From the
Pig and human myoglobin have been engineered to reverse the
positions of the distal histidine and valine (i.e. His
The nature of the axial heme ligand is an important factor
defining the biochemical function of hemoproteins (Dawson, 1988). In
myoglobin and hemoglobin, the fifth coordination site of the heme iron
is occupied by the imidazole group of the proximal histidine, and
exogenous ligands bind to the sixth position (Dickerson and Geis,
1983). There are two highly conserved residues, His(E7) and Val(E11),
located in the distal pocket adjacent to the ligand binding site. The
distal histidine does not directly interact with the heme iron but
forms a hydrogen bond with bound O
Using
site-directed mutagenesis, we have prepared myoglobin in which the
distal histidine and valine are exchanged, i.e. His
In
order to evaluate this model for the coordination structure of
H64V/V68H myoglobin and to determine the effects of these mutations on
the reactivity of the heme, we have measured optical absorption, IR,
EPR, and x-ray absorption spectra; CO binding kinetics; and the
oxidation/reduction midpoint potential of human H64V/V68H myoglobin and
have determined the structure of pig H64V/V68H myoglobin by x-ray
crystallography. Both spectroscopic and crystallographic results
confirm axial coordination of His
Mutagenesis of the wild-type human and pig myoglobin coding
sequences was performed as described in the previous papers
(Varadarajan et al., 1985; Ikeda-Saito et al., 1991;
Smerdon et al., 1991), and the recombinant myoglobins were
expressed in Escherichia coli and purified to homogeneity
following procedures described in these publications.
Light
absorption spectra were recorded using a Hitachi U-3210
spectrophotometer at 20 °C. Extinction coefficients were obtained
by the alkaline pyridine hemochromogen method (Antonini and Brunori,
1971). Oxidation-reduction potentials were determined as described
previously (Ikeda-Saito and Prince, 1985) using a calomel electrode as
the reference electrode. The measurements were carried out in the
presence of 10 µM 2-hydroxy-1,4-naphthaquinone,
2-hydroxy-1, 4-anthraquinone, N-methylphenazine methosulfate,
and N-ethylphenazine ethosulfate plus 20 µM 2,3,4,5-tetramethylphenylenediamine. Potentials are expressed on
the basis of the assumption that the calomel electrode had an E
X-ray absorption
spectra were measured at the Stanford Synchrotron Radiation Laboratory
on beam line 7-3 using a Si (220) double crystal
monochrometer with a 1-mm vertical aperture and no focussing optics.
Twenty 30-min scans were averaged, and the sample was maintained at a
temperature close to 10 K in an Oxford Instruments CF1204 flowing
liquid helium cryostat. Iron-K edge spectra were measured as
fluorescence excitation spectra using a Canberra 13-element detector
(Cramer et al., 1988). The extended x-ray absorption fine
structure (EXAFS)
EPR spectra were recorded on a Bruker ESP-300
instrument operating at 9.45 GHz as described previously (Ikeda-Saito et al., 1992). Experiments were carried out at an
incident microwave power of 1 mW with a field modulation of 0.5
millitesla at 100 kHz. An Oxford flow cryostat (ESR-900) was used for
liquid helium temperature measurements. Microwave frequency was
monitored by a Hewlett Packard 5350B frequency counter, and a Bruker
ER-035M NMR gauss meter was used to determine the magnetic flux
density.
IR spectra were measured by a Mattson Galaxy 6020
spectrometer as described previously (Li et al., 1994). CO
association rates were measured by a flash photolysis apparatus
consisting of two photographic strobe units (Sunpak 544) equipped with
thyristor quenching devices, and CO dissociation rates were determined
by the ligand replacement method using NO in a Gibson-Dionex stopped
flow apparatus (Rohlfs et al., 1990).
