From the Department of Biochemistry and Cell Biology and the W. M. Keck Center for Computational Biology, Rice University, Houston, Texas 77005
Received for publication, September 20, 2000, and in revised form, November 13, 2000
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ABSTRACT |
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The ability of myoglobin to bind oxygen
reversibly depends critically on retention of the heme prosthetic
group. Globin side chains at the Leu89(F4),
His97(FG3), Ile99(FG5), and
Leu104(G5) positions on the proximal side of the heme
pocket strongly influence heme affinity. The roles of these amino acids
in preventing heme loss have been examined by determining high
resolution structures of 14 different mutants at these positions using
x-ray crystallography. Leu89 and His97 are
important surface amino acids that interact either sterically or
electrostatically with the edges of the porphyrin ring.
Ile99 and Leu104 are located in the interior
region of the proximal pocket beneath ring C of the heme prosthetic
group. The apolar amino acids Leu89, Ile99, and
Leu104 "waterproof" the heme pocket by forming a
barrier to solvent penetration, minimizing the size of the proximal
cavity, and maintaining a hydrophobic environment. Substitutions with
smaller or polar side chains at these positions result in exposure of
the heme to solvent, the appearance of crystallographically defined
water molecules in or near the proximal pocket, and large increases in
the rate of hemin loss. Thus, the naturally occurring amino acid side
chains at these positions serve to prevent hydration of the
His93-Fe(III) bond and are highly conserved in all known
myoglobins and hemoglobins.
Three important functions of the globin portion of myoglobin are
to sequester heme, enhance coordination with the proximal histidine,
and inhibit oxidation of the iron atom. In myoglobin, the heme pocket
is surrounded by four of the eight globin helices. The B, C, and E
helices form the top and sides of the porphyrin binding site, and the F
helix forms the bottom (as oriented in Fig.
1). Heme binding causes the globin to
form a more compact structure with an approximately 20% increase in
helicity (1). Three edges of the porphyrin are buried in the protein
interior and stabilized by hydrophobic interactions with apolar side
chains that line the heme pocket (2-4). The fourth edge contains the solvent-exposed heme propionates, which interact electrostatically with
polar amino acid side chains located on the surface of the protein.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Space-filling representation of the proximal
heme pocket of sperm whale myoglobin showing the position of the
Leu89(F4) side chain (dark, space-filling)
relative to the heme (shown in stick representation).
The affinity of free heme for binding a single imidazole is very weak,
whereas the affinity for binding a second base is very high. Thus,
hexacoordinate bis-imidazole complexes are much more stable than the
pentacoordinate intermediate (5-7). Although a much weaker ligand,
water competes effectively against the binding of the first imidazole
base in aqueous solution, causing the apparent equilibrium dissociation
constant to be 10
2-10
4 M (7).
Hemoglobins and myoglobins have evolved to stabilize the mono-imidazole
heme complex to facilitate reversible O2 binding to the
sixth coordination position. This stabilization appears to be
accomplished by removing water molecules from the vicinity of the
proximal coordination site.
The side chains of the proximal amino acids, Leu89(F4),
His97(FG3), Ile99(FG5), and
Leu104(G5), are within 4 Å of the porphyrin ring.
Leu89 is located at the entrance of a hydrophobic cavity
underneath pyrrole B. Xenon gas binds readily in this proximal pocket,
which has been designated the Xe1 site (8). Photodissociated
O2 and CO access both the distal Xe4 site and this Xe1
proximal site on nanosecond to microsecond time scales, suggesting that
these spaces may be functionally significant (9-13,
34). His97 forms a hydrogen
bond with the heme-7-propionate and is a hydrophobic barrier to
solvation of the proximal heme pocket on the other side of the heme
group. Ile99 and Leu104 are located just
beneath the pyrrole rings B and C, respectively. Leu104
forms one of the internal sides of the Xe1 cavity. Hargrove et al. (3) suggested that both these amino acids may be important for
keeping the internal region of the proximal pocket anhydrous and
preventing disruption of the His93(F8)-Fe bond. To test
these ideas, we have examined the effects of mutations at these four
key positions on the crystal structure of recombinant sperm whale myoglobin.
