From the Department of Chemistry, University of
California, Davis, California 95616 and the ¶ Department of
Physiology and Biophysics, Case Western Reserve University School of
Medicine, Cleveland, Ohio 44106-4970 and ** Institute of
Multidisciplinary Research for Advanced Materials, Tohoku University,
Katahura, Aoba-ku, Sendai 980-8577, Japan
Received for publication, November 4, 2002, and in revised form, December 9, 2002
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ABSTRACT |
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The molecular structure and dynamic properties of
the active site environment of HmuO, a heme oxygenase (HO) from the
pathogenic bacterium Corynebacterium diphtheriae, have been
investigated by 1H NMR spectroscopy using the human HO
(hHO) complex as a homology model. It is demonstrated that not only the
spatial contacts among residues and between residues and heme, but the
magnetic axes that can be related to the direction and magnitude of the
steric tilt of the FeCN unit are strongly conserved in the two HO
complexes. The results indicate that very similar contributions of
steric blockage of several meso positions and steric tilt
of the attacking ligand are operative. A distal H-bond network that
involves numerous very strong H-bonds and immobilized water molecules
is identified in HmuO that is analogous to that previously identified
in hHO (Li, Y., Syvitski, R. T., Auclair, K., Wilks, A., Ortiz de
Montellano, P. R., and La Mar, G. N. (2002) J. Biol. Chem. 277, 33018-33031). The NMR results are completely
consistent with the very recent crystal structure of the
HmuO·substrate complex. The H-bond network/ordered water molecules
are proposed to orient the distal water molecule near the catalytically
key Asp136 (Asp140 in hHO) that stabilizes the
hydroperoxy intermediate. The dynamic stability of this H-bond network
in HmuO is significantly greater than in hHO and may account for the
slower catalytic rate in bacterial HO compared with mammalian
HO.
Heme oxygenase (HO)1 is
an The remarkable recent progress in understanding the functional
properties of HO based on mutagenesis and spectroscopic studies (3, 4,
12-14), of a slightly truncated, soluble, and completely active
recombinant mammalian HO, has been considerably enhanced by the
successful x-ray crystallographic characterization of the substrate
complexes of first human HO (hHO), followed by rat HO (15, 16). These
structures shed light on a key determinant of the
Solution 1H NMR characterization of hHO and its substrate
complex has contributed to the understanding of the structure/function relationship of HO (18-22). An annoying, but functionally irrelevant property of the mammalian HOs is that binding of the native substrate, protohemin (PH; R = vinyl in Fig. 1), leads to ~1:1
orientational isomerism about the Thus, both the position of the distal helix in blocking access to other
meso positions (15, 16) and its influence on tilting (19-21, 24) the axial ligand toward the
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical enzyme that carries out the highly stereoselective
conversion of hemin to
-biliverdin, iron, and CO, excising CO from
exclusively the
-meso position (1). In contrast to the
better understood heme peroxidases and cytochromes P450, which pass
through the common ferryl intermediate, the reactive form of HO is a
ferric hydroperoxy intermediate (2-4). In mammals, the ~300-residue
membrane-bound enzyme occurs as an inducible HO-1, whose primary roles
are iron homeostasis and heme catabolism (5, 6), whereas the
constitutive HO-2 has been proposed (7) to generate CO as a neural
messenger. In higher plants, algae, and cyanobacteria, HO generates the
open tetrapyrroles as light-harvesting pigments (8). HO has also been
identified in several pathogenic bacteria, where its role appears to be
the essential "mining" of iron from hemes in the host (9, 10). Plant and bacterial HOs are soluble and somewhat shorter (~200 residues) (9, 10) than mammalian HO (11). Among the characterized bacterial HOs, sequence homology to the more extensively studied mammalian HO varies from relative high (33% sequence identity/70% similarity) for HmuO from Corynebacterium diphtheriae (10)
to low (<25%) for HemO from Neisseria meningitides
(9).
-stereoselectivity, in that the distal helix covers the heme so as
to sterically completely block access to the
- and
-meso positions and partially block access to the
-meso positions (15-17). Although no distal residue that
would stabilize the hydroperoxy unit could be identified, the
occurrence in the crystal of a localized water molecule H-bonded to the
distal helix Asp140 carboxylate, together with the
observation that mutating Asp140 to a non-anionic side
chain abolishes HO activity (12, 14), has led to the proposal that the
water molecule may be sufficiently stabilized in its
crystallographically defined position to serve as the weak H-bond donor
to stabilize the hydroperoxy unit.
