(Received for publication, April 24, 1995)
From the
The crystal structure of Asian elephant cyano-metmyoglobin which
has a glutamine instead of the usual distal site histidine has been
determined to high resolution. In addition to this replacement, the
substitution of a conserved leucine residue in position 29(B10) at the
distal side by a phenylalanine was unambiguously identified based on
the available electron density. The suspicion, that there were errors
in the original sequence which has caused some confusion, is thus
confirmed. Comparison with other myoglobin structures in various
ligated forms reveals an essentially unchanged tertiary structure in
elephant myoglobin despite the two amino acid substitutions in the heme
pocket. Our current structural model shows that the N2
atom of Gln
(E7) has moved with respect to the
corresponding nitrogen position of His
(E7) in the CO
complex of sperm whale myoglobin. The newly assigned residue
Phe
(B10) penetrates into the distal side of the heme
pocket approaching the ligand within van der Waals distance and causing
a much more crowded heme pocket compared to other myoglobins. Kinetic
properties of Asian elephant myoglobin, wild type, and recombinant
sperm whale myoglobins are discussed in relation to the structural
consequences of the two amino acid substitutions H64Q and L29F.
Myoglobin (Mb), ()a small, mostly
-helical,
globular hemeprotein, is most abundant in heart, but skeletal muscles
also contain variable amounts of this protein. Mb is commonly said to
act as oxygen storage, even though it is less present in skeletal
muscles of young animals, which are more active, than older ones.
Furthermore, the amount of myoglobin present in the heart of an animal
is sufficient to supply oxygen only for a few beats(1) . Mb is
also reported to facilitate O
unloading from hemoglobin and
O
diffusion to the mitochondrial surface(2) .
Oxidative phosphorylation appears also to be mediated by
Mb(2) . At our present level of understanding, we cannot decide
with certainty about all possible roles of Mb, and more work is needed
to fully clarify the situation.
In addition to their physiological implications, studies on Mb are also of general relevance to our understanding of the relationships between structures, dynamics, and function in proteins. Mb is the first protein for which a three-dimensional structure has been determined to atomic resolution by means of x-ray crystallography(3) . Its folding is nearly identical with that of hemoglobin subunits, even of highly unrelated organisms like the mollusc Scapharca inaequivalvis(4) or the larvae of the insect Chironomus thummi thummi(5) . Nearly all experimental approaches can be applied to Mb, due to its relatively small size, to the presence of a heme, and its availability in large quantities. Indeed, Mb is probably one of the best characterized proteins and therefore can be used as a reference for comparative analysis on other more complex systems.
The interplay between the overall dynamic properties and the
structural features of the heme pocket in modulating the reactivity of
the heme iron is an important element for our understanding of the
structure, dynamics, and function relationships in hemeproteins. Simple
heme compounds without a protein matrix exhibit a preferred binding to
CO versus O with a ratio of several ten thousands,
while a situation more favorable for oxygen affinity is found in
myoglobins, allowing these proteins to function as efficient oxygen
carriers. Individual binding properties arise from the specific protein
environment in the vicinity of the prosthetic group. Of particular
interest in this respect is the distal site pocket which harbors the
bound ligand. Much attention has been drawn to the role of the distal
residue 64 at the E7 helical position, since it directly interacts with
the bound ligand. In most hemeproteins, ranging from hemoglobin (6) to lignin peroxidase(7) , a histidine residue is
found in this place. Of all known vertebrate myoglobin sequences, only
a few have no histidine occupying position E7, one of them being
elephant myoglobin which has a glutamine(8) . The role of the
distal residue E7 is probably to provide a hydrogen bond to the bound
oxygen ligand, favoring its binding against CO and reducing
autooxidation of the heme iron(9) . Further effects on ligand
access and binding by other distal side amino acids correlate to their
specific steric and electrostatic properties.
The primary structure
determination of Asian elephant myoglobin (8) revealed that the
distal side residues are conserved apart from the E7 Gln substitution.
