled with their ordinary position numbers and the letter A,
those of the
chain with position numbers increased by
600 and the letter D. Panel b, only the negative
values of the difference map are shown (red contours) and the
HbA structure is displayed. The dashed lines represent
hydrogen bonds. Labels as in panel
a.
As shown in Table 5, upon introduction
of a Trp at 38 (C3) the increase in the number of interface
contacts between
and
is negligible
in deoxy, but considerable in oxy-Hb. Conversely the substitution Trp
Thr at
37 (C3) leads to a substantial decrease in the
number of contacts in both oxy- and deoxy-Hb, with loss of a hydrogen
bond between
37 Trp and
94 Asp in the deoxy-Hb (see Fig. 7, panel C). Hence one could deduce that compared
to HbA, the
T38W mutant has a more compact and extensive
interface especially in the R-state; on the other hand, Hb
W37T has a looser
interface, especially in the T-state, with loss of a hydrogen
bond.
Figure 7:
Residues at position C3 (shown in black) and their neighboring amino acids in a sphere of 4
Å for oxy- and deoxy-HbA (Table 5). Residues belonging to
the and
chains are shown in light gray. The labels
are indicated only for the monomer opposite the C3 residue. The dots represent the water accessible surface (probe radius 1.4
Å) within the HbA tetramer. Hydrogen bonds involving the C3 side
chain (either with other amino acid residues or with water molecules)
are represented by a gray dotted line. Oxy-Hb: a,
38Thr from the crystallographic structure (Shaanan,
1983); b,
38Trp modeled starting from a.
Deoxy-Hb: c,
37Trp from the crystallographic structure
(Kavanaugh et al., 1992b); d,
37Thr modeled from c; e,
38Thr from the crystallographic structure
(Kavanaugh et al., 1992b); f,
38Trp form the
crystallographic structure (this paper). It has to be noticed that the
number of residues in a sphere of 4 Å from the amino acid side
chains of interest are fewer when the side chain is smaller and larger
when this is greater; therefore, the amino acids displayed in panels a and b, c and d, and e and f of this figure are not the
same.
Assuming that these effects are additive in the double mutant, one may expect that in this hemoglobin cooperativity will be severely impaired, since the R-state would be favored with a concomitant destabilization of the T-state; this prediction agrees well with the experimental data reported above.
In order to
compare the data obtained on Hb W37T with observations reported in
the literature on Hb Hirose (which bears the substitution
W37S,
Sasaki et al., 1978), we have measured the atoms in a sphere
of 4 Å to a Ser in
C3. As may be seen in Table 5, the
number of contacts in Hb Hirose is reduced, in both oxy and deoxy
state, compared to our mutant
W37T, in agreement with the
experimental data (see ``Discussion'').
Inspection of the
water accessible surface in the proximity of position C3 allows
evaluation of the cavities at the edge of the
interface, and how the shape of the
surface is affected by the mutations. The crystallographic structure of
deoxy-Hb
T38W shows that the Trp in
C3 lies flat on the edge
of the interface, excluding
145 Tyr and
100 Pro from contact
with the solvent, and thus seems to act as a ``hydrophobic
plug'' (Fig. 7, panel F). Modeling the oxygenated
state of the same mutant shows that the side chain of the Trp
introduced in
C3 could also fit nicely in a cavity, in contact
with
145 Tyr.
The substitution W37T, on the other hand,
seems to deepen a cavity at the edge of the opposite side of the
interface, possibly allowing to
141 Arg greater accessibility to the external medium.
Hb T38W, which bears two Trp
residues in the
interface, is a
slightly more stable tetramer than HbA, especially in the liganded form
where the tetramer dissociation constant is decreased 6-fold (Vallone et al., 1993). The three-dimensional structure of this mutant
in the deoxy state shows that the indole side chain of Trp
38 has
been accommodated by expulsion of two water molecules hydrogen-bonded
to
38 Thr in HbA, without significant changes of the adjacent
residues (Fig. 6). Thus, the enhanced stability of the oxy
tetramer is consistent with the increased hydrophobic character of the
interface (which is not perturbed by the bulkier side chain), with the
more extensive contacts of the indole (Table 5), and with the
hypothesis that the most significant contribution to the stability of
the tetramer resides in the pseudo symmetric
FG-
C contact,
which is unmodified. The slightly increased affinity of the T-state observed for Hb
T38W as compared to HbA is not
easily accounted for, given that the constraints in the T-state are not decreased. On the other hand, the lower value
of L
is consistent with the increased stability of R and thus with a (slightly) reduced cooperativity.