/
-axis (18-20), which leads to
spectral congestion and limits both the range and reliability of
structural characterization. Nevertheless, the pattern of dipolar
shifts for the protons on the proximal helix allowed determination of
the orientation of the major magnetic axis, which could be correlated
with a ~20° tilt of the FeCN in the direction of the
-meso position (19, 20). The orientation of ligated azide
in the rat HO·heme·N3 complex confirms such a
steric influence (23).
-meso position
contribute to the stereoselectivity of the reaction. Two-dimensional
1H NMR of a hHO complex with the 2-fold symmetric substrate
2,4-dimethyldeuterohemin (DMDH; R = CH3 in
Fig. 1) (25) allowed sufficiently
definitive and extensive assignments to identify (21) an unusual
distal H-bond networks involving some extremely strong H-bonds (labile proton shifts between 17 and 10 ppm) whose acceptor could be identified in the hHO·PH·H2O crystal structure (15). Moreover, it
was demonstrated that water molecules were in the immediate vicinity
(~3 Å) of each of the strong H-bond donors (22). The strongest of
these H-bonds is between a conserved Tyr58 serving as a
donor to the catalytically critical Asp140. We proposed
that this network has, as one of its primary roles, the stabilization
of the Asp140 side chain and the H-bonded water molecules,
one of which can interact with the heme ligand (4, 21).
View larger version (25K):
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Fig. 1.
Schematic representation of hemin (where
M = methyl, P = propionates, and
R = vinyl (PH) or methyl (DMDH)). The
orientation of the axial His20 ring plane is shown as a
rectangle. The magnetic coordinate system,
x,y,z, is related to the iron-centered
reference coordinate system, x',y',z',
by the Euler angles (
,
,
), where
is the tilt of the
major magnetic axis (z) from the heme normal
(z'),
describes the direction of the tilt in the angle
between the projection of z on the
z',y' axes and the x' axis, and the
rhombic axes (x,y) are related to the reference
x',y' axis by the angle
~
+
.
We report herein on the extension of our 1H NMR
investigation to HmuO, the 216-residue soluble bacterial HO from
C. diphtheriae (10), using hHO as a homology model
(21). Functional (26, 27) and spectroscopic (27, 28) studies, as well
as mutagenesis (29, 30), have confirmed the same mechanism and
stereospecificity as for mammalian HOs, although the turnover rate is
slower (27); the enzyme has been crystallized (31), and the structure
of the substrate hemin complex has been refined to 1.4-Å
resolution.2 Our interests
are to establish the degree to which the available extensive NMR data
on hHO·DMDH·CN (19-22) and the crystal structure of
hHO·PH·H2O (15) can be used to assign the resonances
and to structurally interpret the NMR spectral parameters (32) of HmuO·PH·CN in terms of the orientation of the FeCN vector and the
presence or absence of a distal H-bond network similar to that reported
for hHO·DMDH·CN (21, 22). This 216-residue soluble HmuO enzyme has
His20 as its axial ligand (30) and exhibits extensive
sequence homology to the distal helix and the four fragments of HO
shown (21) to participate in the H-bond network in hHO·DMDH·CN
(Fig. 2). To provide a broader comparison
with the NMR data on hHO complexes (19-22), we explore in parallel
both the disordered HmuO·PH·CN complex and the homogeneous
HmuO·DMDH·CN complex to show that this bacterial HO exhibits
remarkable conservation of the distal steric effects on the axial
ligand and distal H-bond network relative to hHO.
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EXPERIMENTAL PROCEDURES |
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Protein Sample-- HmuO was expressed and purified as reported previously (27). PH was purchased from Sigma. 2,4-Dimethyldeuteroporphyrin was purchased from Mid-Century Chemicals, and the iron was incorporated to yield DMDH by standard procedures (25). PH and DMDH were titrated into apo-hHO to a 1:1 stoichiometry in the presence of a 10-fold molar excess of KCN in a 90% H2O and 10% 2H2O solution buffered at pH 7.4 with 100 mM phosphate. The final concentrations of the HmuO·substrate·CN complexes were ~1.5 mM.
