(Received for publication, January 29, 1997)
From the Program in Molecular Medicine and Department
of Biochemistry and Molecular Biology, University of Massachusetts
Medical Center, Worcester, Massachusetts 01605, the
§ Department of Biochemistry, Molecular and Cell Biology,
Cornell University, Ithaca, New York 14853 and the Department of
Biochemistry and Molecular Biology, Rice University, Houston, Texas
77025, and the ¶ Consiglio Nazionale delle Ricerche Center of
Molecular Biology, Department of Biochemical Sciences, University La
Sapienza, 00185, Rome, Italy
Residue Phe97, which is thought to play a central role in the cooperative functioning of Scapharca dimeric hemoglobin, has been mutated to leucine to test its proposed role in mediating cooperative oxygen binding. This results in an 8-fold increase in oxygen affinity and a marked decrease in cooperativity. Kinetic measurements of ligand binding to the Leu97 mutant suggest an altered unliganded (deoxy) state, which has been confirmed by high resolution crystal structures in the unliganded and carbon monoxide-liganded states. Analysis of the structures at allosteric end points reveals them to be remarkably similar to the corresponding wild-type structures, with differences confined to the disposition of residue 97 side chain, F-helix geometry, and the interface water structure. Increased oxygen affinity results from the absence of the Phe97 side chain, whose tight packing in the heme pocket of the deoxy state normally restricts the heme from assuming a high affinity conformation. The absence of the Phe97 side chain is also associated with diminished cooperativity, since Leu97 packs in the heme pocket in both states. Residual cooperativity appears to be coupled with observed structural transitions and suggests that parallel pathways for communication exist in Scapharca dimeric hemoglobin.
The homodimeric hemoglobin (HbI)1 from the blood clam Scapharca inaequivalvis offers a simple model system for studying communication between two chemically identical subunits. Analysis of the mechanistic details of this intersubunit communication is being pursued to provide insights into the regulation of protein function.
Scapharca HbI binds oxygen cooperatively, with a p50 of 7.8 torr and a Hill coefficient (n) of 1.5 at 20 °C, neither of which changes as pH varies from 5.5 to 9.0 (1). Although the individual subunits of HbI have the same myoglobin fold as mammalian hemoglobins, the quaternary assemblage is radically different (2). Upon oxygen binding by HbI, only small tertiary changes are seen at the subunit interface in contrast to the relatively large quaternary changes observed with mammalian hemoglobins (3). Analysis of structures of this hemoglobin at 1.6 Å for the deoxygenated molecule, 1.4 Å for the CO-liganded form, and 1.7 Å for the oxygenated form has provided a framework for understanding the role of individual side chains and interfacial water in mediating cooperativity (2, 4).
Phenylalanine 97 has a central role in the proposed structural
mechanism of cooperativity (2, 4). The side chain of Phe97
undergoes the largest ligand-linked conformational change in HbI. In
the deoxy state, it is tightly packed in the heme pocket, whereas upon
ligand binding it is displaced from the heme pocket into the subunit
interface. The lowered oxygen affinity of the deoxy state appears to
result primarily from the packing of Phe97 in the heme
pocket, where it restricts movement of the iron atom into the heme
plane and lengthens a hydrogen bond involving the proximal histidine.
Upon ligation, movement of Phe97 into the subunit interface
is coupled with disruption of a well ordered interfacial water cluster
and movement of the heme groups deeper within each subunit. These
effects within a subunit alter the nature of interactions between
subunits, which presumably encourages movement of the
Phe97 side chain (second subunit) into the interface,
allowing the second subunit to attain a high oxygen affinity state
prior to ligand binding (2). In view of its significant role in the proposed cooperative mechanism of HbI, Phe97 is clearly
indicated as a target for mutation. Phe97, four residues
from the proximal histidine at position 101, is analogous to the fourth
residue of the F helix in mammalian hemoglobins, which is a leucine in
all known vertebrate hemoglobins and most invertebrate hemoglobins. The
smaller size of the leucine side chain in these hemoglobins allows it
to remain packed in the heme pocket in both liganded and unliganded
states.
