(Received for publication, July 3, 1996, and in revised form, October 28, 1996)
From the Department of Chemistry, University of
California, Davis, California 95616, the § Department of
Biochemistry, University of Antwerp (UIA), B-2610 Antwerp, Belgium,
the ¶ Department of Zoology, A.M. University, Aligarh, India
202002, and the
Department of Biochemistry, School of Medicine,
Wayne State University, Detroit, Michigan 48201
A two-dimensional 1H NMR study has been carried out on the heme cavity of the extreme oxygen-avid and autoxidation-resistant oxy-myoglobin complex from the trematode Paramphistomum epiclitum, and the residues were identified which potentially provide hydrogen bond stabilization for the bound oxygen. Complete assignment of the heme core resonances allows the identification of 10 key heme pocket residues, 4 Phe, 4 Tyr, and 2 upfield ring current aliphatic side chains. Based solely on the conserved myoglobin folding topology that places the E helix-heme crossover and the completely conserved Phe(CD1)-heme contact at opposing meso positions, the heme orientation in the cavity and the E helix alignment were unambiguously established that place Tyr66 at position E7. Moreover, all eight aromatic and the two aliphatic side chains were shown to occupy the positions in the heme cavity predicted by amino acid sequence alignment with globins of known tertiary structure. The dipolar contacts for the Tyr32(B10) and Tyr66(E7) rings indicate that both residues are oriented into the heme cavity, which is unprecedented in globins. The ring hydroxyl protons for both Tyr are close to each other and in a position to provide hydrogen bonds to the coordinated oxygen, as supported by strong retardation of their exchange rate with bulk solvent. A more crowded and compact structure increases the dynamic stability of the distal pocket and may contribute to the autoxidation resistance of this myoglobin.
Myoglobin and hemoglobin are oxygen binding proteins that reflect extraordinary structural homology in spite of often very limited sequence homology (1, 2). Both are composed of ~150-residue globular proteins containing eight helices (labeled A-H) and an iron-protoporphyrin-IX (heme) bound to the completely conserved His(F8); the only other completely conserved residue is Phe(CD1). Among the vertebrate Hb1s/Mbs, the strongly conserved His, but also Gln, provide the crucial hydrogen bond to stabilize the bound O2 (3-6). Identified natural human Hb mutants with Tyr at position E7 do not bind oxygen but convert to the Met or ferric form under physiological conditions (7). Hbs and Mbs from invertebrates exhibit a much broader range of functionality and show key substitutions at position E7 that are unknown among vertebrates and provide hydrogen bonds to the bound O2 via residues at positions other than E7 (2). Prominent among these globins are the Mb from the sea hare Aplysia limacina, which, with a Val(E7), provides the key hydrogen bond via Arg(E10) (8), and the parasitic trematode, Ascaris suum, HbI, which provides two hydrogen bonds to the bound O2 via a relatively common Gln(E7) and an uncommon Tyr(B10) (9, 10). Tyr(B10) is present in several other trematode Hbs/Mbs (11, 12), among others (13-15).
The Tyr(B10) hydrogen bond has been identified as the source of the extra stabilization that leads to extreme O2 avidity in A. suum HbI through primarily an extremely slow O2 off-rate (9, 12); the autoxidizability is similar to that of common Mbs/Hbs. The Tyr(B10) is present in Lucina pectinata Hbs (13, 14), A. suum Mb(12), and legume Hb (16, 17) but does not result in similarly slow O2 off-rates and likely does not provide a hydrogen bond to the O2. 1H NMR studies of the nematode Dicrocoelium dendriticum Hb had identified a Tyr hydrogen-bonded to the bound ligand (18, 19). Hence it is clear that Tyr at position B10, and possibly E7, can strongly stabilize the Fe-O2 bond in some cases but not in others. Engineering a Tyr(B10) and Gln(E7) into sperm whale Mb to mimic A. suum HbI failed to reproduce the high oxygen affinity for the latter Hb but demonstrated that Tyr(B10) provides H bond stabilization at the bound O2 (20).