Against expectations, kinetic studies on elephant myoglobin show an
unchanged CO association constant if compared to other vertebrate
myoglobins(10) . Therefore, it was concluded that the
interaction of bound ligand with the E7 residue remains essentially
unchanged. The recent progress in molecular biology permits us to probe
the function of certain residues by site-directed mutagenesis. A single
mutant of sperm whale myoglobin with a Gln residue at position E7
instead of the common His has been constructed(11) . The
substitution was expected to produce a kinetic behavior similar to
elephant myoglobin. Instead, a 5-fold decrease of the O binding constant and a 3-fold increase in the rate of
autooxidation were observed. Obviously, this single amino acid change
in the distal side, deduced from the reported primary sequence, is not
sufficient to explain the kinetic properties of Asian elephant
myoglobin which are similar to myoglobins having the usual histidine
E7. Yu et al. (12) and Vyas et al. (13) have reported proton NMR spectra of Asian elephant
myoglobin. These data were interpreted in terms of the presence of an
additional phenylalanine in the heme crevice close enough to the
prosthetic group to interact with the bound ligand. On the basis of the
available sequence of EMb(8) , this feature cannot be explained
unless a major reshuffling of the fold is assumed. Among point
mutations at position 29 (B10), a recombinant L29F SWMb was produced
which is characterized by a dramatic increase in O
affinity
and a significant decrease of autooxidation (14) . A double
mutant L29F/H64Q construct shows rate constants for O
and
CO binding and autooxidation which are very similar to EMb, indicating
that the two effects of the single mutation cancel each other. On the
other hand, these two mutations do cause a 4-fold decrease of the CO
dissociation rate resulting in a significantly increased CO equilibrium
constant (see accompanying paper by Zhao et al.(44) ).
In the absence of detailed three-dimensional structural information on EMb, its peculiar CO kinetics and light absorption properties over broad temperature ranges (15) have been tentatively ascribed to the crowding of its distal heme crevice proposed by the La Mar group(12, 13) . The high susceptibility of the prosthetic group to structural fluctuations of the protein moiety, indicated by the thermal evolution of the kinetic and optical spectroscopy properties of EMb, was also tentatively related to the tight packing of its heme pocket.
The determination of the crystal
structure of elephant myoglobin has been undertaken to clarify the
situation at the distal side residues and to correlate the structural
information with available kinetic and optical spectroscopy data. The
results presented here show that the primary structure reported by Dene et al. (8) contains a crucial error and a
phenylalanine is present at position 29 (B10) of EMb. An additional
small sequencing error was found at position 27 (B8) where a Thr was
observed in our structure instead of a Phe as reported by Dene et
al.(8) . But, this newly assigned Thr residue appears of
no functional consequences. Phe(B10) is in close contact
with the bound ligand and occupies the cavity in which the free CO is
found in the 40 K crystal structure of photolyzed SWMb recently
reported by Schlichting et al.(16) .
Macroseeding was important to obtain large (0.8 0.4
0.3 mm
) single crystals. Seeding crystals have been
obtained by hair-seeding experiments using a rabbit whisker. The
crystals belong to space group P2
2
2
with unit cell dimensions a = 33.51 Å, b = 58.59 Å, c = 70.44 Å
with one molecule per asymmetric unit and a V
value of
2.03 Å
/Da which is calculated according to
Matthews(21) . The crystals diffract to at least 1.70 Å on a conventional x-ray source.
Figure 1:
Plot of the main chain -
angles for Asian elephant cyano-metmyoglobin. Glycine residues are
indicated by squares.
The whole polypeptide chain of 153 amino
acids, 1 protoporphyrin IX molecule, 1 cyanide ligand, and 255 water
molecules were included in the model. During map interpretation, it
became obvious that the originally reported sequence (8) is
erroneous at least for two amino acids. Of most significance here is
that the reported Leu(B10) is rather a phenylalanine as
can unambiguously be seen from a difference omit map (Fig. 2).