Crystals of the
H64V/V68H mutant were prepared as described previously for recombinant
wild-type pig myoglobin (Dodson et al. 1988) except that 5
mM Tween 60 was included in the hanging drops. A single
crystal mounted in a glass capillary in the usual manner was exposed to
CuK
The starting model for the refinement of the
H64V/V68H mutant was the wild-type pig metmyoglobin structure
(1MYG.PDB) from which water molecules had been removed and the side
chains of His
Reaction
of the ferrous H64V/V68H mutants with O
The distal pocket
structure was further examined by measurements of the IR spectrum of
the CO complex. Fig. 2compares the IR spectra of human wild-type
and H64V/V68H myoglobin. The major CO stretching band of the wild-type
protein at 1944 cm
Details of the iron ligation and heme and histidine stereochemistry
are given in I. The electron density for His
The unusual
conformation of the proximal histidine was insensitive to manual
modelling and refinement steps taken to impose a more standard
geometry. Similar imidazole stereochemistry is not seen in the B
molecule even though the main chain atoms of the F helices in the A and
B molecules of the asymmetric unit are closely superimposable. Neither
the unusual imidazole conformation nor the break in the electron
density are observed in wild-type pig myoglobin or in a dozen or so
mutant pig myoglobins that form crystals isomorphous with those of the
H64V/V68H double mutant. One possible explanation of the differences
observed in the Fe-His
An important goal of the present study is to correlate the
spectroscopic properties of the H64V/V68H myoglobin mutant with its
structure as determined by x-ray crystallography. Ideally both sets of
data would be collected on protein from one species. However, human
H64V/V68H myoglobin did not yield crystals suitable for x-ray
structural determination. The crystal structure of pig H64V/V68H
myoglobin was therefore used for interpretation of the spectroscopic
and functional properties of both pig and human H64V/V68H myoglobins.
Optical absorption spectra of the human double mutant are very similar
to those of the corresponding pig protein. The hemochrome type spectrum
is seen in both of the proteins, indicating a hexacoordinate low spin
state in the ferrous forms. The CO association and dissociation rates
of human H64V/V68H are similar to those of the pig mutant. In the
ferric state, the pig and human mutants show virtually identical spin
state compositions, indicating similar ligand field. The g value of low spin complexes is a function of the bound axial
ligand geometry (Walker et al., 1986; Inniss et al.,
1988), and the EPR results show the virtually identical axial ligand
geometry in human and pig H64V/V68H myoglobins. Thus, the heme pocket
structure of human H64V/V68H myoglobin appears to be spectroscopically
identical to that of pig H64V/V68H myoglobin, and comparisons with the
crystal structure of the latter protein should be valid.
The x-ray structure shows the constraints
placed upon the possible orientations of the position 68 imidazole by
the surrounding globin. In x-ray studies with V68F sperm whale
myoglobin, Quillin et al.(1995) have shown that the extra bulk
of the aromatic substituent can be accommodated in the back of the
distal pocket without significant alteration in the tertiary structure.
In H64V/V68H myoglobin, the favorable coordination and hydrogen bonding
of His
Another key feature
of the H64V/V68H structure is that the coordinated His
Sligar and his
co-workers (Sligar et al., 1987) have demonstrated that the
replacement of one of the axial histidine ligands by methionine in
cytochrome b
The Val
The net decrease in CO affinity observed for the double mutant is
due to the requirement for disruption of the Fe-His
H64Y
myoglobin is another well characterized mutant in which a distal
residue coordinates to the ferric heme iron. Spectroscopy and x-ray
crystallography have shown direct binding of the Tyr
Hargrove et al. (1994a) have
shown that the H64V/V68H double mutation markedly inhibits the rate of
hemin loss from myoglobin preventing measurement of this process at
room temperature, pH 7.0. This is another manifestation of direct
coordination between His
All of the data were determined in 0.1 M phosphate buffer, pH 7.0, at 20 °C. Equilibrium constants, K
The crystallographic coordinates and structure factors (code
1MNI) have been deposited in the Protein Data Bank, Brookhaven National
Laboratory, Upton, NY.
We thank Drs. M. Jaskólski and G. Murshudov for
expert assistance in x-ray data collection and structure refinement,
respectively.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(E7)
Val and Val
(E11)
His). Spectroscopic and ligand binding properties have been measured
for human and pig H64V/V68H myoglobin, and the structure of the pig
H64V/V68H double mutant has been determined to 2.07-Å resolution
by x-ray crystallography. The crystal structure shows that the N
of His
is located 2.3 Å
away from the heme iron, resulting in the formation of a hexacoordinate
species. The imidazole plane of His
is tilted relative to
the heme normal; moreover it is not parallel to that of
His
, in agreement with our previous proposal (Qin, J., La
Mar, G. N., Dou, Y., Admiraal, S. J., and Ikeda-Saito, M.(1994) J.