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MATERIALS AND METHODS |
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Preparation of Proteins-- Recombinant wild-type and mutant sperm whale myoglobins were constructed, expressed, and purified as described by Springer and Sligar (15) and Carver et al. (16). Recombinant wild-type myoglobin differs from native myoglobin by the initiator methionine required for bacterial expression and an Asp122 to Asn mutation that was a result of an error in the original sequence determination. These changes do not affect the function of the recombinant protein (17) but do cause differences in crystallization conditions (18). The protein samples were concentrated to about 1 mM in heme, frozen, and stored under liquid nitrogen until ready for use.
Measurement of Hemin Dissociation Rate Constants-- Hemin dissociation rates were measured by monitoring the absorbance changes associated with the transfer of hemin from holoprotein to excess H64Y/V68F apomyoglobin at pH 5.0 and 7.0, as described by Hargrove et al. (19). Briefly, the transfer of hemin from 6 µM metmyoglobin to 30 µM H64Y/V68F apomyoglobin was measured by following the absorbance decrease at 409 nm as "green" H64Y/V68F holo-metmyoglobin was formed. The experiments were carried out with 0.15 M buffer and 0.45 M sucrose at 37 °C in either sodium acetate at pH 5.0 or potassium phosphate at pH 7. The values for the rate of hemin loss for most of the mutants were originally reported in Hargrove et al. (3); the remainder were measured for this report (20).
Preparation of Protein Crystals-- Prior to crystallization, recombinant proteins were purified further by HPLC1 (LDC/Milton Roy). Samples were dialyzed against 20 mM sodium phosphate, 1 mM EDTA, pH 6.5, at 4 °C. The dialyzed samples were loaded onto a preparative weak cation exchange column (Custom LC) equilibrated with the dialysis buffer. The samples were eluted using a linear gradient of 0.0-1.0 M sodium chloride in 20 mM sodium phosphate, 1 mM EDTA, pH 6.5. Fractions containing the recombinant protein were combined and dialyzed against 20 mM Tris-HCl, 1 mM EDTA, pH 9.0, at 4 °C. A few of the myoglobins, particularly the Leu89 mutants, were unstable because of increased rates of heme loss and subsequent precipitation, which made protein purification and crystallization difficult. To overcome this problem, the unstable samples were reduced with sodium dithionite prior to purification to strengthen the covalent bond anchoring the heme group to the globin. Excess reducing agent was removed by passing the sample through a Sepharose column equilibrated with "no salt" HPLC buffer. Crystals of the HPLC-purified recombinant proteins were produced using the hanging drop vapor diffusion method employing a pH and ammonium sulfate gradient, as described by Phillips et al. (18). Briefly, 10 µl of 20 mg/ml HPLC-purified protein was mixed with an equal volume of 2.4-3.0 M ammonium sulfate, 20 mM Tris-HCl, 1 mM EDTA, pH 8.0-9.0, with well solutions having concentrations of 2.4-3.0 M ammonium sulfate. During sample preparation those mutants which were reduced for HPLC purification reoxidized to the met form. Crystals grew in several weeks to sizes from 0.1 to 0.5 mm. The Ile99 to Ser mutation failed to crystallize with these conditions. The other mutations for which heme loss rates were given and no structure is presented were not screened for crystallization (Leu89 to Ala and Ser and His97 to Ala).
X-ray Diffraction Data Collection and Refinement--
X-ray
diffraction data were collected on a Rigaku R-AXIS IIC imaging plate
area detector with a Siemens rotating-anode source. Typically, 60-90
frames were collected at a detector distance of 70 mm with a
30-min/frame exposure time and a oscillation range of 1°/frame.
The data were processed using the XDS and XSCALE software packages
(21).
In all cases, 10% of the reflections were set aside for a test set and
the calculation of Rfree. The X-PLOR package
(22) was used for the initial refinement of the model structures and for the generation of difference maps, using the Engh and Huber (23)
parameter set for stereochemical restraints. The coordinates of
wild-type metmyoglobin (18) were used as a starting model in the
refinement of the mutant structures. An iterative procedure of manually
fitting the model using the molecular graphics program CHAIN (24)
followed by Powell minimization, solvent B factor, and occupancy
refinement, and individual B factor refinement for all atoms was
repeated until Rfree was unchanged, and no
significant differences were observed in electron density maps. The
mutated side chains were changed only after
|2Fo Fc| and
|Fo
Fc| difference Fourier maps indicated clear positions for these altered side chains.