Alignment of the amino acid sequence of the globin from the trematode
Paramphistomum epiclitum clearly reveals Tyr at position B10
and places, unprecedented, Tyr66 rather than
His65 at position E7 (21), as shown in Fig.
1. P. epiclitum Mb has a high oxygen affinity created by a
high "on-rate" and is rather resistant to
autoxidation.2 In order to shed light on
the nature of the active site in this unusual P. epiclitum
MbO2 complex, unequivocally establish the alignment of the
E helix, and determine the potential role of Tyr(E7) and Tyr(B10)
interacting with bound oxygen, we report herein on a solution
1H NMR study of its heme cavity. Our interests are to
assess the degree to which homologous residues on the various helices
make contact with the heme when compared with A. suum HbI
(9, 10) or sperm whale Mb crystal structures (22) and as suggested by the molecular modeling (21). The limited availability of the protein
dictated that a choice had to be made between sample concentration and
sample purity for the desired two-dimensional 1H NMR study.
It was concluded that the needed ~1.5 mM concentration was possible only for a sample ~60% pure, with the remainder one or
more of the minor Mb isoforms. This sample heterogeneity limits the
scope of the study but not the details to which the positions of the
identified heme cavity residues can be placed relative to each other
and to the heme.
P. epiclitum (Platyhel-minthes, Trematoda, Paramphistomatidae) Mb was prepared by ammonium sulfate precipitation and gel filtration chromatography as described elsewhere (21). The resulting preparation is homogeneous in Mr as evidenced by SDS-polyacrylamide gel electrophoresis and equilibrium centrifugation. Separation by isoelectric focusing under native conditions reveals two major isoforms differing in less than 0.1 pH unit which could not be purified further. Isoelectric focusing under denaturing conditions reveals four different globin isoforms. It is not known if these are real isoforms, coded by different genes, or if they are the result of post-translational modifications or artificial modifications during the isolation process. Due to its high oxygen avidity, the purified protein remains in the oxy-Mb form as shown by spectral analysis (21). The final oxy-Mb sample contained ~1.5 mM total heme concentration in 50 mM NaCl and 50 mM phosphate buffer at pH 7.1. The 1H2O solution was subsequently converted to 2H2O solution using an Amicon ultrafiltration cell. Solution pH was adjusted with NaO1H (NaO2H) or 1HCl (2HCl) solution.
NMR Spectra1H NMR data were collected on a GE
Omega-500 spectrometer operating at 500 MHz for protein samples in both
1H2O and 2H2O. The
observed chemical shifts are referenced to
2,2-dimethyl-2-silapentane-5-sulfonate (DSS) via the solvent signal.
Solvent suppression, when necessary, was achieved by direct saturation
during the relaxation delay. Phase-sensitive TOCSY (23, 24) and NOESY
(25) were collected (512 t1 × 2048 t2) at 30 °C in order to identify scalar and
dipolar connectivities among heme and amino acid residues. Spectral
widths of 8.0 kHz and mixing times of 35 ms for TOCSY and 150 ms for NOESY were used. The 90° pulse width was ~8 µs (~25 µs for
TOCSY). 128 scans were collected for each block with a repetition time of 1.2 s. Two-dimensional data sets were processed by FELIX
software on a Silicon Graphics work station. Both NOESY and TOCSY
spectra were processed by a 15°-shifted sine bell-squared apodization and zero-filled to 2048 × 2048 points prior to Fourier
transformation.
The resolved portions of the 500 MHz
1H NMR spectrum of P. epiclitum MbO2
in 90% 1H2O, 10% 2H2O
at 30 °C, pH 7.1, are shown in Fig. 2. Inspection of
both the low field and high field resolved portions of the spectrum reveals a molecular heterogeneity indicative of two obvious compounds, in relative ratio ~2:3. In the low field spectrum, the three likely meso-H candidates differ in intensity with the extreme low field peak
both weaker and significantly broader than the other two equal
intensity resolved low field peaks. Similarly, the high field portion
of the spectrum reveals several ring-current shifted peaks with
intensity for methyl groups for both the major and minor components.