NMR Spectroscopy--
1H NMR data were collected on
a Bruker AVANCE 600 spectrometer operating at 600 MHz. Reference
spectra were collected in both 1H2O and
2H2O over a temperature range of 10-40 °C
at a repetition rate of 1 s1 using a standard one-pulse
sequence with saturation of the water solvent signal. Chemical shifts
are referenced to 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) through
the water resonance calibrated at each temperature. Nonselective
T1 values were determined in both
1H2O and 2H2O at 20, 25, and 30 °C from the initial magnetization recovery of a standard
inversion-recovery pulse sequence. The distance of proton Hi
from the iron, RHi, was
estimated from the relation
RHi = R*Fe(T1*/T1i)1/6,
using the heme for the
-meso-H for H*
(R*Fe = 4.6 Å and T1* = 50 ms) as reference (20, 21, 32). Steady-state NOEs from HmuO·DMDH·CN in 1H2O were recorded with and
without saturation of the solvent resonance for 300 ns using 3:9:19
detection (33). NOESY spectra (mixing time of 40 ms, 10-40 °C) (34)
and Clean-TOCSY spectra (25, 35 °C; spin lock of 15 and 30 ms) (35)
using MLEV-17 (36) were recorded over a bandwidth of 14 kHz (or 28 kHz)
(NOESY) and 14 kHz (TOCSY) with recycle times of 1 s (or 0.33 s) using 512 t1 blocks of 128 and 250 scans, each consisting
of 2048 t2 points. Two-dimensional data sets were processed
using Bruker XWIN software on a Silicon Graphics Indigo work station
and consisted of 30° sine-squared bell apodization in both dimensions
and zero filling to 2048 × 2048 data points prior to Fourier transformation.
Magnetic Axes-- The magnetic axes (Fig. 1) were determined by a least-squares search for the minimum in the error function (21, 32, 37) (Equation 1).
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(Eq. 1) |
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(Eq. 2) |
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(Eq. 3) |
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(Eq. 4) |
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RESULTS |
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The initially assembled HmuO·PH·CN complex exhibits two sets
of hyperfine shifted resonances (data not shown; see Supplemental Material), one set of which loses intensity over several days to yield
an Mi:mi or Hj:hj isomer equilibrium
ratio of ~3:1 (Mi and Hi represent a methyl
and single proton of the major or only equilibrium species,
respectively; and mi and hj reflect a methyl and
hydrogen of the minor equilibrium species, respectively), as
illustrated in Fig. 3B. The
subscript i refers to heme pyrrole substituent
positions 1-8, heme /
-meso positions, or the residue number and proton position. Hence, the substrate is initially bound
disordered about the
/
-meso axis and, like mammalian
HO complexes, equilibrates to a ~3:1 ratio with the more stable heme orientation depicted in Fig. 1 (18-20). This heterogeneity is absent in the complex with the symmetric DMDH substrate (Fig. 3C),
as found previously for hHO·DMDH·CN (21). We will concern ourselves further only with the major isomer of HmuO·PH·CN and the single species HmuO·DMDH·CN.
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The resolved portions of the 600-MHz 1H NMR spectra of
equilibrated hHO·PH·CN and HmuO·PH·CN (19, 20) are compared in
Fig. 3 (A and B, respectively). Similarly, the
traces of HmuO·DMDH·CN and hHO·DMDH·CN (21) are compared in
Fig. 3 (C and D, respectively). The very close
similarity of the pattern of resolved resonances in the hHO and HmuO
complexes is quite apparent. The homologous assignments are connected
by dashed lines between the two hHO and the two HmuO
complexes. The crowded region between 10 and 15 ppm for
HmuO·DMDH·CN in 1H2O is expanded in Fig.
4A. The relevant homologous
portions of the amino acid sequences for the two HOs are illustrated in
Fig. 2. The nonselective T1 values for well
resolved peaks of interest in hHO·DMDH·CN and HmuO·DMDH·CN (as
well as in the PH complexes) are the same. In particular, the low-field
labile proton peaks Gly139 NH and upfield
Ser138 C1H and C
2H exhibit
T1 values indistinguishable from those of
Gly143 NH and Ser142 C
1H and
C
2H (~50, 85, and 50 ms, respectively) reported previously (20, 21).
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Comparison in Fig. 3 of the NMR spectra of the complexes of the two HOs
shows that the patterns of shifts are so similar in the two proteins
that it is highly advantageous to pursue assignments on the basis of
the comprehensive and definitive assignments previously reported for
hHO complexes (20-22). Hence, two-dimensional NMR data are presented
only to define an important distal H-bond network/aromatic cluster as
just recently characterized in hHO. We initially (and trivially) assign
the heme, followed by locating hyperfine shifted protons that arise
from TOCSY-detected side chains placed on sequentially assigned
backbone via the standard Ni-Ni+1, i-Ni+1,
i-Ni+1, ai-Ni+3, and/or
i-
i+3 NOESY connectivities
characteristic of helices (41) as described for hHO (20, 21).
Heme Assignments-- Dipolar contacts were observed in a set of pyrrole substituents on both HmuO·PH·CN and HmuO·DMDH·CN (data not shown) that are identical to those reported in detail for the analogous hHO complexes (21). The remarkably similar hyperfine shifts for a given substituent in the HmuO and hHO complexes are in evidence in the data provided in Table I. The essentially identical shift pattern for the major isomer of the PH substrate is evidence for the same heme orientation in HmuO and hHO (see below).