Peaks in the difference Fourier maps were modeled as water molecules
using the program PEAK2 or
OH2.3
After the refinement converged in X-PLOR, the model was refined further using the conjugate gradient and full matrix least squares method in SHELXL (25). Estimated standard deviations of the atomic positions of the heme iron and the 24 atoms of the pyrrole rings and methionine bridges were calculated from refinement without the imposition of standard distances and planarity restraints, as described in Ref. 25.
Sequence Alignment--
All myoglobin sequences were downloaded
from the National Center of Biotechnology Information. About 460 sequences were obtained through the Entrez Protein search engine, which
included all myoglobin related data from SwissProt, Protein Information
Resource, Protein Data Bank, and Protein Research Foundation entries,
etc. Incomplete myoglobin sequence fragments were immediately
discarded. Most of the myoglobin sequences from the Protein Data Bank
are from known myoglobins, and their mutants were also omitted in the
sequence alignment. The first sequence alignment containing 203 sequences was performed using Clustal W (version 1.8) (26). Duplicate sequences with different entry names were recognized and sorted in a
log file, and extra copies of duplicate entries were discarded. The
final multiple alignment contains 99 myoglobin sequences, and the
nomenclature for helical position was taken from Dickerson and Geis
(e.g. F4 is the fourth amino acid along the F-helix in sperm
whale myoglobin) (27). Similar alignments were used for the and
subunit sequences of vertebrate hemoglobins.
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RESULTS |
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O2 Binding and Rates of Hemin Loss-- Although the focus of this work is a structural interpretation of the effects of proximal pocket mutations on hemin loss (Table I), it is important to note that some of these amino acid replacements do have significant effects on ligand binding. Table II presents a list of the O2 binding properties of all 14 mutants whose structures have been determined by x-ray crystallography. There is a progressive 4-fold decrease in O2 affinity as the size of the amino acid at position 89 increases from Gly to Trp, whereas the mutations at His97 cause less than 2-fold changes in the O2 binding parameters. The Ile99 to Ala and Val replacements cause 2-fold increases in O2 affinity and similar, and sometimes large increases are seen for the Leu104 substitutions (Table II). In all four cases, there is no obvious correlation with hemin loss rate constants. A detailed interpretation of the O2 binding parameters for a more complete set of proximal mutants will be presented elsewhere and has been discussed by Liong (20), Dou (28), and Scott et al. (34). None of these ligand binding effects are large in comparison with the dramatic decreases (>10-fold) observed for the resistance of these mutants to hemin dissociation.
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Native and wild-type recombinant sperm whale myoglobin lose hemin at
rates of 1.0 and 0.01-0.05 h1 at pH 5 and 7, respectively. Table I lists rates of hemin loss from 20 different
proximal pocket mutants of myoglobin. The pH dependence of hemin loss
from myoglobin is due to protonation of both His93, which
facilitates disruption of the proximal imidazole-Fe bond, and
His64, which then rotates out into solvent causing loss of
distal water coordination (29). As a result, hemin loss at pH 5 is
rapid; data collection can be completed within 5 h; and the rate
constants are well defined. At pH
7, protonation of
His93 is greatly inhibited; coordinated water is stabilized
by hydrogen bonding to the neutral form of the side chain of
His64; hemin dissociation is much slower; and the time
courses must be recorded for over 24 h. During these long
incubation times, apoprotein denaturation often occurs, making data
analysis difficult. As a result, the small rate constants observed at
pH 7 (k
H
~0.1 h
1) have much
greater uncertainty (almost a factor of 2) than those for the more
unstable mutants and those obtained from measurements at lower pH values.
Most of the proximal pocket mutations cause 10-fold increases in the
rate of hemin loss (3). Removal of the proximal histidine base, as in
the H93G mutant (30), results in an extremely high rate of hemin
dissociation, k
H = 660 and 140 h
1 at pH 5 and 7, respectively (Table I). This result is
clearly due to the lack of a covalent link anchoring the heme group to the globin. Many of the Leu89 mutants also show dramatic
increases in the rate of hemin loss, with the order being: L89G > L89S
L89A > L89W > L89F
native/wild type.
