(Received for publication, April 24, 1995)
From the
The functional, spectral, and structural properties of elephant
myoglobin and the L29F/H64Q mutant of sperm whale myoglobin have been
compared in detail by conventional kinetic techniques, infrared and
resonance Raman spectroscopy, H NMR, and x-ray
crystallography. There is a striking correspondence between the
properties of the naturally occurring elephant protein and those of the
sperm whale double mutant, both of which are quite distinct from those
of native sperm whale myoglobin and the single H64Q mutant. These
results and the recent crystal structure determination by Bisig et
al. (Bisig, D. A., Di Iorio, E. E., Diederichs, K., Winterhalter,
K. H., and Piontek, K.(1995) J. Biol. Chem. 270,
20754-20762) confirm that a Phe residue is present at position 29
(B10) in elephant myoglobin, and not a Leu residue as is reported in
the published amino acid sequence. The single Gln
(E7)
substitution lowers oxygen affinity
5-fold and increases the rate
of autooxidation 3-fold. These unfavorable effects are reversed by the
Phe
(B10) replacement in both elephant myoglobin and the
sperm whale double mutant. The latter, genetically engineered protein
was originally constructed to be a blood substitute prototype with
moderately low O
affinity, large rate constants, and
increased resistance to autooxidation. Thus, the same distal pocket
combination that we designed rationally on the basis of proposed
mechanisms for ligand binding and autooxidation is also found in
nature.
Myoglobin is a small, 16-18-kDa, globular hemeprotein
which stores molecular oxygen in muscle cells. Because of the
similarity of their tertiary structures, myoglobin is frequently used
as a model for the and
subunits of tetrameric hemoglobin.
The key physiological properties of myoglobin are its high affinity for
oxygen and low rate of spontaneous oxidation to the inactive ferric
form (Antonini and Brunori, 1971). Studies of naturally occurring and
mutant hemeproteins have shown that ligand affinity and autooxidation
are affected markedly by the chemical nature of the amino acids in the
heme pocket. In order to understand these functional properties, the
molecular structures of a large number of myoglobins and hemoglobins
have been determined by x-ray crystallography and solution NMR studies.
These studies have been pursued along two lines. Traditionally,
naturally occurring genetic or species variants with distinctive
functional properties have been investigated (i.e. Bolognesi et al.(1989), Steigmann and Weber(1979), Vainshtein et
al.(1978), Yu et al.(1990), Qin and La Mar (1992), and
Vyas et al.(1993)). More recently, site-directed mutagenesis
has been used either to test structural hypotheses (Olson et
al., 1988; Braunstein et al., 1988; Carver et
al., 1990; Ikeda-Saito et al., 1991; Balasubramanian et al., 1993; Li et al., 1994; Ling et al.,
1994; Huang and Boxer, 1994) or to explore systematically the
physiological roles of individual residues (Rohlfs et al.,
1990; Egeberg et al., 1990; Smerdon et al., 1991,
1993; Carver et al., 1991, 1992; Quillin et al.,
1993, 1995; Lai et al., 1995). A review of the mutagenesis
literature is given in Springer et al.(1994).
Dene et
al.(1980) sequenced Asian elephant myoglobin and reported that it
contains a Gln at position 64 (E7) rather than a His, which is found at
this position in all other mammalian myoglobins. This replacement
appeared to be neutral since the O affinity and
autooxidation rate of elephant myoglobin are very similar to those of
pig, human, and, sperm whale Mb (
)(Table 1,
Romero-Herrera et al., 1981; Springer et al., 1994).
Romero-Herrera et al.(1981) concluded that in elephant
myoglobin, N
of Gln
(E7) forms a hydrogen
bond with bound oxygen which is equivalent in strength to that formed
with His
(E7) in all other mammalian myoglobins. However,
when this idea was tested directly by constructing the His(E7)
Gln mutation in recombinant sperm whale myoglobin, the results
indicated that this substitution is not neutral. The H64Q mutation
causes a 5-fold decrease in K
Studies aimed at understanding why
H64Q sperm whale myoglobin does not mimic the functional and spectral
properties of elephant myoglobin have been pursued by two approaches.