Biol. Chem. 269, 1083-1090). At cryogenic temperatures, the
heme iron is in a low spin state, which exhibits a highly anisotropic
EPR spectrum (g
= 3.34, g
= 2.0, and g
< 1), quite different
from that of the imidazole complex of metmyoglobin. The mean
iron-nitrogen distance is 2.01 Å for the low spin ferric state as
determined by x-ray spectroscopy. The ferrous form of H64V/V68H
myoglobin shows an optical spectrum that is similar to that of b-type cytochromes and consistent with the hexacoordinate
bisimidazole hemin structure determined by the x-ray crystallography.
The double mutation lowers the ferric/ferrous couple midpoint potential
from +54 mV of the wild-type protein to -128 mV. Ferrous
H64V/V68H myoglobin binds CO and NO to form stable complexes, but its
reaction with O
results in a rapid autooxidation to the
ferric species. All of these results demonstrate that the
three-dimensional positions of His
and Val
in
the wild-type myoglobin are as important as the chemical nature of the
side chains in facilitating reversible O
binding and
inhibiting autooxidation.
to stabilize the oxy
form of the globins (Phillips and Shoenborn, 1981; Kitagawa et
al., 1982; Olson et al., 1988; Springer et al.,
1989; Ikeda-Saito et al., 1991). In deoxymyoglobin, N
of His
(E7) forms a hydrogen
bond with a noncoordinated water that must be displacedbefore ligands
can bind to the iron atom, thus inhibiting the rate of ligand entry
into the heme pocket (Quillin et al.,1993; Springer et
al., 1994). The distal histidine also plays apivotal role in
regulating ligand reactivity in metmyoglobin (Brancaccio et
al., 1994). Mutation of Val
(E11) also causes marked
changes in the ligand binding properties in myoglobin (Egeberg et
al., 1990b; Smerdon et al., 1991; Brancaccio et
al., 1994; Quillin et al., 1995). Val
interacts with bound ligands, the His
side chain,
and the noncoordinated water molecule in deoxymyoglobin. Mutations at
this position affect ligand binding by a modulation of these
interactions (Springer et al., 1994). In addition, His
and Val
are now known to play key roles in
apomyoglobin folding (Hargrove et al., 1994a).
(E7)
Val and Val
(E11)
His. Our previous hyperfine-shifted proton resonance studies on this
double mutant have shown that the hemin iron is a thermal mixture of
low spin and high spin states (Qin et al., 1994). The NMR
results also indicated that the imidazole side chains of
His
(F8) and His
(E11) are coordinated to the
hemin iron in both the high spin and low spin forms. The axial bond to
the distal His
appeared to be weakened or strained
compared with that for the proximal histidine in both spin states. A
tilted His
coordination was proposed as the only
conformation in which His
can be sufficiently close to
coordinate to the heme iron in the normal myoglobin tertiary fold. The
distorted Fe-His
coordination was proposed to
produce a weaker ligand field than those in typical
bishistidine-ligated hemoproteins, such as b-type cytochromes,
and hence, to allow significant population of a high spin state.
as proposed in the
previous NMR studies (Qin et al., 1994). The ferrous H64V/V68H
double mutant forms stable CO and NO complexes, but its reaction with
O
results in a rapid formation of ferric species. Thus,
proper stereochemical positioning of His
and Val
is essential for creation of a distal pocket capable of
reversible O
binding. The altered coordination structure of
H64V/V68H myoglobin also serves as a useful model for examining the
role of heme pocket amino acids in determining the spectroscopic and
redox properties of hemoproteins in general.
of +247 mV.
(
)oscillations were
quantitatively analyzed as described previously (George et
al., 1989) using the full multiple scattering code feff (version 6.01) of Rehr and co-workers (Rehr et al., 1991;
Mustre de Leon et al., 1991) and included a total of 49
nondegenerate scattering paths. The x-ray energy was calibrated with
respect to the first inflection energy of an iron metal foil, which was
defined as 7111.3 eV.
radiation generated from a rotating copper anode source, and
diffraction intensities were recorded on a Mar Research image plate.