The rates of hemin loss from the His97 mutants show both a
size and a charge dependence with the order of
k
H being: H97D
H97E
H97A > H97Q > H97V
H97F
native/wild type.
Interestingly, replacing either Leu89 or His97
with a phenylalanine side chain is relatively conservative with respect
of the rate of hemin loss, particularly at pH 5 (Table I).
Replacement of Ile99 with serine results in a large 40-fold
increase in the rate of hemin loss, whereas mutation to alanine and
valine results in only moderate increases. Leu104 mutations
also exhibit increases in the rate of hemin loss. The correlation with
size is weak; however, the L104A mutation does cause almost a 20-fold
increase in kH at both pH values (Table I).
The lack of effect of the polar L104N replacement on hemin loss at pH 5 is even more difficult to interpret without a structure of the mutant.
Placement of an amide side chain in the proximal pocket would be
expected cause an influx of water molecules, which should enhance
disruption of the His93-Fe bond.
X-ray Structures-- Crystals of all but one of the 14 metmyoglobin mutants grew in the hexagonal P6 space group with one molecule/asymmetric unit. The His97(FG3) to Asp mutant crystallized in the orthorhombic P212121 space group, also with one molecule per asymmetric unit. Statistics for data collection and refinement are listed in Table III. The limit of diffraction (dmin) was 2.2 Å or better for all of the crystals, and the quality of the diffraction data, as assessed by the internal agreement of symmetry-related reflections (Rmerge), was acceptable. Refinement of the models converged with reasonable crystallographic R factors (Rcryst and Rfree). There is good agreement between the calculated and observed structure factors, with an average value of 15.8% for Rcryst. The difference between Rcryst and Rfree was 6.1% or less. The deviations from ideal stereochemistry in the refined structures were also within normal limits, as summarized in Table IV.
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The displacement of the iron from the heme plane varied slightly among
the 14 mutants examined (Table V).
However, there appears to be no correlation between Fe3+
displacement and the rate of hemin dissociation from the met forms of
these mutants. Table V also lists the bond length between the proximal
imidazole ring and the heme iron
(His93N2-Fe), showing that this
parameter is virtually the same for all 14 mutants. Furthermore, the
deviations in heme planarity observed in most of these proximal pocket
mutants are of the same magnitude as those observed in wild-type and
distal pocket mutants of sperm whale myoglobin (31). Thus, changes in
heme planarity, imidazole-Fe3+ bond distance and
Fe3+ displacement do not appear to be the underlying causes
of the large increases in the rate of hemin loss.
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The mutations at positions 89, 97, 99, and 104 produce only minor
changes in the overall structure of the globin moiety. The major
alterations are localized in the region of the mutated side chain and
include significant changes in local water structure (Figs.
2-3). These local structural
modifications were examined closely and used to interpret the changes
in kH.
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Leu89 Mutants--
A space-filling model of wild-type
myoglobin shows that Leu89 is packed against the heme
forming a significant part of the outer covering of the proximal
portion of the heme pocket. The His93(F8) is hidden in a
hydrophobic cavity that is protected from the solvent molecules located
on the surface of the protein. Replacing leucine at position 89 with
glycine creates a clear pathway for the entry of solvent into the heme
pocket (Fig. 3B). Electron density indicative of at least three internal water molecules is seen
in the |Fo Fc|
omit map for L89G metmyoglobin (Fig. 2). The three internal water
molecules are observed inside the Xe1 pocket of this mutant and are
connected to four other water molecules located on the protein exterior
(Fig. 3B).
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The hydration and increase of polarity in the heme pocket of
Gly89 metmyoglobin greatly facilitates disruption of the
His93-Fe bond. As a result, the rate of hemin loss from
L89G myoglobin is greater than or equal to that of the H93G mutant
where there is no covalent attachment between the heme and the globin.
A similar dramatic increase in the rate of hemin loss is observed for
the L89S mutation, where the small hydroxymethyl side chain probably also allows an influx of water and increases the polarity of the heme
pocket. The L89A mutation also causes a substantial increase in
kH, but the effect is significantly smaller
than that seen for the L89G and L89S mutants.