Contributions from signals from both components in the composite peak
centered at 0.8 ppm are partially resolved at 38 °C (Fig.
2B). Our interest is in the peak assignment and structure
elucidation of solely the major component whose residue origin in Fig.
2 are labeled by the one-letter amino acid code with the deduced
helical positions given in parentheses. Resonances for any minor
component are simply labeled X1; the possible
relationship of the minor to the major component will be addressed
briefly below. The determination of the alignment of the E helix and
nature of the interaction of distal residues with the bound ligand are pursued by identifying contacts to the heme, with emphasis on resolved
aliphatic residue peaks in the ring-current upfield spectral window and
the aromatic side chains, each of which can be readily identified by
highly characteristic TOCSY cross-peak patterns even in an impure
sample. To guarantee that all assignments relate solely to the major
isomer of interest, we report assignments only when we observe multiple
dipolar contacts between pairs of residues or between a residue and two
or more heme substituents.
Heme Assignment
The use of the heme as a "template" on
which to place the upfield aliphatic and low field aromatic side chains
first requires unambiguous assignment of the heme resonances. The low
field 8-11 ppm portion of the NOESY spectrum in Fig. 3
reveals four nonlabile proton peaks which fail to exhibit TOCSY peaks
and hence must arise from meso-Hs; two are resolved (the heme positions
are labeled in Fig. 4). The four meso-Hs exhibit strong
NOESY cross-peaks to two, one, one, and no likely heme-methyls in the
2.5-3.8 ppm window (Fig. 3B) which also fail to exhibit
TOCSY cross-peaks. These dipolar contacts therefore differentiate the
-meso-H (nuclear Overhauser effects to 1-CH3 and
8-CH3),
-meso-H (no heme methyl neighbor), and the pair
-meso-H,
-meso-H (nuclear Overhauser effect to 3-CH3
or 5-CH3). TOCSY identifies two AMX spin systems at low
field (Fig. 5A) with chemical shift and
spin-coupling patterns diagnostic of two vinyl groups in a diamagnetic
protein. NOESY cross-peaks from one vinyl to one of the
1-CH3, 8-CH3 pair identify 1-CH3
and the 2-vinyl group and, by inference, 8-CH3 and the
4-vinyl group (Fig. 5B). The NOESY cross-peaks of the
2-vinyl group to a meso-H differentiate
-meso-H from
-meso-H and
identify both the 3-CH3 and 5-CH3 (Fig.
5B) The remaining
-meso-H exhibits NOESY cross-peaks to
two sets of spin-coupled protons near 3.5 ppm (Fig. 5B)
which exhibit NOESY cross-peaks to 5-CH3 or
8-CH3. These identify the H
s for the two
propionate groups and complete the heme ring core substituent
assignment in a unique and self-consistent manner (26, 27) that
guarantees that the major component alone is being characterized. The
chemical shifts for assigned heme resonances are listed in Table
I.
|
Assuming the basic Mb fold and a heme
orientation about the ,
-meso-H axis for P. epiclitum
Mb that is the same as in A. suum HbI (9, 28) and sperm
whale Mb (22), the sequence alignment and homology model (21) predict
the following for aromatic side chain contacts to the heme:
Tyr94(F4) with pyrrole A, Phe108(G5),
Phe115(G12) with the pyrrole A/B junction,
Tyr42(C4) with pyrrole B, Phe46(CD1) with
pyrrole C, and, if oriented into the heme pocket, Tyr66(E7)
with the pyrrole C/D junction. In addition, Tyr32(B10) and
Phe36(B14) are expected to contact Phe46(CD1).