First, La Mar and co-workers (Yu et al., 1990; Vyas et
al., 1993) have carried out high resolution H NMR
studies of both diamagnetic carbonyl and paramagnetic cyano-met
complexes of elephant Mb and compared the results to native sperm whale
myoglobin as a standard. The spectra show that, in addition to features
for the expected distal Gln
(E7), there are also signals
which strongly indicate that a phenylalanine side chain is in van der
Waals contact with bound ligand in the elephant protein.
Second,
Olson, Phillips, and co-workers (Carver et al., 1992; Gibson et al., 1992; Springer et al., 1994; Li et
al., 1994) have carried out a systematic study of point mutations
at position 29 (B10) in sperm whale myoglobin. Substitution of Phe for
Leu(B10) in sperm whale myoglobin leads to a dramatic
increase in O
affinity and marked decrease in autooxidation
rate (Carver et al., 1992). The crystal structure of this
mutant shows that C
of the Phe
side chain
makes van der Waals contact with bound ligands. The
Fe
-O-O
complex is
stabilized by favorable electrostatic interaction with the positive
edge of the phenyl multipole, accounting partly for the 15-fold
increase in affinity shown by the L29F mutant. The large Phe
side chain also excludes water from the distal pocket of the
deoxy form of the mutant. (
)The favorable multipole and
water exclusion effects also inhibit protonation of the
Fe
-O-O
complex,
preventing disproportionation into Fe
and
HO
(Brantley et al., 1993).
In
an effort to construct a blood substitute prototype, Olson and
co-workers (Brantley et al., 1993; Olson, 1994) constructed a
double mutant in which Phe was put at the 29 position to inhibit
autooxidation by excluding water from the active site and Gln was put
at the 64 position to lower O affinity by weakening
hydrogen bonding to the bound ligand. Later, it was discovered that
this active site configuration,
Phe
(B10)/Gln
(E7), was predicted to occur in
elephant Mb by Yu et al.(1990) even though the published
sequence for the elephant protein lists a Leu at the B10 position (Dene et al., 1980). To resolve this problem and test the
conclusions of Yu et al.(1990), we have compared in detail the
structural, spectral, and functional properties of elephant myoglobin
and the L29F/H64Q sperm whale mutant. Rate constants for O
and CO binding and autooxidation were determined by conventional
kinetic techniques. Infrared spectra of the CO complexes of wild-type,
L29F, H64Q, and L29F/H64Q sperm whale myoglobins were compared with
that of elephant MbCO. Resonance Raman techniques were used to measure
the
and
bands for the same
set of mutants and native proteins. The structure of the active site of
the cyano-met form of the sperm whale double mutant was determined by
solution
H NMR and compared with that of the single mutants
and elephant myoglobin. Finally, the structure of L29F/H64Q sperm whale
MbCO was determined by x-ray crystallography for comparison with the
active site parameters determined by NMR and with the crystal structure
of native elephant metmyoglobin recently obtained by Bisig et
al.(1995).
For the resonance Raman measurements, the reduced CO forms of the
proteins were prepared by flushing deoxymyoglobin samples with 1 atm of
CO under rigorously anaerobic conditions. The cyano-metmyoglobin
samples were prepared by oxidation with ferricyanide and addition of
potassium cyanide, and then excess KCN and KFe(CN)
were removed by ion exchange chromatography. Approximately 100
µM samples were placed in a quartz spinning cell to
prevent photodissociation in the case of MbCO and photoreduction in the
case of Mb
CN (this was judged negligible as measured
by the absence of any discernible contribution from the
mode of the photoproduct). The resonance Raman spectra were
acquired using a triple spectrograph (Spex 1877) equipped with a liquid
nitrogen cooled 1152
298 pixel charge-coupled device (Princeton
Instruments LN/CCD with an EEV chip) as the detector. Excitation was
provided by the 415.4 nm output of a krypton ion laser (Coherent Innova
200-K3). The laser power was typically 5 milliwatts, and the spectral
resolution was
4 cm
.