Mutation of Leu89 to an amino acid with similar size and polarity, such as L89F, has a much smaller effect on the rate of hemin loss, particularly at pH 5. As shown in Fig. 3C, the aromatic side chain of Phe89 metmyoglobin forms a hydrophobic barrier to solvation which is equivalent to that of the native leucine side chain. Trp89 also provides a steric barrier to solvation (Fig. 3D). However, the L89W mutation causes a 17-fold increase in the rate of hemin loss at pH 5, which is most likely due both to steric hindrance between the large indole side chain and the rigid porphyrin ring and to the increased polarity of the indole side chain.
His97 Mutants--
The location of
His97(FG3) is shown in Fig.
4A where the protein molecule
has been rotated 90° to the right relative to its position in Fig. 1.
Replacing His97 with small (H97A and H97V) or negatively
charged (H97D and H97E) side chains results in large increases in
kH. The crystal structures of H97V and H97D
show that the heme-7-propionate is no longer interacting with this side
chain and that a channel has been created that connects the solvent
exterior to the proximal histidine (Fig. 4, B and
E, respectively). In both mutants, hydration of the heme
pocket is significantly smaller than that seen for the L89G mutation,
which almost completely exposes the proximal histidine to water. The
smaller channel and extent of hydration explain why the effects of H97V
and H97D mutations on hemin loss are less than those observed for the
L89G, A, and S replacements.
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Replacing His97 with glutamine was expected to have little
effect on the metmyoglobin structure because the amide side chain can
still form a hydrogen bond with the heme-7-propionate. However, as
shown in Fig. 4D, the glutamine side chain is not large
enough to completely seal the opening to the hydrophobic cavity,
resulting in significant increases in kH at
both neutral and acid pH (Table I). Phenylalanine can replace
His97 with very little effect on the rate of hemin loss at
either pH. (Table I). In the crystal structure of this mutant, the
phenyl ring is located in the same position as the naturally occurring imidazole ring (Fig. 4C). Phe97 also appears to
provide some electrostatic stabilization through interaction between
the positive edge of the phenyl multipole and the heme-7-propionate.
Thus, the native His97 side chain provides both a steric
barrier to solvation and electrostatic stabilization of one of the
heme propionates.
Ile99 Mutants--
The side chain at the FG5 position
is important in maintaining the exact position of the porphyrin ring in
the heme pocket. Replacing the native isoleucine with a smaller side
chain results in tilting of the B ring toward the proximal side of the
heme pocket because of the loss of the C atom of the
sec-butyl side chain (Fig. 5).
Hargrove et al. (3) suggested that an apolar side chain at
position 99 is important for keeping the heme pocket anhydrous and
observed that the I99S mutation results in a 90-fold increase in hemin
loss at pH 5. Mutation to alanine results in a more moderate 20-fold
increase. Other than changes in the tilt of porphyrin ring, the crystal
structures of I99A and I99V do not show significant alteration of the
protein or hydration of the heme pocket with well defined internal
water molecules. Thus, the causes of the high rates of hemin
dissociation from the I99A, I99V, and I99S mutants are more ambiguous
and could be due to the increase in internal space and/or changes in
the tilt of the heme which weaken the Fe-His93 bond.
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Leu104 Mutants--
Replacing Leu104(G5)
with alanine and valine causes moderate to small increases in the rate
of hemin loss (from 20- to 5-fold, respectively). Ordered water
molecules are found in the proximal portion of the heme pocket in the
L104A and L104N mutants (Fig. 6A) and almost certainly
account for the increases in kH for these
proteins. The presence of these interior water molecules was
unexpected. There is no obvious connection to the solvent phase because
Leu89 still blocks direct access to solvent, making
visualization of the internal waters difficult (Fig.
6A).
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There are two possible pathways for entry of the water molecules seen in the L104A and L104N mutants. Solvent could enter the heme pocket through the distal histidine gate and then migrate to the proximal Xe1 site as is seen for the apolar ligands, O2 and CO (9-13, 34). Alternatively, water molecules could diffuse into the protein via multiple transient channels created by thermal fluctuations in the protein matrix (32). There are two well defined surface water molecules within hydrogen bonding distance of Gln105(G6), and when Leu104 is replaced with Asn, a chain of three water molecules is found in this area and extends from the exterior surface of the amide side chain out into the solvent phase along the G helix.