110 images were collected in 1° oscillations at 19 °C. Data
were processed with the DENZO package of programs (Z. Otwinowski, Yale
University). The crystals are in the monoclinic space group, C2, with two molecules in the asymmetric unit. These two
molecules are related by a noncrystallographic 2-fold axis of symmetry,
which is perpendicular to the crystallographic 2-fold symmetry axis. As
a result, the diffraction pattern exhibits pseudo I222 symmetry. As for wild-type and a series of mutant myoglobin
crystals analyzed previously, we chose to process the data in the
nonstandard space group I2
in which the
angle is close to 90 °C rather than in the C2 cell in
which
= 128 °C and the
dimension is 157 Å
(see ).
and Val
truncated in both
molecules of the asymmetric unit. Initially, cycles of rigid body
refinement were carried out as described by Derewenda(1989). This
lowered the R
from 35.2 to 30.2%. Cycles of
least squares refinement in the program PROLSQ (CCP4, 1979) in which
the weighting of the x-ray terms to the geometric terms was 1:2 reduced
the R
to approximately 22%, at which point
2F
- F
and F
- F
electron density maps were calculated
and displayed graphically using the program O (Jones et al.,
1991). These maps contained electron density peaks for the missing
Val
and His
side chains. There was a
noticeable break in the electron density between His
and
the heme iron in the A molecule in 2F
- F
maps contoured at
3
. The mutated side chains were built into the model, which was
subsequently refined further in cycles of the automated refinement
procedure (Lamzin and Wilson, 1993). In this procedure, water
molecules, which satisfy distance criteria, are introduced
automatically in successive cycles into peaks in F
- F
electron density maps. These water molecules are
automatically removed in subsequent cycles if they do not refine
satisfactorily. In these intermediate cycles of refinement, the root
mean squared deviations of atomic positions from noncrystallographic
symmetry were restrained to 0.05 and 2 Å for main and side chain
atoms, respectively. The noncrystallographic symmetry restraints were
removed in subsequent cycles of least squares minimization refinement
procedures in the program PROLSQ (CCP4, 1979). Electron density maps
were inspected periodically during refinement, and manual adjustments
were made to the model in the program O (Jones et al., 1991).
Spectroscopic and Functional Properties of H64V/V68H
Myoglobin
Fig. 1shows the light absorption spectra of
ferric and ferrous forms of human and pig H64V/V68H myoglobins (toppanels) and its ferrous CO and NO forms (bottompanels). The ferric form of the human mutant myoglobin
has absorption bands at 563 and 525 nm in the visible region and a
Soret band at 412 nm, together with a small band around 630 nm. The
spectrum of the ferric pig mutant myoglobin is very similar to that of
the human mutant, as seen in the rightpanel of Fig. 1. The absorption bands at 563 and 525 nm are attributable
to a ferric low spin species, and that at 630 nm is an indication of
the presence of a high spin state (Eaton and Hofrichter, 1981). Both
human and pig ferric H64V/V68H mutants are mixtures of low spin and
high spin species in agreement with the NMR results on the human
mutant, which indicated a thermal equilibrium between a low spin (70%)
and a high spin state (30%) (Qin et al., 1994). When cyanide,
azide, imidazole, and fluoride (up to 50 mM concentration)
were added to the ferric H64V/V68H mutants, there were no detectable
changes in the light absorption spectrum. Ferric H64V/V68H myoglobin
either has very weak ligand affinities or cannot bind these exogenous
ligands. Upon reduction by dithionite, both of the mutant myoglobins
showed a typical hemochrome-type absorption spectrum with peaks at 563,
529, and 427 nm, indicating that ferrous H64V/V68H myoglobin is a
hexacoordinate low spin species. The ferric/ferrous couple of the
oxidation-reduction potential of the H64V/V68H mutant is -128 mV
at 20 °C in 0.1 M phosphate buffer, pH 7, which is
markedly lower than the +54 mV potential of the wild-type protein.
Figure 1:
Light
absorption spectra of human (left panels) and pig (right
panels) H64V/V68H myoglobins in 0.1 M phosphate buffer,
pH 7, at 20 °C. Toppanels, spectra for ferric (solidline) and ferrous (brokenline) forms. Bottompanels, spectra for
ferrous CO (solidline) and NO (brokenline) forms.