All the H NMR spectra were collected on the GE Omega 500 MHz
spectrometer. To effectively observe rapidly relaxing signals, the
slowly relaxing diamagnetic envelope was suppressed by the WEFT pulse
sequence (Gupta, 1976). Nonselective T
s for the
resolved strongly relaxed protons were measured via the
inversion-recovery experiment. Steady-state NOEs were recorded as
described in detail previously (Emerson and La Mar, 1990a); the ratio
of the steady-state NOEs to a common proton upon saturating the
1-CH
and 8-CH
groups was obtained from the
ratio of the amplitudes of the NOEs in the difference traces
(Rajarathnam et al., 1993). The phase-sensitive TOCSY
(Braunschweiler and Ernst, 1983; Davis and Bax, 1985; Rance 1987),
NOESY (Jeener et al., 1979), and magnitude COSY (Bax, 1982)
measurements employed the same parameters, and were processed in the
same fashion, as described in detail previously (Rajarathnam et
al., 1992, 1993, 1994; Qin and La Mar, 1992).
Figure 1:
Orientation of the coordinate system, x, y, z, for the magnetic susceptibility
tensor , relative to the symmetry (x-ray crystallographic)
coordinate system x`, y`, z`. A,
tilt of the major magnetic axes, z, from the heme normal, z`, given by the angle
; note that since the z axis is positive on the proximal side, the Fe-CN tilt is defined
by the -z axis. B, projection of the tilt of
the z axis on the heme plane, defined by the angle
from
the symmetry coordinate system x` axis, and location of the
rhombic magnetic axes, x and y, whose projection on
the heme plane is related to the symmetry axes, x`, y`, by the angle
≅
+
, where
,
, and
are the Euler angles that transform the symmetry
coordinates into the magnetic coordinates. The angle
and modulus r define lateral movement of the E-helix. The arrows labeled a, b, c, d, and e correspond to the Fe-CN orientation (-z direction)
in WT- H64V-, H64Q-, L29F-, and L29F/H64Q-cyano-metmyoglobin,
respectively.
,
are axial and
rhombic anisotropies, and
is the observed chemical
shift referenced to 2,2-dimethyl-2-silapentane-5-sulfonic acid.
is the shift in the isostructural diamagnetic MbCO
complex (Dalvit and Wright, 1987; Chiu, 1992) or calculated for protons
whose
are not available by using the equation (Qin et al., 1993):
where is the shift of an amino acid proton
typical for
-helices,
-strand, coils, etc. (Wishart et
al., 1991), and
is the heme-induced ring
current shift of the proton based on the WT coordinates by using the
eight-loop model (Cross and Wright, 1985). Minimizing the error
function F/n in was performed over three
parameters,
,
,
, using available
and
, or extended to all five parameters
to yield both the Euler angles and anisotropies as described in detail
previously (Rajarathnam et al., 1992).
where a known proton, i.e. His(F8) NH
with R
(a) = 5.0 Å and T
= 31 ± 3 ms, yields R
(b) when T
is measured.
The molecular modeling was carried out on a Silicon Graphics personal
IRIS for the MbCO structure using the MIDAS program.
The coordinates of wild-type
MbCO were used as the starting model in the refinement of the double
mutant structure (Protein Data Bank entry 1MGK, Brookhaven National
Laboratory; Quillin et al. (1993)). Initial difference maps
were calculated using the program X-PLOR (Brunger et al.,
1989). The Gln(E7) and Phe
(B10) side chains
were then introduced into the difference map using the molecular
modeling program CHAIN (Sack, 1988). Constrained least-square
refinements and map calculations were performed by X-PLOR using the
Engh and Huber parameter set for the ferrous myoglobins (Engh and
Huber, 1991). After several cycles of refinement, manual refitting, and
solvent placement, the crystallographic R-factor converged to
17.3% with root mean square bond deviations of 0.02 Å.
Coordinates for the L29F/H64Q MbCO structure have been deposited in the
Brookhaven Protein Data Bank (1MCY).
Figure 2: Fourier transform infrared spectra of CO-myoglobin complexes at pH 7, 25 °C.