Also, residue 27 (B8), a phenylalanine according to Dene et
al. (8) , is now assigned to a threonine. The difference
omit map for this residue (Fig. 3) shows density for either a
valine or a threonine. The proximity of the threonine hydroxyl group to
a carbonyl atom and to a neighboring water molecule, permitting the
formation of hydrogen bonds, imposes the assignment of the latter
residue type. A few other instances were encountered where the side
chain density was very diffuse or absent for some of the terminal side
chain atoms. Whether this is due to disorder or additional sequencing
errors could not be decided, especially since these side chains belong
to large, charged amino acids, like Lys, which are situated at the
surface of the protein.
Figure 2:
Phe(B10) in its electron
density map ((2
F
-
F
)
) contoured at 2.0 standard deviations. The
contribution of this residue for the phase calculation was
omitted.
Figure 3:
Thr(B8) in its electron
density map ((2
F
-
F
)
) contoured at 1.5 standard deviations. The
contribution of this residue for the phase calculation was
omitted.
Figure 4:
Ribbon plot showing the fold of Asian
elephant myoglobin. -Helices are represented by spirals and labeled with uppercase letters. This plot was created
using the program MOLSCRIPT(43) .
Figure 5:
C stereo plot of EmMbCN (thick lines) superimposed with SWMb (thin
lines).
Figure 6: B-factor distribution of Asian elephant cyano-metmyoglobin. Presented are the mean isotropic temperature factors for the main chain atoms and for all atoms of a residue.
Figure 7:
Electron density map ((2
F
-
F
)
) of the heme pocket of Asian elephant
myoglobin. Gln
(E7) forms a hydrogen bond to the carbon
atom of the CN ligand. The ligand is 3.5 Å away from
Phe
(B10).
Despite the two
amino acid replacements (H64Q and L29F) in the heme pocket, the
conformational changes are small if compared to aquo-met-SWMb. High
structural similarity is found for those residues which are identical (Fig. 8). Even in the case of the distal residue 64 (E7) where a
glutamate substitutes the commonly found histidine, the length of the
hydrogen bonds formed by the N2 atom and the N or O
counteratom, respectively, of the ligand differs only marginally (2.87
Å versus 2.78 Å). Therefore, it appears that His
and Gln stabilize the bound ligand in a similar manner. On the other
hand, Lys
(CD3) in EmMbCN being replaced by an Arg in SWMb
forms a hydrogen bond between its N
atom and the
O
1 atom of the distal residue Gln
(E7). No
hydrogen bond between Arg
(CD3) and His
(E7) is
found in SWMb. Residue Arg
(CD3) in the carbonmonoxy sperm
whale myoglobin x-ray structure shows two conformational
states(34) . We did not detect any evidence for an additional
conformation of the Lys
(CD3) side chain in the electron
density.
Figure 8:
Stereo picture of the superposition of the
heme pocket of Asian elephant cyano-metmyoglobin (thick lines)
and sperm whale aquo-metmyoglobin (thin lines). The distance
between the N2 atom of the distal residue 64 (E7) and
the ligand is very similar. Residue 45 (CD3) being a lysine in EmMbCN
forms a hydrogen bond (dashed line) to atom O
1
of the distal residue. Arg
(CD3) in SWMb instead forms a
hydrogen bond (dashed line) to a heme propionate
group.
The most significant finding in our crystal structure
concerns the amino acid replacement of Leu at position 29 (B10) by a
phenylalanine which causes drastic steric restraints on the movement of
the ligand. This residue is situated in a hydrophobic patch formed by
Phe(B10), Leu
(B13), Phe
(B14),
Leu
(BC5), Phe
(CD1), Phe
(CD4),
Leu
(E4), Val
(E11), and Ile
(G8) (Fig. 9). The side chain of Phe
(B10) penetrates
into the distal side of the heme pocket approaching the CN ligand up to
3.5 Å. This situation causes the crevice to be more crowded than
in myoglobins having a leucine at position 29 (B10).
Figure 9:
Stereo picture of the heme pocket in Asian
elephant myoglobin. The hydrophobic residues forming the upper part of
the distal side are depicted. Phe(B10) penetrates further
into the heme pocket than leucine which is usually found at this
position in myoglobins.