Light absorption spectra of CO and NO forms of ferrous H64V/V68H
human and pig myoglobins are similar to those of the corresponding
wild-type complexes. The NO and
NO complexes
of ferrous H64V/V68H human myoglobin exhibit typical six-coordinate
nitrosyl EPR spectra, indicating that NO binds to the sixth
coordination site of the heme iron with the imidazole of His
as a trans axial ligand (data not shown). CO and NO
appear to assume the normal His
-Fe-L (L = CO or NO) coordination structure.
resulted in a rapid
oxidation of the iron, and the spectrum of the putative oxy form was
not detected in a conventional spectrophotometer with a mixing time of
10-30 s. The CO binding parameters for the double mutants are
compared in with those for wild-type and H64V human and
pig myoglobins. These functional results show that the effects of
mutagenesis are virtually identical in the myoglobins from pig and
human muscle. In contrast to the single H64V mutant, the double
H64V/V68H mutant shows a marked
10-fold reduction in the CO
association rate constant and a
4-fold reduction in the
dissociation rate constant compared with the wild-type protein. The net
result is a 2-3-fold reduction in the CO affinity for the double
mutant compared with the 3-fold increase in the CO affinity observed
for the single His
Val mutant.
is shifted to 1967
cm
in the H64V/V68H double mutant. The CO stretching
band of H64V/V68H myoglobin is thus similar to those of the human, pig,
and sperm whale H64V single mutants, which have major bands at
1965-1967 cm
(Li et al., 1994;
Balasubramanian et al., 1993). The order of the CO bond is
coupled to the surrounding electric field, and thus the vibrational
properties of the bound CO are sensitive to the polarity of the distal
pocket (Park et al., 1991; Sakan et al., 1993; Li et al., 1994). Thus, the electrostatic field around the bound
CO appears to be the same in both H64V/V68H and H64V myoglobins. This
suggests that once the Fe
-imidazole bond is broken,
the His
side chain rotates away from the bound ligand
taking up a conformation similar to that of the aromatic side chain of
Phe
in the crystal structure of V68F myoglobin (Quillin et al., 1995).
Figure 2:
IR spectra of recombinant human CO
myoglobin in 0.1 M phosphate buffer, pH 7.0, with 1 atmosphere
of CO recorded at room temperature (22
°C).
The EPR spectra of human and pig ferric
H64V/V68H myoglobins are shown in Fig. 3. A residual high spin
signal at g = 6, and a ``large g type'' low spin signal (g
=
3.34, and g
2.0) are seen in the human and
pig mutant myoglobins. The optical absorption (Fig. 1) and the
EPR results show that iron atoms in the human and pig mutants have
virtually identical electronic structures. In the low spin signal, the g
feature is well defined, the g
feature is still visible, but the g
feature
is not visible under the experimental conditions. The hemin iron is
predominantly in a low spin state at cryogenic temperatures, indicating
that it is the ground state. The H64V/V68H low spin EPR is somewhat
similar to that of b-type cytochromes of mitochondria,
neutrophils, and chloroplasts and some model compounds that have a
strong g
> 3.3 as the major observable
spectral feature (Tsai and Palmer, 1983; Salerno, 1984; Salerno and
Leigh, 1984; Nitschke and Rutherford, 1989; Walker et al.,
1986; Miki et al., 1992). In contrast, cytochrome b
and the imidazole complex of metmyoglobin
exhibit EPR signals with three well defined components at g
3, g
2.2, and g
1.4 (Blumberg and Peisach, 1971; Hori,
1971; Ikeda-Saito et al., 1974).
Figure 3:
EPR
spectrum of human (top) and pig (bottom) ferric
H64V/V68H myoglobins in 0.1 M phosphate buffer, pH 7, recorded
at 6 K.
Fig. 4, A and B present the iron-K edge x-ray absorption edge
spectrum and the analyzed EXAFS of ferric human H64V/V68H myoglobin at
10 K. Shiro et al.(1990) have suggested the 1s 3d pre-edge feature at approximately 7112
eV is an indicator of the coordination state of the heme iron. An
increase in its intensity indicates a decrease in the molecular
symmetry around the iron atom as observed for five coordinate
hemoproteins (Shiro et al., 1990). The relatively weak feature
at 7112 eV observed for the double mutant is characteristic of a
hexacoordinate low spin species and is consistent with the notion that
His
becomes a heme ligand.
Figure 4:
A, iron-K edge x-ray absorption
spectrum of human ferric H64V/V68H myoglobin recorded near 10 K. B, EXAFS Fourier transform of human ferric H64V/V68H myoglobin. The
transform was phase-corrected for the first-shell Fe-N scattering. The inset shows a curve-fitting analysis (brokenline) of the EXAFS data (solidline).