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Proximal and Distal Helices--
Standard backbone NOESY
connectivities (data not shown; summarized in Fig. 2) among
TOCSY-detected side chains locate two helical fragments (I and II) for
which numerous side chains exhibit moderate-to-large hyperfine shifts.
Fragment I is
Alai-Zi+1-Alai+2-AMXi+3-Zi+4-Zi+5-Alai+6-Zi+7 (Z > four spins; AMX = three-spin system), which is unique for Ala17-Glu24 on the proximal helix. Consistent
with the assignments are the large low-field contact shift for
AMXi+3 of the axial His20
(His25 in hHO), the low-field dipolar shifts for
Ala17 (Lys22 in hHO) and Ala23
(Ala28 in hHO), and the high-field dipolar shifts for
Glu24 (Glu29 in hHO). The NOESY slices through
the 3-CH3 (M3) of HmuO·PH·CN and
hHO·PH·CN are compared in Fig. 5
(A and B), where it is clear that the contacts
are remarkably conserved (21). Although only a part of
Glu24 could be resolved, it exhibits similar shifts and
NOESY cross-peaks to 3-CH3 in the two complexes. The
conservation of the interaction of the proximal helix with the heme is
apparent in the comparison of the NOESY slices through
Ala23 CH3 (M23
;
Ala28 in hHO) of the two PH complexes in Fig. 5
(C and D), which interacts in a similar fashion
with the 2-vinyl/2-CH3 substituents; and the expected
strong Ile211 contacts (21) in hHO (Fig. 5D) are
replaced with a contact by the NH2 group (Fig.
5C) of the homologous His205.
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A moderately relaxed and weakly shifted aromatic ring in close contact
with His20 and 8-CH3 (Phe207 in
hHO) (20, 21) must arise from a similarly placed Phe201.
The failure to detect a NOESY cross-peak between Phe201 and
Ala23, as observed in hHO (20, 21), indicates that
Phe201 is slightly shifted away from Ala23.
Finally, note that 3-CH3 in HmuO·PH·CN exhibits weak
NOESY cross-peaks to Phe208 (which also exhibits
strong NOESY cross-peaks to the 2-vinyl group) (data not shown),
whereas the small Phe214 cross-peak to 3-CH3 is
not seen in hHO·PH·CN (20, 21) (but the strong 2-vinyl cross-peak
to Phe214 is observed). This indicates that the conserved
Phe208/Phe214 at the pyrrole A/B junction is
slightly closer to position 2 in HmuO than in hHO. Moreover, NOESY
cross-peaks of the labile protons for NH2 of
Gln38 in hHO (Fig. 5B) (20, 21) are absent in
the HmuO complex (Fig. 5A), but strong contacts with some
aliphatic protons are present as expected because of the
Gln38 Leu33 replacement in HmuO. The
chemical shifts of the two HmuO complexes, as well as of the two hHO
complexes (20, 21), for these assigned residues are compared in
Table II, where we
also include the predicted dipolar shifts for the residues in the hHO
complex. The observed inter-residue and heme-residue dipolar contacts
are summarized schematically in Fig.
6.
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The NOESY and TOCSY data (data not shown; summarized in Fig. 2)
indicate that helical fragment II is represented by
Vali-AMXi+2-Leui+3-Ni+4-AMXi+5-Nai+6-AMXi+7-Glyi+8 (Fig. 2), where AMXi+2 is in contact with a
two-spin aromatic ring; Vali, Leui+3, and
AMXi+7 exhibit moderate-to-large high-field dipolar
shifts; and Glyi+8 exhibits strong low-field
dipolar shifts. Both the sequence and the dipolar shift pattern
identify (20, 21) this as a key portion of the distal helix
Val131-Gly139 (analogous to
Thr135-Gly143 in hHO). As shown in the slices
through 8-CH3 in HmuO and hHO (Fig.
7, A and B), the
strong contact with CH of Val131 is
conserved (relative to C
H of Thr135).
However, the weak contacts between 8-CH3 and NH of the
adjacent conserved Leu134 and Gly135 in HmuO
(residues 138 and 139 in hHO) observed in the hHO complex (Fig.
7B) (20, 21) are not detectable in HmuO (Fig. 7A)
and indicate a small movement of the distal helix near its kink away from 8-CH3 in HmuO relative to hHO. Finally, slices through
the similarly relaxed (T1 ~ 85 ms)
Ser138 C
1 in HmuO (Fig. 7C) and
Ser142 in hHO (Fig. 7D) indicate that the Ser is
slightly farther from heme 6-H
s in HmuO relative to the
hHO complex. The chemical shifts for helix II residues, together with
data from hHO complexes (20, 21), are listed in Table II. The observed
inter-residue and heme-residue contacts are illustrated schematically
in Fig. 6.