In general, water molecules are not seen in the proximal and distal cavities of myoglobin because these spaces are lined with hydrophobic amino acids. However, in the L104A mutation, the proximal cavity is probably large enough to sequester two internal water molecules that appear to hydrate the interior edge of the porphyrin ring and the Xe1 pocket. The net result is a ~20-fold increase in the rate of hemin dissociation.
The amide side chain in the L104N mutation is highly polar. Two
internal water molecules are attached to this side chain in the crystal
structure of the metmyoglobin mutant. Remarkably, this mutation causes
little increase in the rate of hemin loss at either pH 5 or 7. The
water molecules in the L104N mutant appear to be attached strongly to
each other and to the amide side chain by strong hydrogen bonds with
O-O and N-O distances of 2.6 to 3.1 Å (Fig. 6B). Thus,
the hydroscopic nature of the Asn side chain appears to prevent these
internal water molecules from solvating the heme group and disrupting
the proximal histidine-iron bond. In contrast, the more mobile water
molecules present in the L104A mutant do facilitate disruption of the
Fe-His(F8) bond (Table I).
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DISCUSSION |
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The four proximal pocket amino acids examined appear to
"waterproof" the heme pocket. They maintain a favorable
apolar environment for the heme group and prevent the hydration of the
proximal imidazole base. Leu89(F4) seals the hydrophobic
cavity underneath pyrrole B, preventing solvation of the Xe1 site in
the proximal portion of the heme pocket. Decreasing the size or
increasing the polarity of the side chain at position 89 results in
dramatic increases in the rate of hemin loss. There appears to be
strong selective pressure to seal off the heme pocket as judged by the
high degree of conservation at the F4 amino acid. The frequency of
leucine at this position is 87% in the 99 known myoglobin sequences,
and the remaining major variation is conservative (7% Phe). Leu(F4) is
even more conserved in vertebrate hemoglobins showing a frequency of
99% in both the (279 sequences) and
(224 sequences) subunits.
His97(FG3) is part of the extensive hydrogen-bonding network found at the exterior portion of the proximal pocket of myoglobin. Disruption of the hydrogen-bonding network by mutation of His97 to smaller, apolar, or negatively charged side chains has little effect on the ligand binding properties of the protein (10, 20, 34). However, loss of favorable electrostatic interactions with heme-7-propionate or opening of a channel to the proximal histidine does accelerate the rate of hemin loss up to 40-fold.
Both Ile99 and Leu104 are located in the interior region of the heme pocket beneath the heme prosthetic group and are important in maintaining the position of the porphyrin ring, which in turn influences the reactivity of the heme iron toward ligands (10, 20, 34). Mutations at both positions have significant effects on the rate of hemin dissociation. Substitution with an alanine at position 104 enlarges the proximal pocket enough to allow the entry of water molecules that promote hemin loss. In the L104N mutant, solvent also enters the proximal cavity, but the water molecules are sequestered by the Asn side chain. In effect, the amide group acts as a hydroscopic agent to keep the Fe-His93 "dry," preventing a large increase in the rate of hemin dissociation.
All of these results show that the proximal environment of the heme group must be kept anhydrous and that direct exposure to solvent must be minimized. Tilton et al. (8) have shown that there are internal cavities at both the proximal and distal sides of the heme group and visualized them by adding Xe to crystals and determining the binding sites. The most stable Xe site(Xe1) is circumscribed by Leu89, His93, Leu104, and Phe138. In the native protein, this cavity is too small and hydrophobic to accommodate water molecules. A small fraction of the apolar ligands O2 and CO do reach this location on ns to µs time scales after laser photolysis or under high pressure (9-13, 34). However, if the Xe1 cavity is opened to solvent (L89G), enlarged (L104A), or made more polar (L104N), water does enter and destabilize the proximal His93-Fe bond.
The idea that solvation of the heme pocket facilities hemin loss was
first proposed to account for the instability of Hemoglobin Boras ( Leu(F4)
Arg), which exhibits abnormally high rates of hemin loss,
formation of semihemoglobin, and precipitation (14, 27, 33). The
structures presented here show directly and convincingly that
Leu89 does protect the proximal pocket from hydration and
subsequent loss of the heme prosthetic group. Clearly, there is strong
selective pressure to prevent hydration of the heme pocket, as can be
seen by the high degree of conservation of large apolar side chains at
the Leu89(F4), Ile99(FG5), and
Leu104(G5) positions in myoglobin. At the FG5 position, the
frequencies are 83% Ile, 12% Val, 3% Leu, and 2% others in the 99 known myoglobin sequence; those at the G5 position are 78% Leu, 17%
Phe, and 5% other. In contrast, there is much more variability at the
His97(FG3) position. In most cases, large side chains are
present at FG3 to seal off the proximal pocket, and when polar side
chains are present, they interact with the heme propionates.