In this paper, we present functional and high resolution crystallographic studies of the Leu97 HbI (F97L) mutant. This mutation results in increased oxygen affinity and diminished cooperativity that appear correlated with observed ligand-linked structural transitions. Our results confirm the crucial role of residue Phe97 in modulating oxygen binding by HbI, while demonstrating the persistence of residual cooperativity and suggesting that parallel pathways exist for information transfer between subunits.
Escherichia coli strain TG1 was used as the host strain for propagating recombinant bacteriophage M13mp18 vector and for in vitro site-directed mutagenesis. E. coli strain W3110lacIq L8 was the host used for overexpression of recombinant HbI (5).
MutagenesisIn vitro oligonucleotide-mediated,
site-directed mutagenesis was used to generate the F97L mutant. The
initially constructed gene for HbI bearing 5 and 3
termini
complementary to KpnI and SacI sites was cloned
into the polylinker region of a M13mp18 bacteriophage vector (New
England Biolabs, Beverly, MA), which had been cut previously with the
same two restriction endonucleases. Following several rounds of plaque
purification using TG1 cells as hosts, recombinant single-stranded DNA
was isolated (6). Subsequently, a 19-mer oligonucleotide (University of
Massachusetts Medical Center DNA synthesis facility) complementary to
the region of the gene coding for residue Phe97 was used in
a site-directed mutagenesis protocol (Sculptor in vitro
mutagenesis system, Amersham Life Science Inc.) to mutate residue 97 to
leucine. Following three rounds of plaque purification, single-stranded
DNA was isolated and the changes in DNA sequence were confirmed by
dideoxy sequencing using the Sequenase kit (U. S. Biochemical Corp.).
Double-stranded DNA of the replicative form of the vector was then
prepared and subjected to AccI and SacI restriction cleavage to isolate a segment of the HbI gene that contains
the leucine 97 mutation. This fragment was then subcloned into a
similarly digested wild-type HbI gene borne on a pCS26 expression
vector that, as reported earlier, has allowed for significant overexpression of recombinant, wild-type HbI protein (5).
Recombinant F97L was overexpressed in E. coli and purified as described for wild type (5).
Spectroscopic CharacterizationSpectroscopic measurements
were carried out at 20 °C with a Cary 3 spectrophotometer at a
protein concentration of about 6 × 105
M (heme) in 0.1 M phosphate buffer at pH 7.0. Oxygen equilibria were measured using a tonometer, according to
Rossi-Fanelli and Antonini (7), with a reducing system to minimize
protein oxidation (8).
Circular dichroism spectra were measured at 20 °C in 0.1 M phosphate buffer at pH 7.0 in a Jasco J710 spectropolarimeter equipped with a Jasco J700 processor in the range 380-650 nm. The deoxygenated derivative was obtained by addition to the oxygenated protein of a small volume of a 1.0 M sodium dithionite solution prepared in deoxygenated buffer.
Kinetic MeasurementsKinetic measurements using flash photolysis were performed using a YAG laser giving a 9-ns flash of 25 mJ at 532 nm. The beam was telescoped to give a parallel beam 2 mm in diameter colinear with the observing beam supplied from a 75-W xenon arc passing through a blue filter and imaged at the sample within the photolysis beam. The observing beam continued through a Spex 250-mm monochromator to a photomultiplier and amplifier (Teledyne-Philbrick 1321). The output from the amplifier was digitized by a DAS-50 12-bit A/D converter (Metrabyte) and transferred to an IBM personal computer. The combination had an overall response time of 2 µs.