The EXAFS analysis indicates
that the first shell Fe-N distance is 2.02 Å (with the usual
EXAFS accuracy of ± 0.02 Å). This is typical of ferric low
spin bisimidazole complexes regardless of low spin EPR type (Walker et al., 1986; Inniss et al., 1988). The EXAFS
resolution, as determined by the k range of the data, is
approximately 0.11 Å, and we conclude that the Fe-N distances of
the histidine ligands differ by less than 0.11 Å from the mean
distance of 2.02 Å in the ferric H64V/V68H double mutant. Outer
shell Debye-Waller factors were semiempirically approximated using a
correlated Debye model assuming a sample temperature of 10 K and a
Debye temperature of 1200 K, the latter being determined from the first
shell Fe-N Debye-Waller factor of 0.0025 Å. A planar
heme ring was assumed with final (refined) distances of 3.05, 3.40, and
4.24 Å for the
-,
-, and
-carbons, respectively.
No improvements in the fit were obtained by allowing the histidine
rings to move from a position normal to the heme, and we conclude that
in this case EXAFS lacks the sensitivity to detect such displacements
in the presence of the intense backscattering from the porphyrin
carbons.
X-ray Structure of Pig H64V/V68H Myoglobin
The
proposed bisimidazole coordination in H64V/V68H myoglobin has also been
examined by x-ray crystallography. Initial attempts to crystallize the
human H64V/V68H mutant resulted in small needle shaped crystals that
did not provide useful x-ray diffraction. Fortunately, pig H64V/V68H
myoglobin formed single crystals suitable for x-ray analysis, and the
structure of the ferric form has been determined and refined at
2.07-Å resolution. The data collection and refinement statistics
are listed in . The refined model, which includes all
protein atoms except Gln and Gly
in both
molecules of the asymmetric unit, comprises 2,653 atoms including 200
water molecules. The final R
is 17.5% (free R = 22.7%) for 24,848 reflections in the resolution
range of 10.0-2.07 Å. The overall structure of pig
H64V/V68H myoglobin is closely similar to that of wild-type pig
myoglobin. Following least squares overlap of the A and B molecules of
the mutant myoglobin with the corresponding molecules of wild-type pig
myoglobin, the root mean square deviation in main chain atomic
positions is 0.24 and 0.25 Å, respectively. The root mean square
deviation in the positions of main chain atoms following overlap of the
A and the B molecules of the H64V/V68H asymmetric unit is 0.16 Å.
is very clear in both molecules of the asymmetric unit and
provides unambiguous evidence of ligation of the imidazole side chain
to the iron (Fig. 5). However, the orientation of the imidazole
side chain with respect to the heme plane is far from optimal for
overlap of the bonding orbitals of the imidazole's N
and the heme iron. As a result, the
Fe-N
distance in this bond is rather long (2.3
Å). The N
-H group of His
is ideally
placed to make a strong hydrogen bonding interaction (2.7 Å) with
the carbonyl oxygen of Val
. Superposition of the double
mutant and wild-type myoglobin structures by least squares minimization
of the root mean square differences in the main chain atomic positions
shows a significant change in the position of the heme group relative
to the protein backbone (Fig. 6). In both A and B molecules of
the mutant, the movement can be described by a lateral translation of
the heme group of
0.5 Å toward the EF corner and about an
8° rotation of the heme about an axis through the
- and
-meso carbon atoms. The effect of this change, coupled with a
small movement of the protein backbone, is to bring the C
atom of residue 68 0.6 Å closer to the iron in the
H64V/V68H double mutant than in the wild-type myoglobin.
Figure 5:
Stereoviews of the heme pocket of pig
H64V/V68H myoglobin. 2F - F electron density
contoured at 1.8 is displayed on the heme and surrounding
residues. These are shown for the A and B molecules of the asymmetric
unit in the top and bottompanels,
respectively.
Figure 6:
Stereo view of the superposition of the
heme environments of pig wild-type myoglobin (thin lines) and
H64V/V68H myoglobin (thick lines). The overlap minimizes the
root mean squared deviation in the positions of main chain atoms of
residues 1-151 in the chains of the respective molecules. These
are shown for the A and B molecules of the asymmetric unit in the top and bottompanels, respectively. The
coordinated water molecule in the wild-type structure and solvent
molecules in both structures have been omitted for
clarity.