The results described in this report confirm experimentally the
necessity of a hydrophobic pocket to bind and retain the heme prosthetic group in myoglobin and show directly by crystallography the
presence of water molecules around the heme when this hydrophobic pocket is disrupted. Furthermore, the character and contributions of
particular proximal side chains on heme binding and retention have been dissected.
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ACKNOWLEDGEMENTS |
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We thank Eileen Singleton for construction, expression, and purification of the mutant proteins, Bog Stec for help with the figures, and Mark Hargrove for encouraging determination of the structures of these mutants by x-ray crystallography.
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FOOTNOTES |
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* This work was supported by Robert A. Welch Foundation Grants C-1142 (to G. N. P.) and C-612 (to J. S. O.); National Institutes of Health Grants AR40252 (to G. N. P.), HL47020 (to J. S. O.), and GM35649 (to J. S. O.), Texas Advanced Technology Program Grant 003604-025 (to G. N. P.), a traineeship from the Houston Area Molecular Biophysics Predoctoral Training Grant GM08280 (to E. E. S.), and funds from the W. M. Keck Center for Computational Biology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Biophysics Group (P-21), MS D454, Los Alamos
National Laboratory, Los Alamos, NM 87545.
§ Present address: Dept. of Pharmacology and Toxicology, UT Medical Branch at Galveston, 301 University Blvd., Galveston, TX 77555.
¶ To whom correspondence should be addressed. Present address: Dept. of Biochemistry, University of Wisconsin-Madison, 433 Babcock Dr., Madison, WI 53706. E-mail: phillips@biochem.wisc.edu.
Published, JBC Papers in Press, November 17, 2000, DOI 10.1074/jbc.M008593200
1 E. E. Scott, Q. H. Gibson, and J. S. Olson, submitted for publication.
2 M. L. Quillin, unpublished data.
3 M. B. Berry, unpublished data.
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ABBREVIATIONS |
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The abbreviation used is: HPLC, high pressure liquid chromatography.
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REFERENCES |
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1. | Griko, Y. V., Privalov, P. L., Venyaminov, S. Y., and Kutyshenko, V. P. (1988) J. Mol. Biol. 202, 127-138[Medline] [Order article via Infotrieve] |
2. | Hargrove, M. S., and Olson, J. S. (1996) Biochemistry 35, 11310-11318[CrossRef][Medline] [Order article via Infotrieve] |
3. | Hargrove, M. S., Wilkinson, A. J., and Olson, J. S. (1996) Biochemistry 35, 11300-11309[CrossRef][Medline] [Order article via Infotrieve] |
4. | Hargrove, M. S., Barrick, D., and Olson, J. S. (1996) Biochemistry 35, 11293-11299[CrossRef][Medline] [Order article via Infotrieve] |
5. | Cole, S. J., Curthoys, G. C., Cole, and Magnusson, E. A. J. (1970) J. Am. Chem. Soc. 92, 2991-2996[Medline] [Order article via Infotrieve] |
6. | Cole, S. J., Curthoys, G. C., Cole, and Magnusson, E. A. J. (1971) J. Am. Chem. Soc. 93, 2153-2158 |
7. | Brault, D., and Rougee, M. (1974) Biochemistry 13, 4591-4597[Medline] [Order article via Infotrieve] |
8. | Tilton, R. F., Jr., Kuntz, I. D., Jr., and Petsko, G. A. (1984) Biochemistry 23, 2849-2857[Medline] [Order article via Infotrieve] |
9. | Scott, E. E., and Gibson, Q. H. (1997) Biochemistry 36, 11909-11917[CrossRef][Medline] [Order article via Infotrieve] |
10. | Scott, E. E. (1998) Apoglobin Stability and Ligand Movements in Mammalian Myoglobins.Ph.D. Thesis , Rice University, Houston, TX |
11. | Chu, K., Vojtchovsky, J., McMahon, B. H., Sweet, R. M., Berendzen, J., and Schlichting, I. (2000) Nature 403, 921-923[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Brunori, M.,
Vallone, B.,
Cutruzzola, F.,
Travaglini-Allocatelli, C.,
Berendzen, J.,
Chu, K.,
Sweet, R. M.,
and Schlichting, I.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
2058-2063 |
13. | Ostermann, A., Waschipky, R., Parak, F. G., and Nienhaus, G. U. (2000) Nature 404, 205-208[CrossRef][Medline] [Order article via Infotrieve] |
14. | Bunn, H. F., and Forget, B. G. (1986) Hemoglobin: Molecular, Genetic, and Clinical Aspects , W. B. Saunders, Philadelphia, PA |
15. | Springer, B. A., and Sligar, S. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 8961-8965[Abstract] |
16. |
Carver, T. E.,
Brantley, R. E., Jr.,
Singleton, E. W.,
Arduini, R. M.,
Quillin, M. L.,
Phillips, G. N., Jr.,
and Olson, J. S.