Stopped flow measurements were performed with the apparatus described by Gibson and Milnes (9) in the slightly modified version of the Durrum Corp. (Palo Alto, CA). Data were digitized and recorded as described for flash photolysis using a tungsten lamp as light source. The dead time of the stopped flow experiment (2-cm path) was 2.4 ms. For the low oxygen experiments the apparatus was filled with a dilute solution of sodium dithionite and allowed to stand overnight. It was then washed out repeatedly with buffer bubbled with pure nitrogen before introducing the working solutions. Low oxygen concentrations were obtained by mixing air-equilibrated buffer with nitrogen-bubbled buffer in a syringe. For the oxygen-pulse experiments a strong (5 mM) solution of deoxygenated hemoglobin was diluted into nitrogen-bubbled buffer and solid dithionite was added (10).
A kinetic equivalent of the two-state model was then developed to simulate the progress of oxygen saturation in oxygen-pulse and other kinetic experiments using a two-step Runge-Kutta process (11). To make comparisons with experimental data, a set of oxygen-pulse experiments with a series of initial concentrations of oxygen was performed and corresponding simulations were run. The early stages of both the experiments and the simulations were then discarded and the remaining time courses, which do not depart from a single exponential, were fitted using a nonlinear least squares program. In addition to the kinetic parameters for the two-state model it is necessary to supply values for the removal of oxygen from solution by dithionite. This was done empirically with attention directed to reproducing the peak fractional saturation observed with the experiment.
CrystallizationDeoxy and carbonmonoxy F97L crystals were grown as described in Ref. 2, except that both crystal forms were grown from solutions of 40-50 mg/ml hemoglobin. Microseeds obtained by crushing corresponding wild-type HbI crystals were used to nucleate mutant HbI crystal growth. The morphologies of both crystal forms were similar to those of wild-type recombinant HbI.
Collection of Diffraction DataX-ray diffraction data were collected from crystals at room temperature on a R-AXIS IIC imaging plate system mounted on a Rigaku RU 200 rotating anode generator (Molecular Structure Corp., The Woodlands, TX). Data were processed using software supplied by the manufacturer.
The F97L-CO crystals were essentially isomorphous to native crystals,
showing the symmetry of monoclinic space group C2, and cell constants
a = 93.25 Å, b = 43.98 Å,
c = 83.50 Å, and = 122.03°. A total of 116 frames was collected from two crystals with an oscillation range of 1.5 to 1.8° for each frame. The overall Rmerge for
40,595 unique reflections with I/
> 1 was 5.52%
(Rmerge =
I
<I>
/
I, where I is the measured intensity
of each reflection and <I> is the mean value for the
corresponding unique reflection). This data set represents 93.5% of
the predicted unique reflections to Bragg spacings greater than
1.6 Å and 88.8% to 1.5 Å.
Deoxygenated F97L HbI crystals showed the symmetry of orthorhombic
space group C2221, with cell constants of a = 91.99 Å, b = 44.27 Å, and c = 143.83 Å, essentially isomorphous to native deoxy crystals. The deoxy
F97L HbI data set represents diffraction data from a single crystal
that was collected in two different orientations. Initially the large
c axis (143.8 Å) was aligned horizontally (perpendicular to
the spindle axis). The close spacings between reflections along the
c* axis necessitated a small oscillation range (0.9°) in
this orientation for the 34 oscillation frames collected. In a second
set of frames, the crystal was reoriented with its c axis
vertical (parallel to the spindle axis). This allowed for the use of a
larger oscillation range of 2.5°. A total of 30 oscillation frames
was recorded from this crystal orientation. The overall
Rmerge for 27,590 unique reflections derived
from 95,839 independent observations with I/ > 1 was 7.33%. These represent 84.1% of the predicted unique data to Bragg spacings greater
than 1.7 Å.
The starting models for both CO-liganded and deoxy Leu97 HbI structures were the native HbI structures (2) in which residue Phe97 was mutated to leucine using the macromolecular modeling program "O" (12). Also, the water molecules associated with these structures were deleted and alternate conformers of protein side chains eliminated from the Protein Data Bank coordinate files (entry codes 3SDH and 4SDH).