A curious
feature of the maps is a break in the 2F- F
electron density between
the iron atom and the proximal histidine in the A molecule. This was
observed in the initial maps and persists in F
maps and in F
- F
maps calculated after the heme and
His
and His
had been omitted from the
coordinate set for several cycles of refinement and omitted from the
subsequent phase calculation. In the refined model, the imidazole side
chain of the proximal histidine (His
) in the A molecule
has an altered conformation with respect to the plane of the pyrrole
nitrogens, and the Fe-N
His
distance is
unusually long (2.3 Å). The principal structural change is the
increase in the His
angle, from
60° in the B molecule to 78° in the A molecule.
stereochemistry between the A
and B molecules is the different crystal environments inhabited by the
two molecules. As mentioned earlier, however, the crystal form is
pseudo-I222, and the intermolecular contacts made by the A and
B molecules of the asymmetric unit are closely similar. One difference
of note may be the side chain amide of Gln
, which in the B
molecule is involved in crystal contacts with main chain groups of
residues 149 and 150 in an adjacent A molecule; these interactions are
not matched in detail by Gln
in the A molecule. Whether
and how this influences the structure of the side chain at position 93
is difficult to evaluate with data that extend to 2.1-Å spacing
because the magnitudes of the main chain atomic positional changes are
so small (0.1-0.2 Å). It is noticeable, however, that there
is greater symmetry in the iron coordination in the A molecule than in
the B molecule with the N
-H of His
making
stronger interactions with Ser
-OH and Leu
>C=O (I). These interactions may make
favorable energetic contributions that compensate for what appears to
be a weaker Fe-His
coordinate bond in the A
molecule.
Axial Coordination and Electronic Structure in the H64V/V68H
Mutant
The crystal structure of pig H64V/V68H myoglobin
demonstrates coordination of the imidazole of His to the
sixth position of the ferric iron to form a bisimidazole heme complex.
A salient feature is that the imidazole plane of His
(E11)
is not perpendicular to the heme ring. This is because the conventional
myoglobin fold does not allow the E11 imidazole side chain to approach
the iron in a conformation perpendicular to the heme. The x-ray
crystallographic result verifies the heme ligand geometry predicted by
NMR (Qin et al., 1994). On the basis of the heme methyl
contact shift patterns and the significantly smaller contact shift
observed for His
N
H than for the
His
N
H, Qin et al.(1994)
concluded that the Fe-His
bond is weaker than the
Fe-His
bond in both low spin and high spin
complexes. The tilted His
and the longer Fe-imidazole
N
bond observed in the x-ray structure of the pig
double mutant also indicate a weaker ligand field when compared with
more conventional bis-histidine ligated proteins such as
ferricytochrome b
(Ikeda-Saito et al.,
1974; Lee et al., 1990). This weakening accounts for 30%
population of the ferric high spin state at ambient temperatures (Qin et al., 1994).
to the iron and Val
>C=O,
respectively, appear to drive heme reorientation and small adjustments
of the protein main chain. As a result, the iron atom is 0.6 Å
closer to the C
atom of residue 68 in the double mutant
protein than it is in the wild-type myoglobin. This reorientation is
facilitated by the absence of the large polar side chain of
His
, which is replaced by valine.
imidazole plane is significantly displaced from being parallel to
the imidazole plane of the proximal His
. This is quite
different from the geometries found in cytochrome b
or the imidazole complex of metmyoglobin, where the planes of the
axial ligands assume nearly parallel orientations (Durley and Mathews,
1994; Lionetti et al., 1991). The nearly
perpendicular orientation of His
and His
imidazoles appears to be the primary cause of the large g
low spin EPR signal in the H64V/V68H double
mutant. Walker, Huynh, Scheidt, Strouse, and their co-workers (Walker et al., 1986; Inniss et al., 1988)
demonstrated that large g
low spin EPR signals
are associated with a perpendicular orientation of the imidazole
ligands. The tilted Fe-His
bond, which reduces the
ligand field, thereby decreasing the tetragonal splitting, also favors
a large g
low spin EPR signal.
modulates the spin and coordination
states of the ferric cytochrome and that properties of the abnormal
subunits of M-type hemoglobins can be modeled to some extent in
engineered myoglobin mutants with altered axial ligation (Egeberg et al., 1990a). However, designing and engineering a
hemoprotein with a highly anisotropic low spin EPR signal has not been
done before. Choma et al.(1994) and Robertson et
al.(1994) reported the design and synthesis of heme-peptide
complexes. Although the redox potentials of the heme-helix complexes
were between -200 and -100 mV and similar to that of
H64V/V68H myoglobin, these model compounds exhibited low spin EPR
spectra typical of ordinary bisimidazole heme complexes (g
3.0). To our knowledge, H64V/V68H
myoglobin is the first mutant with a large g
low
spin EPR signal and will serve as a good model for understanding the
origin of this type of EPR signal in naturally occurring hemoproteins.