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H-bond Network/Aromatic Cluster--
The HmuO
complexes, like the hHO complexes (21, 22), exhibit a set of strongly
low-field shifted labile proton peaks (Figs. 3 and 4A),
which (with the unique exception of HmuO Gly139 NH/hHO
Gly143 NH) exhibit negligible paramagnetic relaxation, so
their strong low-field bias must be attributed to strong hydrogen bonds
(42). Three sequential fragments are easily recognized (summarized in Fig. 2) by their remarkably similar arrangements compared with the
three characterized fragments labeled IV-VI in hHO·DMDH·CN (21).
The analogous fragment III could not be recognized as easily in the
HmuO complex (but see "Discussion"). The helical fragment IV,
Zi-Glyi+1-AMXi+2-AMXi+3, exhibits strong low-field NH shifts for Glyi+1 and AMXi+2 (Fig. 8,
B and D; summarized in Fig. 2), as found for
Ala165-Phe166 in hHO (21). In agreement with
the assignment of Leu159-Tyr161 to fragment
IV, a three-spin TOCSY ring makes contact with
AMXi+2 (Fig.
9D), and two spins of a
three-spin aromatic ring make contact with AMXi+3
(Fig. 9C), which must arise from Tyr161. The
three TOCSY/NOESY peaks of the AMXi+3 side chain
exhibit the unusual pattern (Fig. 9, B-D) that one
cross-peak becomes narrower, whereas the other broadens as the
temperature is elevated. This behavior is consistent with a Tyr ring
that reorients sufficiently slowly to resolve the individual
CH or C
H, but leaves the two
C
H shifts averaged. The NOESY cross-peak to its own
backbone, as well as the cross-peak to a new low-field shifted labile
proton with no TOCSY connectivity, identifies Tyr161 OH
(Fig. 9D). Tyr161 C
H exhibits a
weak-to-moderate intensity NOESY cross-peak to NH of Gly139
(data not shown; as also observed for the homologous Phe166
Gly143 in hHO) (20, 21). A strong dipolar contact of
Phe166 NH with a Met51 methyl in hHO (21) is
lost in HmuO, but is replaced by a dipolar contact of
Phe160 NH with an NH2 group (data not shown),
which sequence comparison identifies as Gln46. A strong
contact between the Tyr161 ring and a low-field,
dipolar-shifted three-spin aromatic group (Fig. 9C) is
analogous to the Phe167-Phe47 contact in hHO
(21) and is readily rationalized by the homologous Tyr161-Phe42 contact observed here. This
assignment is consistent with the observation (data not shown) of a
Gln46 NH2 contact with Phe42. The
dipolar contact for this fragment with the distal helix (II) and
Phe42/Gln46 are summarized schematically in
Fig. 6.
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The extreme low-field labile proton exhibits no TOCSY peak, but
displays strong NOESY cross-peaks to a two-spin aromatic ring (Fig.
9E) whose AMX backbone is readily identified.
Ni-Ni+1 and -Ni+1
(Fig. 8A; summarized in Fig. 2) locate the adjacent residue
as Thr, which identifies the Tyr53-Thr54
segment with shifts and dipolar contacts analogous to fragment V in hHO
(21). The Tyr53 ring exhibits the NOESY cross-peak to the
Phe160 ring (Fig. 9B) that was observed between
the homologous Tyr58 or fragment V and Phe166
or fragment VI in hHO (21). The Tyr53 ring exhibits
NOESY cross-peaks to N
H of Arg132 and
OH of Tyr133 (data not shown) on the distal helix II in the
same fashion as observed for the homologous residues in hHO
(schematically shown in Fig. 6) (21). The dipolar contacts of fragment
IV are summarized in Fig. 6.
Finally, the other two extreme low-field peptide NH groups are
part of a 5-residue fragment,
Zi-Alai+1-Zi+2-Vali+3-Zi+4 (Figs. 2 and 8, A, B, and D), which
the sequence identifies as Arg79-Leu83, with
the low-field NH groups occurring from the homologous residues i (Arg79) and i+1 (Ala80)
in HmuO (as observed for Arg85 and Lys86 in
hHO) (21). The contacts between fragment VI and the distal helix II and
fragment V, viz. Asn78 and Arg79 to
Tyr133 and Tyr53 (Fig. 6), expected on the
basis of the contact in hHO, are clearly observed, as summarized in
Fig. 6. Attempts to assign the fragment in HmuO analogous to fragment
III on the basis of the NMR studies on hHO·DMDH·CN (21) failed,
although the 1H NMR data available can be used to infer
that the fragment is similarly highly conserved in HmuO relative to hHO
(see below). The peak at 14.1 ppm exhibits equally intense cross-peaks
to two non-labile protons in the aromatic window indicative of a His ring NH, which, by analogy to hHO (19, 22), is
His128; the expected strong NOESY cross-peak to
Ala200 is observed (data not shown).