(1992)
J. Biol. Chem.
267,
14443-14450 |
17. | Springer, B. A., Sligar, S. G., Olson, J. S., and Phillips, G. N., Jr. (1994) Chem. Rev. 94, 699-714 |
18. | Phillips, G. N., Jr., Arduini, R. M., Springer, B. A., and Sligar, S. G. (1990) Proteins 7, 358-365[Medline] [Order article via Infotrieve] |
19. |
Hargrove, M. S.,
Singleton, E. W.,
Quillin, M. L.,
Ortiz, L. A.,
Phillips, G. N., Jr.,
Olson, J. S.,
and Mathews, A. J.
(1994)
J. Biol. Chem.
269,
4207-4214 |
20. | Liong, C. E. (1999) Structural and Functional Analysis of Proximal Pocket Mutants of Sperm Whale Myoglobin.Ph.D. Thesis , Rice University, Houston, TX |
21. | Kabsch, W. (1988) J. Appl. Crystallogr. 21, 916-924[CrossRef] |
22. | Brunger, A. T. (1992) X-PLOR, version 3.1 , Yale University Press, New Haven, CT |
23. | Engh, R. A., and Huber, R. (1991) Acta Crystallogr. A 47, 392-400[CrossRef] |
24. | Sack, J. S. (1988) J. Mol. Graphics 6, 244-245 |
25. | Sheldrick, G. M., and Schneider, T. R. (1997) Methods Enzymol. 276, 319-343[CrossRef] |
26. | Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-4680[Abstract] |
27. | Dickerson, R. E., and Geis, I. (1983) Hemoglobin: Structure, Function, Evolution, and Pathology. Benjamin/Cummings Series in the Life Sciences , Benjamin/Cummings Publishing Company, Menlo Park |
28. | Dou, Y. (1997) Protein Engineering Reveals Specific Roles of Amino Acid Residues in Function and Stability of Myoglobin.Ph.D. Thesis , Case Western Reserve University, Cleveland, OH |
29. | Yang, F., and Phillips, G. N., Jr. (1996) J. Mol. Biol. 256, 762-774[CrossRef][Medline] [Order article via Infotrieve] |
30. | Barrick, D. (1994) Biochemistry 33, 6546-6554[Medline] [Order article via Infotrieve] |
31. | Quillin, M. L., Li, T., Olson, J. S., Phillips, G. N., Jr., Dou, Y., Ikeda-Saito, M., Regan, R., Carlson, M., Gibson, Q. H., Li, H., and Elber, R. (1995) J. Mol. Biol. 245, 416-436[CrossRef][Medline] [Order article via Infotrieve] |
32. | Benson, E. S., Rossi Fanelli, M. R., Giacometti, G. M., Rosenberg, A., and Antonini, E. (1973) Biochemistry 12, 2699-2706[Medline] [Order article via Infotrieve] |
33. | Antonini, E., and Brunori, M. (1971) Hemoglobin and Myoglobin and Their Reaction with Ligands , North Holland Publishing Co., Amsterdam |
34. |
Scott, E. E.,
Gibson, Q. H.,
and Olson, J. S.
(2001)
J. Biol. Chem.
276,
5177-5188 |