These models were then refined using the XPLOR package (13). Prior to the commencement of refinement, 10% of randomly selected reflections from each data set were designated as test reflections for use with the Free-R cross-validation method (14). Two cycles of simulated annealing were performed, with the first cycle incorporating noncrystallographic symmetry restraints upon the model. The protocol for simulated annealing that was used called for "heating" the molecule to 1000 K followed by 50 steps of 0.5-fs molecular dynamics. After each set of 50 steps, the temperature was lowered by 25 K and 50 new dynamics steps were performed. This procedure was continued until a temperature of 300 K was reached and was followed by 60 cycles of conjugate gradient minimization combined with B-factor refinement.
Addition of water molecules to the models was initiated at this stage
using the programs PEAKS and LOCATE written by Wayne Hendrickson
(Columbia University, New York). Peaks of electron density greater than
3.5 in Fo
Fc maps
and 0.8
in 2Fo
Fc
maps that satisfied hydrogen-bonding criteria were identified as
locations for water molecules. This procedure was reiterated, resulting
in the addition of five and four shells of water to the CO-liganded and
deoxy structures, respectively. Each model was subjected to 120 cycles
of conjugate gradient minimization combined with B-factor refinement
using XPLOR following the addition of each shell of water molecules.
Water molecules with refined B-factors greater than 50 Å2
were deleted.
The model was then examined in conjunction with
2Fo Fc and
Fo
Fc maps to locate
poorly placed protein side chains and missing water molecules, which
were then built. This was followed by 120 cycles of conjugate gradient
energy minimization combined with B-factor refinement.
Mutation of phenylalanine 97 to leucine (F97L) in Scapharca HbI resulted in increased ligand affinity and diminished cooperativity as observed by both equilibrium and kinetic measurements. The p50 value for oxygen binding, based on equilibrium measurements, decreased from 7.8 to 1.0 torr, and the Hill coefficient decreased from 1.5 to 1.16 ± 0.05. The statistical analysis of all the data sets (n = 12) indicated that the Hill coefficient was significantly higher than 1.0, such that cooperativity, although drastically reduced, was not abolished.
A summary of kinetic experiments on ligand binding is presented in Table I. The increased oxygen affinity observed in equilibrium measurements was primarily due to a decrease in the oxygen dissociation rate, particularly at low O2 saturation. Oxygen-pulse and CO-replacement experiments (15) showed a greater than 10-fold decrease in the dissociation rate at low oxygen saturation compared with HbI. In contrast to HbI, there was little or no change in the oxygen-dissociation rate for F97L as a function of varying levels of oxygen saturation (Fig. 1). Additionally, measurements of CO binding to F97L by both stopped flow and flash photolysis showed a clear increase in combination rates compared with wild type.
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The absorption spectra of the oxygenated and CO-liganded derivatives of F97L were almost identical to those reported for native HbI (1). In contrast, significant differences were observed for the deoxygenated derivative. In the Soret region, the band line shape was wider and more symmetric and the absorption maximum was shifted to lower wavelengths by 4 nm. In the visible region, the unusual shoulder present at 590 nm (Qo band) in wild-type HbI was absent (data not shown).
The circular dichroism spectra of deoxy F97L (Fig. 2)
confirmed the peak shift observed in the absorption spectra and
displayed additional interesting features: (i) the overall molar
ellipticity values were decreased by 20-25% with respect to HbI, a
value that is beyond experimental error; (ii) the ellipticity of the
Qo band was reduced; and (iii) the splitting of the Soret
band (Bo band), which gives rise to the characteristic
negative peak at 419 nm in HbI, was greatly diminished in F97L.
Upon substitution of Phe97 with the smaller leucine side
chain, the strong Qo band at 590 nm, which is diagnostic of
proximal strain in wild-type HbI (1, 16), was reduced. In addition, the
asymmetry of the energy and polarization of the electronic transition
moments of the components of the Bo band, which gives rise
to splitting of the Soret band, was diminished in the mutant in which
symmetry was restored. The overall decrease in molar ellipticity in
F97L supports the coupled oscillator theory of Hsu and Woody (17),
since the removal of an aromatic ring from within 4 Å of the heme
plane is expected to decrease the rotational strength of the coupled
-
* transitions of aromatic residues and heme, thus decreasing the
intensity of the Cotton effect.