The low-field peak at 11.4 ppm exhibits properties consistent with its
arising from NH of Trp50 (which replaces
Tyr55 in hHO) in NOESY cross-peaks to a TOCSY-detected
(only two cross-peaks are resolved) aromatic ring and a weak NOESY
cross-peak of the ring (Trp50 ring) to the rings of both
Phe160 and Tyr161 (data not shown). Hence, we
tentatively label it N
of Trp50. It should
be noted that there remains one strongly low-field shifted NH (12.2 ppm; labeled a in Fig. 4A) that cannot be
assigned at this time and that has no analog in hHO (21).
Acceptors for Strong H-bond Donors--
Having demonstrated for
HmuO a remarkably conserved arrangement on the distal side of the heme
for three of the four fragments involved in the H-bond network/aromatic
cluster in hHO (21), one can speculate that the acceptors for these
strong H-bond donors in hHO are also homologous in HmuO. Thus, sequence
comparison indicates that the carboxylate of Glu57
(homologous to Glu62 in hHO) is the acceptor for NH groups
of Arg79 and Ala80 and that the carboxylate of
Asp136 (homologous to Asp140 in hHO) is
acceptor for Tyr53 OH. Similarly,
His132 N
H as donor to Glu202 in
hHO (22) indicates that Glu196 is the acceptor for
His128 N
H in HmuO. Even though fragment III,
identified in hHO (21), could not be assigned in HmuO, the sequence
homology suggests that the Asp86 carboxylate (homologous to
Asp92 in hHO) (Fig. 2) is the acceptor for the NH groups of
Gly159 and Phe160, as depicted in Fig. 6.
Finally, the acceptor for the completely new strong H-bond involving
Tyr161 O
H (Phe167 in hHO) cannot
be identified by comparison with hHO. The relative positions of the
donors and acceptors in the strong H-bonds in HmuO and hHO (21) are
compared in Table III.
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Labile Proton Exchange--
Comparison of the resolved low-field
1H NMR trace of HmuO·DMDH·CN in
1H2O in Fig. 4A with that of the
complex 20 min (Fig. 4B) and 4 h (Fig. 4C)
after transfer into 2H2O shows that, whereas
Tyr53 OH has a half-life <5 min, other strong H-bonding
protons exhibit long exchange half-lives not only for peptide NH groups
(30 min for Arg79, ~30 days for Phe160, and
2 h for peak a), but also for side chain labile protons (~4 h for NH of His128 and ~2 h for
N
H of Trp50). Moreover, comparison of the
3:9:19 trace (33) without (Fig. 4A) and with (Fig.
4B') saturation of the bulk water resonance shows
significant magnetization transfer to the low-field peaks (22, 43), as
shown in the difference trace in Fig. 4C'. The magnetization transfer to the four labile protons shown to
exchange slowly with water must arise from NOEs between these labile
protons and "immobilized" water molecules (43), as we previously
observed for hHO·DMDH·CN (22). The magnitude of the NOEs is ~10%
for His128 N
H and ~25% for
Phe160 NH, Trp50 N
H, and peak
a. For the other peaks that exhibit magnetization transfer
from water, it is not possible at this time to differentiate between
chemical exchange and NOEs as the origin of the magnetization transfer
(43).
Magnetic Axes and Cyanide Tilt--
The completely conserved
pattern of large dipolar shifts for proximal helix residues (Table II)
and conserved contacts with the heme (Fig. 6) in HmuO relative to the
hHO complexes can arise only if HmuO·PH·CN and hHO·PH·CN
possess very similar orientation for the major magnetic axis
(32). A direct determination of the magnetic axes for HmuO·PH·CN
using the present 1H NMR data and the recently available
HmuO crystal coordinates2 leads to = 234 ± 16,
= 18 ± 2, and
= 47 ± 12, which can be
compared with reported values of
= 234 ± 12,
= 20 ± 3, and
= 25 ± 13 for hHO·DMDH·CN (21).
The magnetic axes for both complexes yield similarly excellent
correlation between the
dip(obs) and
dip(calc) for the input proximal side residues (data not
shown; see Supplemental Material). In each case, z is tilted ~20° toward the
-meso position. The
z direction is oriented toward the proximal side (20, 32);
hence, the FeCN vector (
z direction) is tilted
toward the
-meso position. Therefore, a direct
contribution to stereoselectivity from the tilt due to direct distal
steric interactions with the ligand in the direction of the
-meso position is operative in both hHO and HmuO.