Hb
W37T, which has no Trp residues in the
interface, dissociates into dimers
more readily than HbA, in both the unliganded and liganded derivatives,
as shown by results from analytical ultracentrifuge and flash
photolysis to be published elsewhere, indicating that Trp at
C3
contributes to the stability of the
interface in both quaternary structures. In spite of the
destabilization of this interface, cooperativity of
W37T is
reduced but preserved, not only because the contacts in the
C-
FG switch region (
97 His,
41 Thr,
44 Pro,
and
38 Thr) are preserved, but also because Thr at
37(C3) can
to some extent fulfill the role of Trp in the flexible joint, as
indicated by modeling (Table 5). The remarkable but limited
competence of Thr at
37 residue is reflected in a small increase
of the O
affinity of the T-state (Table 1)
and a somewhat greater increase of the rate constant for CO binding to
deoxy-Hb (Table 2); thus, the constraints of the T-state
are partially released by mutation of
C3 and essentially
maintained by mutation of
C3.
The interesting behavior of the
double mutant Hb T38W/
W37T may be understood qualitatively on
the basis of the properties of the two single mutants. Although the
double mutant, like HbA, has only one Trp residue at the
interface, ligand binding and
tetramer dissociation are both remarkably different from wild type.
This is the best evidence that the effect of mutations at topological
position C3 is asymmetric and that perturbing the flexible joint and
the switch indeed has different consequences. This double mutant
dissociates more readily into dimers in both the liganded and
unliganded derivatives, which we attribute largely to the flexible
joint mutation
W37T. However, cooperativity is also reduced, even
when compared to that of the two single mutants, as shown by the
increased O
affinity of the T-state (Table 1). These properties may be understood on the basis of two
synergistic effects, i.e. a significant release of the
interface constraints due to
W37T, leading to a more relaxed T-state (hence the smaller K
), and an
increase in the stability of the R-state tetramer, related to
mutation
T38W. As a result the energy difference between the two
allosteric states is reduced, as indicated by the value of the
allosteric constant L
, which is smaller than HbA
by
15-fold.
As already stated, there is no known natural mutant
of position 38 (Huisman, 1992); however, two site-directed mutants
of this residue, namely Hb
T38S and
T38V, have been expressed
and characterized by Hashimoto et al.(1993). The functional
properties of both these Hbs are by and large similar to those of HbA.
However, the substitutions in these case are semiconservative, and it
is easily conceivable that the residues effectively replace the wild
type Thr.
As to position 37, our data may be compared with
those of the natural mutants Hb Hirose and Hb Rothschild. Comparison of
Hb
W37T with Hb Rothschild (
37 Trp
Arg; Gacon et
al.(1977)) is complex, because of arginine's positive
charge. Nonetheless, consistent with the other
37 mutants, Hb
Rothschild displays reduced cooperativity and an increased tendency to
dissociate into dimers. However, the structure of the deoxygenated
derivative (Kavanaugh et al., 1992b) shows a novel and strong
chloride binding site, which affects the functional properties of this
Hb.
Hb Hirose (37 Trp
Ser) displays extremely low (if
any) cooperativity, very rapid CO binding by flow, and a high tendency
to dissociate into dimers in both the presence and the absence of
oxygen (Sasaki et al., 1978). Hb
W37T, on the other hand,
although extensively dissociated into dimers when liganded, is fully
associated as the deoxy derivative (Fig. 5) and maintains a
higher cooperativity than Hb Hirose, confirming that Thr but not Ser is
a partially competent substitute for
37 Trp at the flexible joint.
The computer-simulated structures of oxy- and deoxy-Hb
W37T (Fig. 7) indicate that the methyl group of Thr
37 makes
contacts with
95 Pro and
140 Tyr in the oxy derivative and
with
140 Tyr in the deoxy derivative; these contacts are lost or
less extensive with Ser (Table 5).