Resonance Raman spectra of the CO-myoglobin
and cyano-metmyoglobin complexes in the regions of the Fe-ligand
stretching vibrations ( and
)
are shown in Fig. 3and 4, respectively (for a review of these
modes in hemoglobins and myoglobins see Yu and Kerr(1988)). The
assignments for the
and
modes were confirmed via experiments with
CO and
CN
(viz. Ling et al. (1994)). The single L29F mutation causes an increase in
from 509 for wild-type MbCO to 525
cm
, and the single H64Q replacement causes a small
decrease to 507 cm
. When both substitutions are
present, as in the double mutant and elephant myoglobin,
is 513 cm
. Similar results are
observed for the Fe-CN complexes (Fig. 4). The L29F mutation
causes a 7 cm
increase in
; the
H64Q replacement causes a 7 cm
decrease; whereas the
values for the double mutant and elephant
myoglobin are roughly equal to that of wild-type sperm whale myoglobin.
Taken together, the spectral results in Fig. 2-4 argue
strongly that elephant myoglobin contains a Phe
(B10)
residue which interacts with bound ligands in a manner identical with
that observed in the L29F/H64Q double mutant of sperm whale myoglobin.
These data also emphasize the sensitivity of the Fe-CO and Fe-CN bond
orders to amino acid substitutions in the distal pocket.
Figure 3: Soret-excitation resonance Raman spectra of CO-myoglobin complexes at pH 7, 25 °C.
Figure 4: Soret-excitation resonance Raman spectra of cyano-metmyoglobin complexes at pH 7, 25 °C.
Figure 5:
Hyperfine-shifted portions of the 500-MHz H NMR reference spectra. A,
H64Q-cyano-metmyoglobin at 35 °C at pH 8.6 in
H
O; B, L29F-cyano-metmyoglobin at 35
°C at pH 8.6 in
H
O; C,
L29/H64Q-cyano-metmyoglobin at 35 °C at pH 8.6 in
H
O; D, in
H
O; E, fast inversion recovery spectrum of
L29F/H64Q-cyano-metmyoglobin in
H
O collected at
12 s
to emphasize broad, efficiently relaxed proton
signals. F, WEFT-NOE difference spectrum of
L29F/H64Q-cyano-metmyoglobin upon irradiating 1-CH
in
H
O at 20 °C. G, super WEFT-NOE
spectrum of L29F/H64Q-cyano-metmyoglobin in
H
O
at 35 °C at a repetition rate of 29
s
.
The H NMR spectrum of
L29F/H64Q-cyano-metmyoglobin in
H
O is shown in Fig. 5D. Two hyperfine shifted and strongly relaxed
labile proton resonances are found in the low field window by
comparison to the trace in
H
O shown in Fig. 5C. One of these signals is apparent only in a
partially relaxed spectrum in
H
O which reveals
a strongly relaxed NH proton overlapping the 1-CH
signal
that is absent in the same spectra in
H
O (Fig. 5E). The super-WEFT trace of
L29F/H64Q-cyano-metmyoglobin in
H
O shown in Fig. 5G suppresses the diamagnetic envelope, retains
the resolved strongly relaxed proton signals for C
H of
Phe
(B10), C
H, C
Hs of
Phe
(CD1) and C
H of Ile
(FG5),
and reveals strongly relaxed protons under the aromatic window, i.e. C
H of His
(FG3) and the ring
CHs of His
(F8).