The models for deoxygenated and CO-liganded F97L have been refined against the x-ray data to conventional R-factors of 18.3 and 18.6%, respectively, with excellent stereochemistry (Table II). An electron density map of the heme region of one subunit is shown in Fig. 3. Both the unliganded and liganded structures of F97L were found to be strikingly similar to the corresponding structures of wild-type HbI, with significant differences confined to the disposition of residue 97 side chain, F-helix geometry, and the interfacial water structure. Evidence of similarities in the overall structure is presented in Fig. 4.
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As shown in Tables III and IV, the overall B-factors were slightly higher in deoxy F97L than in deoxy wild type and were nearly the same for the CO-liganded structures. In contrast to the similarity in overall B-factors, the core interface water molecules showed significantly higher B-factors in F97L than in wild type. This indicates that these water molecules, which have been implicated in cooperative function in wild-type HbI (18), are less well ordered in F97L.
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In the
deoxy F97L structure, the disposition of the side chain of residue 97 was very similar to that in the deoxy wild-type structure, where it is
packed in the heme pocket (Figs. 4 (top) and
5). Unlike the phenylalanine side chain, which is packed
tightly between the heme group and the proximal histidine (residue
101), the smaller Leu97 side chain appeared to be
accommodated without strain. This was reflected in differences in heme
conformation, position of the heme iron, and subtle changes in F-helix
geometry compared with deoxy wild-type HbI, as discussed further
below.
Upon ligand (CO or O2) binding to wild-type HbI, the Phe97 side chain is displaced into the subunit interface, since its tight pocket is reduced when the iron moves into the heme plane (2). In contrast, the leucine side chain remained in the heme pocket in F97L upon ligand binding (Fig. 4, bottom).
F-helix GeometryIn deoxy wild-type HbI, there is a sharp
bend in the F-helix (residues 87-103) (Figs. 4 (top) and 5)
that was postulated to result from wedging of the Phe97
side chain in the heme pocket (2). Unexpectedly, this bend, although
attenuated, persisted in F97L, where the smaller leucine side chain was
readily accommodated in the heme pocket. Subtle differences in F-helix
geometry were present including a movement of the backbone toward the
heme group, most markedly seen involving the C- of residue
Leu97 (0.5 Å), but also involving Lys96 (0.3 Å) and Glu95 (0.3 Å), as shown in Fig. 5. This change
presumably resulted from loss of the tight packing of Phe97
between the F-helix backbone and the heme group. Additionally, the
distances between the carbonyl oxygens of residues 96 and 97 and the
amide nitrogens of residues 100 and 101, respectively (4.5 and 3.9 Å),
were slightly less than the corresponding distances in the wild-type
deoxy structure. These effects reflect a subtle straightening of the
sharp bend observed in the F-helix of deoxy wild-type HbI. Also, the
hydrogen bond between the main chain carbonyl oxygen of residue 97 and
the N
of the proximal histidine, which is hypothesized to play a
role in determining oxygen affinity of the heme iron (2, 19, 20), was
optimized in F97L (Tables V and
VI).
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Coupled with ligand binding, a movement of the heme group deeper into each subunit and away from the dimer interface is observed with wild-type and F97L HbI. This movement was previously suggested to result from extrusion of the tightly packed Phe97 side chain from the heme pocket (2). Our structural results argue against such a determinative role for Phe97. Thus, other interactions between the heme group and protein must be sufficient for this heme movement. In this regard, interactions of protein side chains with the domed deoxy heme as well as intra- and intersubunit interactions with the heme propionates likely contribute to this effect.
Interface Water StructureThere exists an elaborate network of hydrogen-bonded water molecules in the subunit interface of wild-type HbI. These water molecules have been proposed to play an important role in the cooperative mechanism by providing stabilizing hydrogen-bond interactions to the alternate allosteric states of HbI (2, 18).