Iron Reactivity
The His
Val/Val
His double mutation causes marked decreases
in the reactivity of the iron atom with exogenous ligands in both the
ferric and ferrous oxidation states. This is clearly due to direct
chelation of His
with the iron atom. The single His
Val mutation increases markedly the association rate
constants for azide, CO, NO, and O
binding by creating a
larger pathway into the heme pocket, by preventing direct water
coordination in the ferric state, and by maintaining a completely
apolar, anhydrous binding site in the ferrous state (Rohlfs et
al., 1990; Quillin et al., 1993; Brancaccio et al., 1994). In contrast, the distal imidazole-iron bond in
the H64V/V68H double mutant must be broken before ligand can bind to
either oxidation state, causing marked decreases in the rate constants
for association.
His substitution also
causes a decrease in the rate of CO dissociation relative to either
wild-type protein or H64V myoglobin. The decreased size of the distal
pocket due to His
is expected to inhibit movement of
dissociated ligands away from the iron atom, as is observed for
Phe
sperm whale myoglobin (Egeberg et al. 1990a;
Carver et al., 1990). The latter mutant shows lowered
dissociation rate constants, increased extents of geminate
recombination, and markedly decreased quantum yields for complete
photodissociation into the solvent phase (Carver et al., 1990;
Quillin et al., 1995). Presumably similar phenomena occur in
the H64V/V68H double mutant accounting for the lower CO dissociation
rate constant and extremely low quantum yield for NO photodissociation.
bond before CO can bind. This effect is substantial,
10
fold, when K
for the double mutant is compared
with that of H64V myoglobin, but it is more moderate, 2-3-fold,
when compared with that of the wild-type protein. In the native
protein, a distal pocket water molecule has to be displaced before
ligands can bind, whereas in the H64V protein no water is present near
the iron atom in the deoxy structure (Quillin et al., 1993;
Cameron et al., 1993; La Mar et al., 1994).
phenol group at the sixth position of the ferric heme iron
(Egeberg et al., 1990a; Hargrove et al., 1994b;
Maurus et al., 1994). In the H64Y mutant, azide and cyanide
can still bind to the iron by displacing the coordinated Tyr
(Brancaccio et al. 1994). Ferric H64Y myoglobin is in a
high spin state, whereas H64V/V68H myoglobin is largely in the low spin
state (Egeberg et al., 1990a; Qin et al., 1994). The
ligand field in the H64V/V68H mutant is stronger than that in the H64Y
mutant. A stronger Fe-His
bond in the double mutant
effectively prevents azide or cyanide binding to the ferric iron atom.
H64Y deoxymyoglobin is pentacoordinate with His
as a
proximal ligand. In contrast, H64V/V68H deoxymyoglobin is
hexacoordinate with His
bound to the iron atom. However,
once CO is bound, the imidazole side chain probably adopts a
conformation pointing away from the iron atom since
of the bound ligand in the H64V/V68H mutant indicates an apolar
environment (Fig. 2).
and the iron atom since the
single H64V mutation increases the rate of hemin loss 10-20-fold.
However, once hemin is removed, the H64V/V68H apomyoglobin unfolds into
a random coil due to the presence of the polar His
residue
in the normally apolar region between the B, E, and G helices (Hargrove et al., 1994a).
Conclusion
Our present work demonstrates
unequivocally that the imidazole group of histidine at position 68
forms an axial bond with the heme iron. Unlike His,
His
cannot form a hydrogen bond with bound ligands. As a
result the double mutant has lowered reactivity toward both ferric and
ferrous ligands in comparison with wild-type myoglobin. Rapid
autooxidation of the ferrous form and the unusual instability of
H64V/V68H apomyoglobin (Hargrove et al., 1994a) further
demonstrate that the stereochemical positioning of histidine at
position 64 is essential for myoglobin function. Reversal of the distal
valine and histidine produces a protein incapable of O
transport.
Table: Rate
and equilibrium constants for CO binding for human and pig myoglobin
recombinants
, were calculated as k
/ k
.
Table: Data collection and refinement statistics for
pig H64V/V68H myoglobin
Table: 1953066862p4in
Angles between the
normals to the specified pair of planes.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.