Complete sets of resonances for the
heme groups in all three mutants were assigned as described by Qin and
La Mar(1992). Two series of sequential backbone NOESY cross-peaks
uniquely locate the segments
Leu-Thr
(F4-FG1) and
Lys
-Ala
(E6-E17) and the hyperfine
shifted side chain signals (except for Gln
) are located by
TOCSY and COSY (Qin and La Mar, 1992; Qin et al., 1993a,
1993b). Ile
(FG5) is identified by COSY and its NOESY
contacts to the 4-vinyl and 5-CH
. His
(FG3)
ring protons are identified by the characteristic short T
(
20 ms) for C
H and the
dipolar contact of C
H to 6H
. A strongly
relaxed (T
20 ms for C
H)
AMM`XX` spin system with NOESY cross-peaks to 5-CH
locates
the hyperfine shifted Phe
(CD1). TOCSY and MCOSY locate
three weakly hyperfine shifted Phe side chains which characteristic
NOESY cross-peaks to Phe
(CD1), Gln
(E7)
C
H, and 2-vinyl/1-CH
identify
Phe
(B14), Phe
(CD4), and
Phe
(H15), respectively (Emerson and La Mar 1990a;
Rajarathnam et al., 1993). NOESY cross-peaks for 2-H
and 3-CH
to an upfield methyl peak at -0.44 ppm
with apparent COSY cross-peaks to 1.0 and 0.6 ppm locate the
C
H
-C
Hs of
Ile
(G8). Parallel studies on the two single chemical
shifts for the heme model of L29F/H64Q cyano-metmyoglobin, as well as
more limited data on the two single mutants, are listed in Table 2. (
)
The proximal residues,
Leu(F4)-Ala
(F9), His
(FG3),
Ile
(FG5), and His
(H15), of the double mutant
exhibit heme-residue and inter-residue NOESY cross-peak patterns and
paramagnetic induced relaxativities that are unchanged from those
observed for wild-type cyano-metmyoglobin (Emerson and La Mar, 1990a;
Rajarathnam et al., 1992). Thus, the structure on the proximal
side of the heme group is highly conserved in all three mutants. For
the most part, the unchanged distal residues also display intra- and
inter-residue NOESY cross-peak patterns and paramagnetic relaxativity
very similar to those observed for wild-type sperm whale myoglobin. The
exceptions represent small structural changes required to accommodate
the mutated E7 and B10 residues.
Figure 6:
NMR spectra of of
L29F/H64Q-cyano-metmyoglobin. A, the portions of the MCOSY
spectrum in H
O, at 35 °C showing spin
connectivities for the Phe
(B10) and Gln
(E7).
The MCOSY data are are processed by applying an unshifted
sine-bell-squared window over 512 T
512 T
points prior to zero-filling to 2048
2048 data points and Fourier transformation. B, the portions
of the NOESY spectrum (
= 50 ms) of
L29F/H64Q-cyano-metmyoglobin
H
O, pH 8.6 at 35
°C, illustrating the cross-peaks of Phe
(B10) and
Gln
(E7). The data were processed by applying a
30°-shifted sine-bell-squared window over 1024 T
256 T
points prior to zero-filling
to 2048
2048 data points and Fourier transformation. The box indicates that the cross-peak is observed in the other
dimension while the triangles stand for the cross-peaks
observed at a lower contour level.
The spectra of both L29F and
L29F/H64Q cyano-metmyoglobins exhibit two additional relaxed and
low-field hyperfine shifted signals with an AMM`XX` spin system for a
rapidly reorienting Phe ring that must arise from Phe(B10) (Fig. 6A). The relaxation properties yield R
of 4.9 ± 0.2 (T
= 27 ± 3 ms) for C
H of
Phe
(B10) in both mutants, with the mean R
for C
Hs 5.8 ± 0.2 Å (T
= 72 ± 7 ms) in the L29F mutant
and 6.2 ± 0.2Å (T
= 115
± 15 ms) in the L29F/H64Q double mutant. Phe
(B10)
in L29F/H64Q cyano-metmyoglobin does not exhibit NOE connectivities to
heme resonances but does exhibit NOESY cross-peaks to
Gly
(E8), Ile
(G8), Phe
(B14), and
Val
(E11) (Fig. 6B), as well as a
steady-state NOE to Gln
(E7) (not shown). These NOE
connections are expected from the crystal structures of both the single
L29F (Carver et al., 1992) and double L29F/H64Q MbCO mutants (Table 4) and are similar to those displayed by the
Leu
(B10) residue in native and wild-type MbCO (Dalvit and
Wright, 1987; Quillin et al., 1993). These contacts among the
distal pocket residues are described schematically in Fig. 7.
Figure 7: The distal heme pocket structure of L29F/H64Q-cyano-metmyoglobin and NOE connectivities. Dashed and solid lines indicate heme-residue and inter-residue NOE/NOESY contacts, respectively.