Lastly, the upfield shifted E helix position residues
Leu70(E11) and Ala73(E14) are expected to make
contact to the pyrrole A/B junction. The expected dispositions relative
to the heme for the eight aromatic side chains, Leu70(E11),
and Ala73(E14) is shown schematically in Fig. 4. It is to
be noted that, although the assignments for amino acid residues and
their placement near the heme and each other are presented for
simplicity as confirmations of aspects of the homology model (21), the
conclusion on the E helix alignment and conserved folding topology are,
in fact, determined independently by 1H NMR data.
The TOCSY spectrum (Fig. 6A) for the upfield
methyl peak at 1.1 ppm reveals its origin as a Leu, which has dipolar
contacts to the heme 1-CH3 (Fig. 6B), 2-vinyl
(Fig. 5D), and
-meso-H (Figs. 3C). The TOCSY
map (Fig. 6A) also identifies a complete Ala spin system
with similarly strong NOESY cross-peaks to the heme 1-CH3, 8-CH3 (Fig. 6B), and
-meso-H (not shown). The
upfield ring current shifts and contacts to the heme 1-CH3
and 8-CH3 are characteristic of Leu70(E11) and
Ala73(E14) in the normal Mb fold if the heme is oriented
like in sperm whale Mb (22) or A. suum Hb (9,
28). While the peptide NHs for both residues could be located, the
small chemical shift dispersion and the resultant spectral congestion
precluded tracing the backbone to uniquely elucidate the sequence
origin of the two residues; however, the expected NOESY cross-peaks
from Ala(E14) C
H3 to Leu(E11)
C
H and C
H3 (Fig.
6B) confirms their i, i + 3 positions on a helix.
The TOCSY spectrum in Fig. 5A for the aromatic spectral
window exhibits all the cross-peaks for four three-spin (rotationally averaged Phe rings) and four two-spin (rotationally averaged Tyr rings), all of which can be demonstrated to arise from the major isomer
for which the heme has been assigned. NOESY cross-peaks to both
5-CH3 (Fig. 5C) and -meso-H (Fig.
3A) for two ring protons identify Phe46(CD1),
whereas the two ring protons of the other Phe rings yield NOESY
cross-peaks to
-meso-H (Fig. 3A) 3-CH3 (Fig.
5C), and
-meso-H (Fig. 3A) and 2-vinyl (Fig.
5B), thereby uniquely identifying Phe115(G12)
and Phe108(G5). Both protons of two Tyr rings exhibit NOESY
cross-peaks to 3-CH3 (Fig. 5C), 4-vinyl (Fig.
5B), and 1-CH3 (Fig. 5C), identifying Tyr42(C4) and Tyr94(F4), respectively. Both
protons of one of the two remaining Tyr rings fail to exhibit NOESY
cross-peaks to any heme substituent but display such cross-peaks to the
Phe46(CD1) ring (Fig. 5B) and
Leu70(E11) methyls (Fig. 5D) which uniquely
label it as Tyr32(B10). The remaining Phe ring exhibits
NOESY cross-peaks to both the Phe46(CD1) and
Tyr32(B10) rings (Fig. 5B), as is characteristic
of Phe36(B14). The remaining Tyr ring exhibits NOESY
cross-peaks to both the heme 6-H
s (not shown) and
Leu70(E11) methyls (Fig. 5D), and hence must
arise from Tyr66(E7). Other expected and observed intra
residue cross-peaks are between Tyr94(F4) and
Ala73(E14) (not shown) and Tyr32(B10) ring and
Leu70(E11) methyls (Fig. 5D). Important
cross-peaks are also observed between both Phe108(G5) and
Phe115(G12) ring protons and the Leu70(E11)
methyls (Fig. 5D). The intra-ring TOCSY/NOESY cross-peaks for the Phe46(CD1) ring are significantly weaker than for
the other Phe rings (Fig. 5, A and B), which can
be traced to a significantly larger line width for the partially
resolved two proton H
peak (Fig. 2A); the
peak is much narrower at elevated temperature (Fig. 2B), indicating that this residue is exhibiting line broadening due to
relatively slow flipping of the aromatic ring (29).