A comparison
with the carbonmonoxy structure of sperm whale myoglobin (Brookhaven
Protein Data Bank entry 1MBC), where the Fe-CO unit
is iso-structural with the Fe
-CN unit in EmMbCN,
reveals even more structural similarities. The proximal sides show
virtually no differences in the two structures, especially since the
proximal histidines are practically superimposed. On the distal side,
the N
2 atom of Gln
(E7) in EmMbCN has moved
0.9 Å away from the corresponding nitrogen atom of
His
(E7) in SpWCO toward the propionate of the A-pyrrole
and at the same time closer to the heme plane (Fig. 10). Even
so, the combined movement results in an unchanged distance between the
heme iron and the N
2 atom in both structures (Table 2). In addition, the distances between the two
N
2 atoms of the distal residues and the ligand atoms N
or O, respectively, are virtually the same due to the different ligand
bent angles.
Figure 10:
Stereo view of the superposition of the
heme environment of Asian elephant myoglobin (thick lines) and
sperm whale CO myoglobin (thin lines). The N2
atom of residue 64 (E7) has moved with respect to the corresponding
nitrogen position in the CO complex of sperm whale myoglobin. Even so,
the distance to the heme iron remains
unchanged.
A more interesting and appropriate myoglobin structure
for comparison is the double mutant H64Q/L29F of SWMb due to the
presence of the same distal side residues as in EmMbCN. The
carbonmonoxy mutant crystal structure has been determined recently and
was kindly supplied to us by Dr. George Phillips at Rice University,
Houston, TX. Zhao et al. (44) describe some functional
and spectroscopic properties of the double mutant and of the two single
mutants (L29F and H64Q) and compare them with those of the wild type
and EMb. Residues Gln(E7) and Phe
(B10) have
been tested earlier by site-directed mutagenesis for their effects on
ligand binding and
autooxidation(11, 14, 35, 36) . The
models of EmMbCN and of the double mutant were superimposed revealing a
very similar structure. The two Phe
(B10) practically lie
on top of each other while the Gln
(E7) residues show
marginal differences in their positions. In both myoglobin structures,
the tilt angles of the ligands are very much alike (Table 2). The
bend angle is smaller in the H64Q/L29F model with respect to the
situation in EmMbCN reducing the distance to Phe
(B10) in
the former structure.
Of special interest regarding ligand binding
and dissociation is the recently published crystal structure of the
photolyzed carbonmonoxy-SWMb at 40 K(16) . This structure (the
coordinates of the CO-photolyzed, CO-ligated, and deoxy-SWMb structures
at 40 K were kindly provided to us by Joel Berendzen, Los Alamos
National Laboratory, Los Alamos, NM) allowed us to investigate the
relative position of Phe(B10) in EmMbCN with respect to a
fictitious dissociated ligand as in sperm whale myoglobin. Fig. 11depicts a space-filling model of the photodissociated
carbonmonoxy ligand of SWMb in the EmMbCN environment which shows the
dissociated ligand colliding with the side chain of
Phe
(B10). This indicates that, besides electrostatic
effects on the Fe-C bond, an additional steric interaction, introduced
by Phe
(B10), influences ligand binding. Particularly
relevant in this respect are the results of photolysis measurements and
molecular dynamic simulations performed by Gibson et al.(37) on mutagenized SWMb at position 29 (B10). They show
that this residue strongly influences geminate ligand recombination.
Figure 11:
Stereo picture of a space-filling model
of the photodissociated carbonmonoxy ligand of sperm whale myoglobin in
the environment of Asian elephant myoglobin. The fictitious CO ligand
is colored in cyan. The dissociating CO ligand collides with
the side chain of Phe(B10).
The originally reported primary sequence of Asian elephant
myoglobin by Dene et al.(8) has caused some confusion
about the structure-function relationships concerning the distal side
residues of myoglobins. Despite the replacement of the essential His
residue at position 64 (E7) by a Gln, this protein displays essentially
unchanged O binding and autooxidation behavior. Based on
the assumption that only this single substitution has taken place on
the distal side and the fact that a H64Q point mutant of sperm whale
myoglobin exhibits much lower oxygen affinity and greater
susceptibility against autooxidation, the findings above were not
explainable.