The orientation of Gln(E7) in L29F/H64Q
cyano-metmyoglobin was determined by searching for rotation angles,
,
,
, which most
accurately reproduced the magnetic axes of the single and double mutant
and the observed dipolar shifts for the proton j on the fragment
CH
as the i-j bond was rotated (). Starting
with the
-
bond, each subsequent search for C
Hs
(j =
,
, etc) was carried out at the optimized angle
for the previous search as described in detail by Qin et al. (1993a). The orientation of Gln
(E7) in the double
mutant is nearly identical with that determined for the single mutant
and places N
H
4.2 Å from the iron atom, a
distance which is consistent with that obtained independently by
relaxation data. Since this orientation determined by
H NMR
differs inconsequentially from that found in the two crystal
structures, a more global search to optimize the conformation was not
pursued.
Both dipolar shifts () and relaxation data () were used as independent constraints to estimate the
orientation of Phe(B10) in L29F/H64Q cyano-metmyoglobin.
As shown in Fig. 8(solid squares), T
(C
H) = 25 ± 3 ms and R
(C
H) = 4.9 ± 0.2
Å indicate a
value of
-98°,
while the dipolar shifts suggest a value of
-95° (not
shown). The optimized value for
when
is fixed at -95° is
0° (open circles, Fig. 8). The dipolar shift was found to be much less sensitive
to
, but also yielded values consistent with those
obtained from relaxation data.
Figure 8:
Distance versus and
plots. The distance of the affected
proton(s) from iron,
<R
> for the
averaged Phe
(B10) C
Hs as a function of
(solid squares, lower scale) and
(open circles, upper scale) for
Leu
(B10)
Phe substituted in the sperm whale WT MbCO
crystal structure, with the E-helix movement laterally over the heme by
0.8 Å toward the iron. The values of
and
are consistent with the <R
>
obtained from T
data, as shown by dashed
boxes.
Figure 9:
Crystal structures of L29F/H64Q sperm
whale and elephant myoglobins. A, electron density map for the
distal pocket of L29F/H64Q sperm whale CO myoglobin. The benzyl ring of
Phe is in a position to interact directly with the bound
ligand. The N
atom of Gln
is in a position
to hydrogen-bond, albeit weakly, with the bound ligand and occupies a
position similar to that of a corresponding atom in His
which is found in almost all mammalian myoglobins. B,
stereoplot comparison of the structures of the distal pockets of
L29F/H64Q sperm whale myoglobin (black) and asian elephant
myoglobin (white, Bisig et al.(1995)). The
arrangements of the side chains are very similar in both proteins which
serves to explain their similar functional properties. The drawings
were made with MOLSCRIPT (Kraulis, 1991).
Finally, Bisig et al.(1995) have just determined the high resolution crystal structure of native Asian elephant metmyoglobin. As shown in Fig. 9B, a Phe residue is present at the 29 position in the elephant protein, and the conformations of the E7 and B10 side chains are almost identical with those in the sperm whale double mutant.
Solution H NMR studies of elephant myoglobin
indicate the presence of an additional phenylalanine side chain in the
distal portion of the heme pocket, designated Phe
, which is
not seen in other mammalian myoglobins. The signals associated with
Phe
indicate that C
makes van der Waals
contact with the bound ligands (Yu et al., 1990; Vyas et
al., 1993). The decrease in size of the distal cavity and its
enhanced apolar character was proposed to be the origin of the normal,
low autooxidation rate of elephant myoglobin. Detailed NMR
characterization of Phe
indicated that it is located at the
B10 position (Vyas et al., 1993). The difficulty with this
interpretation is that the published sequence of elephant Mb lists Leu
at this position, as is the case in all other known mammalian myoglobin
sequences (Dene et al., 1980).