Comparison of NOESY maps in 1H2O and
2H2O reveals the presence of two labile protons
with a weak NOESY cross-peak between them (Fig. 5E), for
which the absence of any TOCSY cross-peaks indicates that they likely
arise from two Tyr ring hydroxyls (labeled OH1 and OH2 in Fig. 4). OH1
exhibits moderate intensity NOESY cross-peaks to the rings of
Tyr32(B10) and Tyr66(E7), as well as
cross-peaks to the ring (CH) of Phe46(CD1)
(Fig. 5B), and to a C
H3 of
Leu70(E11) (Fig. 5D), as shown schematically in
Fig. 4. OH2 exhibits moderate intensity NOESY cross-peaks to both the
ring of Tyr66(E7) (Fig. 5E) and the
C
H3s of Leu70(E11) (Fig.
5E). These NOESY patterns identify OH1 and OH2 as the side
chain hydroxyl protons of Tyr32(B10) and
Tyr66(E7), respectively, and places both labile protons
well within the heme cavity and in the vicinity of the ligand. The
hydroxyl protons of Tyr94(F4) and Tyr42(C4)
could not be located, either because of spectral congestion and/or
rapid exchange with solvent. The absence of detectable magnetization
transfer to the Tyr32(B10) and Tyr66(E7)
hydroxyl protons upon saturating the solvent resonance dictates that
the labile proton lifetime,
OH, are much longer (by
>10) than the expected T1 for the resonances
(~250 ms), leading to
OH
2 s at pH 7.0 (29).
The positions of the 10 identified heme pocket residues relative to the
heme and to each other and the observed NOESY cross-peak patterns are
depicted schematically in Fig. 4, and the chemical shifts are listed in
Table II.
|
Sufficient assignments are pursued in
order to establish whether the minor component (~40%) arises from
the heme reoriented 180° about the ,
-meso axis (30, 31), from
another polypeptide chain (isoform), or from damage or modification
during isolation. The upfield TOCSY map reveals three minor component
methyl peaks (labeled X2,
X3, and X4 in Fig.
2A) which exhibit spin topology indicative of three
CH2-CH3 fragments (Fig. 6A) of three
different Ile, two of which exhibit NOESY cross-peaks between each
other (Fig. 6B), to the low-field resolved meso-H (Fig.
3C) and its adjacent methyl, as well as to an aromatic side
chain (not shown). The presence of upfield Leu signals for the major
component and Ile signals for the minor component in solution indicates
that the heterogeneity is due to the presence of two different
polypeptide chains and not due to either heme orientational isomerisms
or damage to the major component. Lastly, the significantly broader resolved meso-H signal and strong intensity of NOESY cross-peaks for
the minor relative to the major component suggests that the minor
component may be oligomeric. Assignments were not pursued further for
the minor component.
The unique assignment of the heme for the major isoform of P. epiclitum MbO2, together with the observation of all aromatic ring contacts to the heme and among each other, as expected by amino acid sequence homology with globins of known tertiary structure, provide direct evidence that the heme pocket architecture is very similar to that of other typical Mbs/Hbs. These data also establish both the alignment of the E helix as proposed (21), with a Tyr66 at position E7, and the orientation of the heme as found in sperm whale Mb and A. suum HbI. The residues Tyr32(B10), Phe36(B14), Tyr42(C4), Phe46(CD1), Leu70(E11), Ala73(E14), Tyr94(F4), and Phe108(G5) generally occupy the same positions in the heme pocket as do the homologous residues in other globins. The position of Tyr32(B10) over the heme is supported by both its upfield ring current shifted side chain protons and its NOESY pattern to surrounding residues. The Tyr32(B10) ring labile proton also exhibits NOESY cross-peaks to a series of peripheral residues (Phe46(CD1), Leu70(E11)) that both confirm its position and support the presence of a hydrogen bond between Tyr32(B10) and the bound oxygen. The present failure to detect the Tyr94(F4) hydroxyl proton is consistent with the predicted orientation (21) of this ring toward the surface rather than in the pocket and parallel to the His98(F8) as found for Phe94(F4) in Aplysia limacina Mb (8).