A site-directed mutant of
position 37, in which a Phe substitutes the Trp, has been obtained
by Ishimori et al.(1992). In this case the Phe would be
expected to provide a more effective replacement for Trp than either Hb
Hirose or our mutant. Unfortunately the authors did not measure the
tetramer-dimer equilibrium and kinetics on their mutant Hb. As with
W37T, Hb
W37F displays high oxygen affinity and low
cooperativity, even though the
H NMR spectrum of this
mutant is consistent with that typical of T-state HbA.
Interestingly, Table 3shows that the rate constant of reassociation of
unliganded dimers and the effect of NaI are the same (within a factor
of two) for all four hemoglobins. Thus, the effect of interface
mutation(s) on the stability of the deoxygenated Hb tetramer is only
very slightly (if at all) reflected in its rate of association, in
agreement with Ackers et al.(1992); the same conclusion holds
for the dissociating effect of NaI. These observations imply that
formation of the productive dimer-dimer complex is marginally affected
by one or two conservative changes at the contact interface. This is
not wholly surprising in view of the large surface buried in the
contact between two dimers (
1500 Å
).
On the other hand, the effect of mutations on the rate of
dissociation of the deoxy tetramer is more marked and follows an
interpretable trend (Table 4). In deoxy-Hb T38W there are 8
intersubunit contacts compared to 7 in deoxy-HbA, consistent with just
a 30% decrease in the rate of tetramer dissociation (Table 4).
The
37 mutants, on the other hand, both dissociate faster than Hb
A, which we attribute to the loss (i) of the contact between
141
Arg and
37 Trp with perturbation of the network of salt bridges
present in the T-state, and (ii) of a hydrogen bond between
37 Trp and
94 Asp (Fermi and Perutz, 1981). Therefore, the
reduced number of contacts (Table 5) agrees well with the
increased rate of dissociation of the
37 mutants (which can be as
much as 25-fold).
However, it should be pointed out that the rate
constants for dissociation of deoxy-Hb W37T and Hb
T38W/
W37T are quite different. This implies that the effect
of the two mutations is not additive; otherwise, the double mutant
would dissociate as fast as Hb
W37T. This deviation from simple
additivity suggest that the interface may be also distorted by long
range effects. This is not unreasonable, given that perturbation of a
single residue even at the
interface
leads to a large destabilization of the T-state and loss of
cooperativity (see, for example, Amiconi et al.(1989), Weber et al.(1993), and Zhang et al.(1996)). A thorough
interpretation of non additive effects necessarily implies taking into
account long range interactions, which indeed are well documented.
However, we would rather to defer this analysis until the
crystallographic structure of these mutants (now in progress) is
available.
On the other hand, the effect
of I on the rate of dissociation of the deoxy
tetramer depends in a rational way on the amino acid residue at
position C3. This is evident comparing the ratio of the rate constants k` and k reported in Table 4. With a Trp in the
switch region as in Hb
T38W, I
accelerates
dissociation only 3-fold compared to 13-fold in HbA. We interpret the
reduced effect as resulting from the more hydrophobic character of the
interface and from the position of Trp, which seems to act as a
hydrophobic plug, shielding from contact with the solvent
145 Tyr
and
100 Pro and forming a barrier to the penetration of this ion.
When Trp is removed from the flexible joint as in Hb W37T,
I
accelerates dissociation approximately 13-fold,
just as in HbA. In deoxy-Hb,
37 Trp is on the edge of the
interface ( Fig. 1and Fig. 7), and its orientation is such that
95 Pro and
140 Tyr are also exposed to the external medium; mutation of Trp
with Thr therefore does not result in exposure to the solvent of side
chains previously buried in the interface. Thus, the similarity in the
effect of I
on the dissociation rate constants of HbA
and Hb
W37T mutant is fully consistent with the proposal that
access of the anion to the
interface
is toward the switch region, near position
38, and therefore not
affected by substitution in the flexible joint region. In support of
this hypothesis, the effect of I
on the double mutant
is again smaller and similar to that of Hb
T38W, because of the
hydrophobic plug effect. Thus, we conclude that the effect of
I
on the stability of the deoxy tetramer is linked to
binding of this anion to the interface near position C3. This
prediction may be checked by examination of the crystallographic
structure of deoxy-HbA in the presence of NaI.
The atomic coordinates (1GLI) and structure factors (R1GLISF) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
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