The H NMR
spectrum of L29F/H64Q cyano-metmyoglobin is essentially identical with
that of elephant cyano-metmyoglobin (Table 2). This
correspondence between elephant myoglobin and the L29F/H64Q sperm whale
mutant has been examined quantitatively. The NOE contacts for
Phe
(B10) in L29F/H64Q cyano-metmyoglobin are identical
with those previously reported for Phe
in elephant
cyano-metmyoglobin (see Fig. 7; Vyas et al. (1993)). In
addition, the magnetic axes for elephant cyano-metmyoglobin are
indistinguishable from those of L29F/H64Q cyano-metmyoglobin and
correctly predict the dipolar shifts for both Gln
(E7) and
Phe
when they are placed at position B10 in elephant
myoglobin. The orientations predicted for the elephant protein are also
identical with those determined experimentally in the crystal structure
of the L29F/H64Q mutant of sperm whale MbCO (Table 4). Finally,
the presence of Phe
(B10) in elephant myoglobin has now
been confirmed directly by Bisig et al.(1995) who have
determined the structure of Asian elephant aquo-metmyoglobin. Thus,
there is no longer a controversy; a phenylalanine is clearly at
position 29 (B10) in Asian elephant myoglobin.
Figure 10:
Mechanism for O and CO
binding. The scheme was taken from Springer et al. (1994).
The effects of mutagenesis are
readily explained in terms of this electrostatic mechanism. In
Gln(E7) sperm whale myoglobin, the N
atom
of the amide side chain is farther from bound ligands and shows greater
disorder in its position than the corresponding atom in the His
side chain of the native protein (Quillin et al., 1993).
As a result, O
affinity is 5-fold smaller due to weaker
hydrogen bonding. The large phenyl side chain in Phe
(B10)
sperm whale myoglobin displaces bound water in the deoxy structure and
places another partial positive charge near the bound ligand (Carver et al., 1992). Both of these effects cause a marked
enhancement of O
binding. In both the L29F/H64Q double
mutant and elephant Mb, the weakened hydrogen bonding due to the
presence of Gln
is almost compensated by the presence of
the Phe
side chain. A possible explanation for the reduced
k
The currently accepted mechanism of autooxidation is shown in Fig. 11(Brantley et al., 1993). Under aerobic
conditions, the dominant pathway for autooxidation is protonation of
the Fe(II)-O complex and dismutation into Fe(III) and the
neutral superoxide radical HO
. In native
oxymyoglobin, the hydrogen bond between the neutral side chain of
His(E7) and the bound ligand decreases the rate of autooxidation by
preventing net protonation of the Fe-O
complex since the
pK
for forming the imidazolate anion is
12. This same interaction also enhances O
affinity by
roughly the same amount, accounting for the tight inverse coupling
between K
Figure 11: Mechanism for autooxidation. The scheme was taken from Brantley et al.(1993).
Gln(E7) and
Phe
(B10) in L29F/H64Q cyano-metmyoglobin occupy positions
similar to those of His
(E7) and Leu
(B10) in
the wild-type protein. The smaller bulk of Gln relative to His may be
responsible for the small lateral movement of the E-helix. However, a
comparable shift of the E-helix is observed in the crystal structure of
the single L29F mutant (Carver et al., 1992). The orientation
of the Gln
(E7) side chain is indistinguishable when
comparing both the solution and crystal structures of single and double
mutants. In each case, the N
H is in a position to
provide a hydrogen bond to either bound cyanide in the
cyano-metmyoglobin complex or O
in the oxy complex.
The
side chain of Phe(B10) has a similar orientation in the
single and double mutants of sperm whale myoglobin and in native
elephant myoglobin. In all cases, C
H is close enough to
the iron (
5 Å) to be in van der Waals contact with the bound
ligand. The aromatic ring in the NMR solution structure of the double
mutant appears to be rotated when compared with that in the single
mutant in solution. This structural difference is suggested by the
weaker paramagnetic relaxation of the Phe
(B10)
C
Hs signal (relative to C
H) in
L29F/H64Q cyano-metmyoglobin (T
(C
Hs)
115 ms, R
= 6.2 Å) than that observed in
L29F cyano-metmyoglobin (T
(C
H)
= 72 ms, R
= 5.8 Å). This
difference in
between the double and single mutants
is not observed in the crystal structure (Table 4). However, more
H NMR data are needed to substantiate this small difference
between the solution and crystal structures.