In the deoxy wild-type structure, of the 219 total water molecules, 17 are located at the central core of the dimer interface. These water molecules are symmetrically distributed about a noncrystallographic dyad (2-fold), even though this restraint was not imposed in model building or refinement, and are very well ordered, with an average B-factor of 19.6 Å2. Of these, the lowest B-factors are seen for a cluster of 4 water molecules that are located at the bottom of the interfacial crevice and that are hydrogen-bonded to E-helix residues that remain largely unaltered by ligand binding.
In comparison, 174 water molecules were independently built into the
deoxy F97L structure during the course of model refinement. As shown in
Fig. 6 (top), the interfacial water
architecture for this structure was found to be virtually identical to
that of wild type, with 17 water molecules that were structurally
equivalent (<0.60 Å) to the waters described above. These water
molecules, however, were significantly less well ordered than in wild
type, with an average B-factor of 29.5 Å2. The deepest
cluster of 4 water molecules maintained the lowest B-factors, whereas
those closer to the bulk solvent were increasingly disordered. The
stability of water molecules correlated with the extent to which they
were hydrogen-bonded to protein groups as opposed to other water
molecules. Water molecules whose sole hydrogen-bonding partners were
other water molecules exhibited the highest B-factors. For instance,
the 5 water molecules that hydrogen-bond only with other water
molecules showed an average B-factor of 32.7 Å2 in F97L
compared with 19.1 Å2 in HbI. The changes observed for the
deoxy F97L structure with regard to water molecules therefore
suggest that the subtle changes in the F-helix conformation resulted in
a marked loosening of the interfacial water network.
In contrast to the deoxy form, striking differences in the interfacial water architecture were observed between the CO structures of F97L and wild-type HbI. Upon ligand binding to HbI, the displacement of each Phe97 side chain into the subunit interface results in the direct expulsion of 3 water molecules (Fig. 6, middle). The liganded wild-type interface thus has fewer and less well ordered water molecules than the deoxy interface (Tables III and IV). As shown in Fig. 6 (bottom), loss of the Phe97 side chain resulted in several discrete changes in the interfacial water distribution for the F97L-CO structure. First, 4 of the 6 water molecules that are normally lost due to displacement of the Phe97 side chain were now preserved, since the Leu97 side chain remained in the heme pocket. The other water molecules, including those participating in additional aspects of ligand-linked transitions, however, resembled wild-type HbI-CO water molecules. Second, the interfacial water molecules in the F97L-CO structure were significantly more disordered, with average B-factors of 34.5 Å2 compared with 24.7 Å2 for wild-type HbI-CO. The interfacial water architecture for F97L-CO thus appeared to resemble a hybrid version of the deoxy and liganded HbI water distribution. Another difference is that the distribution of water molecules in the mutant CO interface was asymmetric, in contrast to the wild-type structures. This asymmetry was seen to persist in simulated annealing omit maps.
HbI represents a simple allosteric system that is useful for the
investigation of intersubunit communication. The experiments described
here were undertaken to test the hypothesis that Phe97 is
critical for cooperativity. Based on a comparison of high resolution
crystal structures of liganded and unliganded HbI, Phe97
was suggested to play a key role in both regulation of oxygen affinity
and transfer of information between subunits (2). By virtue of its
tight packing in the heme pocket of deoxy HbI, Phe97 was
thought to lower oxygen affinity by (i) acting as a wedge, restricting
movement of the heme iron into the heme plane and (ii) lengthening and
weakening a hydrogen bond between the N of proximal histidine and
the main chain carbonyl oxygen of Phe97. Furthermore, it
was suggested that its movement into the interface upon ligand binding
would enhance the oxygen affinity of the second subunit through two
pathways: (i) by a direct effect of its side chain in the interface and
(ii) by triggering a movement of the heme group deeper into its subunit
that would alter the hydrogen bonding of the propionates at the
interface. As a result, one would predict that mutation of
Phe97 to Leu would result in a substantial increase in
oxygen affinity and a loss of cooperativity.