H NMR investigations of the heme cavity (12) have been interpreted in such a way that even a dramatic
rearrangement of the CD corner, a movement which brings the conserved
residue Phe
(CD4) in a position only about 5 Å away
from the heme iron, has been postulated. Other investigations,
including site-directed mutations on sperm whale myoglobin, have been
undertaken which finally led to the assumption that a second, so far
unidentified, amino acid replacement had to be considered, but a
definite answer could not been given. All these attempts to explain the
peculiar functional properties of EMb were obviously not sufficient to
construct a sound picture of the distal side pocket.
The crystal structure of Asian elephant myoglobin was determined to resolve this confusion. Recently, the presence of an additional phenylalanine has been suspected based on NMR data (13) which has been confirmed by our work. The assumption that introducing an additional large hydrophobic side chain into the active site causes more crowding was confirmed by our structure, but not the assumed structural perturbation of the standard globin fold(12) . Superpositioning of the EmMbCN model with other myoglobins reveals only minor alterations of the backbone. These deviations appear to be structurally negligible and are probably caused by different crystal contacts(38) . It appears that individual ligand binding properties are the result of local amino acid substitutions rather than a modulated fold in EMb.
The CN ligand in EmMbCN possesses an almost perpendicular
orientation with respect to the heme plane, whereas the bend angles (Table 2) of the CO ligands in the wild type SWMb and the
H64Q/L29F double mutant are nearly identical and deviate significantly
from 180°, suggesting that its orientation is directed by the
chemical nature of the ligand. Furthermore, the comparison between the
elephant myoglobin and the double mutant allows us to draw the
conclusion that the different position of the residue 64 (E7)
N2 atoms in EmMbCN and SWMbCO are not caused by the
hydrogen bond between Gln
(E7) and Lys
(CD3).
We report here on the first cyano-met complex of a native myoglobin
solved by x-ray crystallography. The unfavorable contact between the CN
ligand and the highly conserved Val(E10) is imposed by the
almost perpendicular orientation of the ligand with respect to the heme
plane. Such a close contact is not found in the CO and O
complex of sperm whale myoglobins which might indicate that the
role of residue 68 (E10) is to discriminate between various ligands.
On the distal side, the additional Phe residue could be located at position 29 (E10) as unequivocally interpreted from its corresponding electron density. This residue penetrates into the distal side, pointing toward the heme iron which causes crowding, therefore hindering ligand access.
Interestingly enough, a situation
remarkably similar to that observed at cryogenic temperatures in the
time courses for CO recombination to EMb after photolysis, is also
present in lignin peroxidase, where again a Phe residue is present at
the distal site, in van der Waals contact with the bound
ligand(7, 41) . Further support to the above
interpretation of the low temperature CO binding kinetics comes from
the results obtained by Gibson et al.(37) on SWMb
mutants in which Leu(B10) is replaced by Ala, Val, or Phe.
Based on laser photolysis measurements of the NO and O
derivatives on picosecond and nanosecond time scales, these
authors have shown that the extent and rate of geminate ligand
recombination is determined by the size of residue 29 (B10).
Furthermore, as recently directly observed by x-ray crystallography at
40 K (16) and depicted in Fig. 11, the molecular dynamic
simulations (37) show that immediately after photolysis the
ligand moves toward residue 29 (B10). This implies that motion of the
photolyzed ligand away and toward the heme iron is determined by this
residue.
The structure determination of Asian elephant myoglobin has clarified the situation of the heme pocket in this peculiar protein which has caused much confusion in the past. Obviously, nature is able to achieve solutions for a given biological problem by different approaches. It appears that even a conserved amino acid composition in an active site environment does not have to be the ultimate solution to achieve a certain vital protein function. An interesting result of this study is also that various chemical changes do not have necessarily the effect of overall structural alterations. Rather, the combination of several local chemical modifications leaves the functional properties of a protein conserved.
The atomic coordinates and structure factors of EmMbCN (code 1EMY) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.