The computer model suggests two alternate orientations of Tyr66(E7), one turned out and another turned into the heme pocket (21) within hydrogen-bonding distance of the bound oxygen and in contact with both Phe115(G12) and Leu70(E11). The proposed sequence alignment for the E helix and the present confirmation by 1H NMR place an unprecedented Tyr at position E7 but do not answer whether it is oriented into the pocket or into solution. The monomeric Chironomus Hbs possess a His(E7), but it is oriented into solution in all functional forms (32). Strong and moderate intensity NOESY cross-peaks from the Tyr66(E7) ring to the Leu70(E11) methyls (Fig. 5D), one of which is close to the heme 1-CH3 and both of which exhibit strong NOESY cross-peaks to the Phe115(G12) ring over the pyrrole A/B junction (Fig. 5D), clearly place the Tyr66(E7) ring well within the distal heme pocket. The NOESY cross-peaks from the Tyr66(E7) hydroxyl proton to the Leu70(E11) methyls and the Tyr32(B10) hydroxyl proton place it in a position where it could provide the second hydrogen bond to the bound oxygen, as well as form a hydrogen bond to the Tyr(B10) hydroxyl oxygen to stabilize the optimal orientations of both Tyr32(B10) and Tyr66(E7) for hydrogen bonding to O2. The role of both Tyr32(B10) and Tyr66(E7) hydroxyl protons in forming hydrogen bonds to the ligand is independently supported by the very slow exchange rate with bulk water, with estimated lifetime ~2 s at pH 7.1, some 104 slower than for a free Tyr at this pH (29). This is the first case where a Tyr is demonstrated not only to occupy position E7 but to orient into the heme pocket of a functional globin in a manner common to His(E7) in mammalian Mbs and Hbs.
The present study, however, does not shed much light on the
extraordinary resistance of P. epiclitum MbO2 to
autoxidation when compared with Hbs of other nematodes such as
Isoparorchis hypsolbargi, (21) and nematodes such as
A. suum (9). Autoxidation of Mb is strongly dependent on
hydrogen bonding to the bound ligand by the distal residue and the size
and hydrophobicity of the distal side of the heme pocket (5). NMR data
on P. epiclitum MbO2 indicate the formation of
two hydrogen bonds by Tyr32(B10) and Tyr66(E7)
to the bound oxygen and establish the presence of an unprecedented number of bulky hydrophobic residues in the crowded distal heme pocket
(21). A more crowded distal pocket for P. epiclitum Mb than
other Mbs, Hbs is supported by the observed line broadening of the
Phe46(CD1) ring protons. In spite of its close proximity to
the heme, 1H NMR has shown that this highly conserved
aromatic ring undergoes rapid 180° ring reorientation that completely
averages the environments of the two H (and two
H
) ring protons in common Mbs and Hbs (27, 33, 34). The
significant broadening of the Phe46(CD1) H
resonance (Fig. 2,A and B) (and cross-peaks; Fig. 5B) in P. epiclitum MbO2 is
indicative of slow ring reorientation and suggests a more compact and
dynamically more stable distal pocket than in other NMR characterized
Hbs and Mbs, which likely contribute to the resistance to
autoxidation.
It is clear, however, that the presence of certain residues in the heme pocket (i.e. Tyr(B10)) is insufficient information for predicting oxygen binding and autoxidation resistance. This is illustrated by the A. suum Mb and A. suum HbI, both with Tyr(B10) and Glu(E7), but with differences of a factor 50 in koff for oxygen (9, 10, 12), and by the P. epiclitum and I. hypselobargi Mbs, which exhibit very different autoxidation rates in spite of both possessing Tyr(B10) and Tyr(E7) (21). Indeed, the complete tertiary structure is involved in fine tuning the orientations of residues to their critical positions. Crystallization experiments with different trematode Mbs are in progress.
K. A. R. thanks the University of Antwerp for a postdoctoral fellowship.