F97L exhibited an 8-fold increase in oxygen affinity over that of wild
type. From the kinetic results, it appeared that the increased oxygen
affinity results primarily from an increased affinity of the deoxy
state relative to the deoxy state of wild-type HbI. This was reflected
in the large decrease in oxygen dissociation rates for F97L,
particularly at low oxygen saturation, and a modest increase in ligand
combination rates, especially for CO (Fig. 1, Table I). An altered
deoxy state was also suggested by spectral results and was clearly
shown in the crystallographic analysis. The crystal structures
demonstrated that the smaller leucine side chain is accommodated
without strain in the heme pocket of both deoxy and F97L-CO. This
permitted the heme iron unrestricted movement into the heme plane (Fig.
5, Tables V and VI). Additionally, the hydrogen bond between the main
chain carbonyl oxygen of residue 97 and N of the proximal histidine,
which has been proposed to increase oxygen affinity, was nearly optimal
in both the deoxy and CO structures. Thus, our analysis of the F97L
mutant strongly supports the proposal that Phe97 plays the
central role in determining oxygen affinity.
Cooperativity in F97L was largely decreased due to a loss of the ligand-linked movement of the side chain of residue 97 into the subunit interface. Kinetic measurements showed no distinct change in the oxygen dissociation rate for F97L as a function of varying oxygen saturation, in sharp contrast to wild-type HbI. These observations are consistent with diminished cooperativity resulting from the loss of displacement of Phe97 into the interface. Perhaps surprisingly, cooperativity was not fully lost in this mutant. This appears to be associated with our structural finding that the ligand-linked movement of the heme groups continued to occur in this mutant, refuting the earlier hypothesis that this movement was linked to the disposition of the Phe97 side chain. Thus, other interactions with the heme are responsible for this movement, which results in communication via an alternative pathway that presumably supplements the primary Phe97-based pathway in wild-type HbI.
Royer et al. (18) have recently compared the ligand binding parameters of wild-type HbI and the mutant T72V, which exhibits very high oxygen affinity (p50 = 0.2 torr) in combination with high cooperativity (n = 1.7). The functional behavior of these proteins was analyzed by defining the properties of ideal T and R states of HbI within the context of a two-state model (21). These model properties can never be fully expressed in the experimentally accessible ligand binding behavior of the protein but can be simulated to interpret the experimental data for the liganded and unliganded proteins. That analysis suggested that HbI and T72V have very similar T and R states but that large functional distinctions result from a very different balance between T and R states, as reflected in the allosteric constant L, the ratio between T and R states of the unliganded protein. A similar treatment of data for F97L that assumes no fundamental change in the affinities of the T and R states and that alters the value of L alone does not work. The results from oxygen-pulse and CO-binding experiments agree in indicating more R than T state for singly liganded F97L than predicted by such a simulation as well as greatly diminished cooperativity. The experimental results for oxygen binding data with F97L, however, may be well approximated by large changes in the value of both L and c (c is defined as the ratio between KT and KR for unliganded protein, where KT = Ton/Toff and KR = Ron/Roff). Fig. 1 shows the agreement between experimental results and calculated values based on reducing L by 25-fold and decreasing Toff by 12-fold relative to wild type. This interpretation is in agreement with our evidence from the deoxy crystal structure, spectroscopy, and kinetic data that the T state of F97L is significantly different from that of wild type.
Previous work has shown that the well ordered water molecules in the subunit interface are crucial for stabilization of the deoxy state of HbI and suggested that these water molecules act as allosteric mediators in the cooperative mechanism (18). In this regard, analysis of the water network in the interface of F97L HbI reveals that quite subtle changes in the deoxy conformation result in a destabilization of the interface water cluster. This suggests that the deoxy protein conformation of wild-type HbI is finely tuned to maintain its very well ordered interface water cluster.
The atomic coordinates and structure factors (2HBI and 3HBI) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
We thank Dr. Emilia Chiancone for helpful discussions and Dr. Wayne Hendrickson for computer programs.