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DISCUSSION |
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PH Orientation--
The orientation of PH in both hHO (19,
20) and HmuO complexes is similarly rotationally disordered about the
/
-meso axis in the initially formed complex, with very
similar ~3:1 ratios at equilibrium and with the same heme
orientation dominating in each complex in solution. Notably, the heme
orientation found in the HmuO·PH·H2O crystal
structure2 is the same as the dominant isomer in solution,
whereas that in the hHO·PH·H2O crystal structure (15)
is the minor form in solution (19, 20). The resolution upon using DMDH
rather than PH for HmuO is less dramatic than for hHO because, in
contrast to hHO (20, 21), there are no detectable changes in the
intrinsic line width of the signals in the DMDH relative to the PH
complexes of HmuO, only the loss of the second set of minor compound
signals. The narrower lines for the HmuO complex compared with the HO
complex are attributed in part to a reduction in size (216 versus 265 residues) relative to hHO. An example of the
narrower line widths in the HmuO complex compared with the hHO complex
is the detection of the complete Ser138 TOCSY connections
(data not shown; see Supplemental Material), including the
NH-C
H correlation missing in hHO·DMDH·CN (21), despite unchanged paramagnetic relaxation.
Utility of hHO as a Homology Model--
The similarity in the
1H NMR spectra of hHO·PH·CN and HmuO·PH·CN in Fig.
3 is completely confirmed by the remarkable similarities in not only
the positions of the various secondary structural elements represented
by the homologous fragments I, II, and IV-VI, as reflected in dipolar
contacts among each other and with the heme (Fig. 6), but also the
pattern of paramagnetic relaxation and hyperfine shifts (Table II). The
similar heme methyl contact shifts (Table I) reflect a similarly
oriented axial His imidazole plane, and the conserved pattern of
dipolar shifts for the proximal helix (Table II) confirms an ~20°
steric tilt of the FeCN toward the -meso position in both
mammalian HO (20, 21) and this bacterial HO.
The close similarity of the environments of the individual heme methyls
not only reflects the numerous completely conserved contacts, but
allows the ready identification of the HmuO residues whose nature has
been dramatically altered compared with hHO residues, i.e.
hHO Ile211 HmuO His205 in Fig. 5
(C and D) and hHO Gln38
HmuO
Leu33 in Fig. 5 (A and B). Fragment
III (Fig. 2) could not be located in analogy with hHO because fragment
III possesses three aromatic residues (Phe95,
Trp96, and Tyr97) in hHO that could be easily
identified (21) by their contacts with fragment IV, and these residues
on fragment III are substituted by aliphatic residues
(Lys89, Leu90, and Asn91) in HmuO.
The cluster of aromatic side chains, i.e. those of HmuO/hHO Tyr53/Tyr58,
Phe160/Phe166,
Tyr161/Phe167,
Tyr130/Tyr134,
Tyr133/Tyr137, and
Phe42/Phe47, are again largely conserved in the
two HO proteins (Fig. 6). Finally, the acceptor for all but one
(Tyr161 O
H) of the assigned strong H-bond
donors could be identified in HmuO complexes solely on the basis of
sequence homology. We therefore conclude that a structurally
characterized HO complex will serve as a valuable homology model to
facilitate the assignment of residues involved in many details of the
active site structure in a related HO. The sequence homology between
HmuO and hHO is relatively high (33% identity and 70% similarity if
conservative substitutions are included) (10). Other bacterial HOs,
such as HemO from N. meningitides (44), exhibit less
sequence homology to mammalian HOs, but still exhibit a structure (45)
that is related to that characterized in the two mammalian HOs (15, 16)
and one bacterial HO (45) and exhibit different details of the active
site. To date, 1H NMR data on HemO (44, 45) have not been
reported to allow comparisons.
Comparison with the HmuO·PH·H2O Crystal
Structure--
The 1H NMR data are consistent with the
crystal structure2 for HmuO to the same degree previously
found for the same hHO complexes (20, 21). The proximal helix is
strongly conserved, but the distal helix exhibits dipolar shifts that
deviate from those predicted by the relatively robust magnetic axes in
the same fashion as found for hHO (see Supplemental Material) (20, 21).
The loss in solution of dipolar contacts between the NH groups of
Leu134 and Gly135 and 3-CH3 and the
weakening of contacts between Ser138
CH and 6-H
(Fig. 7B) relative
to predictions based on the crystal structure2
suggest possibly only a small (0.5-1.0 Å) movement of the distal helix near its kink. However, similar differences in the distal helix
position have been observed in the two non-equivalent molecules in the
hHO·PH·H2O crystal (15) and may simply represent the intrinsic mobility of the distal helix.
The distal H-bond network in the HmuO complex,2 as in the
case of hHO·PH·H2O, is not readily discerned in the
crystal structure (14, 15). However, once the donor NH and OH groups
have been identified by 1H NMR, the crystal structure
readily identifies the probable acceptors. The proposed acceptors for
the strong H-bonds in HmuO are the Glu57,
Asp86, Asp136, and Glu196
carboxylates, based solely on the homology to hHO·DMDH·CN NMR data
(21) and the hHO·PH·H2O crystal structure (15)
and completely confirmed in the HmuO crystal structure (Fig.
6).2 The crystallographic geometry (distance and
angle)2 for these H-bonds in HmuO is summarized in Table
III, where they can be compared with similar data on hHO·DMDH·CN
and hHO·PH·H2O. Their dispositions are far from ideal
(42) to allow the strong H-bond so obvious in the 1H NMR
data, as shown in Table III. This non-ideal orientation of the donors
and acceptors may be simply the result of the intrinsic uncertainties
in the crystallographic positions of the two interacting units. The
acceptor for the new strong H-bond from Tyr161
OH is identified in the crystal2 as the side
chain of Gln46 (Fig. 6). Similar strong H-bonds, as yet
unassigned, appear in the 1H NMR spectra of apo-HO in both
mammals and bacteria,3
indicating that the H-bond network plays a key role in the structures of both the apo-HO and substrate complexes.
A structural difference of possible significance between the HmuO
cyanide-ligated complex and the crystal structure of the aquo-ligated
complex2 is the relative position of fragment IV relative
to the distal helix II. This same difference was previously observed in
the hHO complex (20, 21). Thus, although moderate intensity NOEs are
observed between the Tyr161 ring and Gly139 NH
(i.e. rij ~ 4 Å), the crystal
structure indicates rij > 6 Å. This same
difference was previously observed for the analogous hHO complexes
involving the homologous Phe167 ring and Gly143
NH (20, 21). Thus, the aromatic cluster appears to move ~2-3 Å closer to the distal helix in the cyanide complex in solution compared
with the aquo complex in the crystal. This difference may be due to the
different ligands used in the alternate studies in solution
(CN, a H-bond acceptor) and in the crystal
(H2O, a H-bond donor).
Ordered Water Molecules--
NOEs indicative of nearby
immobilized water molecules (Fig. 4, A,
B', and C') (43) are observed for the NH groups
of Arg79 and Phe160 and the NH
groups of His128 and Trp50. Water molecules are
indeed observed close to the NH groups in the crystal
structure.2 Similar water NOEs are observed for the NH
groups of the homologous Arg85 and Phe166 in
hHO and His132 N
H (the Tyr homolog of
Trp50 was not assigned in hHO) (22). Hence, both HOs are
characterized by ordered water molecules, particularly in the distal
pocket. However, even these preliminary data indicate differences
between the two HOs in the organization of these water molecules. Thus, the NOE for His N
H is weaker in HmuO
(His128) than in hHO (His132) (21), but the
NOEs for the NH groups of Arg85 and Phe160 are
significantly larger (~25-30%) in the HmuO complex (Fig. 4C') than in the hHO complex (~10-15%) (22). These
differences could be the result of differences in water-NH distances,
the number of nearby water molecules, and/or the mobility of the
ordered water molecules (43). The crystal structure2 of
HmuO·PH·H2O reveals numerous water molecules in the
distal side of the heme at positions similar to those detected by
1H NMR in the hHO complex (22). The different stages of
refinement for the hHO and HmuO crystal structures and the availability
of only preliminary NMR data in solution suggest that a discussion of
differences in the occupation of water molecules be deferred until
water NOEs can be more effectively studied in 15N-labeled
HO.
Comparison of Dynamic Properties of HmuO and hHO--
The very
close structural homology between HmuO and hHO apparent in both the
solution 1H NMR data and crystal structures is, however, in
contrast to the highly differential dynamic properties of the two
enzymes. On the one hand, the rate of exchange with
2H2O of homologous labile protons involved in
the strong H-bonds differs significantly for the two enzymes, with HmuO
exhibiting significantly reduced rates. Comparison of the homologous NH
groups shows that the half-lives for a residue are ~4 h for HmuO
His128 NH and 45 min for hHO
His132 N
H (22), 30 min for HmuO
Arg79 NH and 25 min for hHO Arg86 NH (22), and
~700 h for HmuO Phe160 NH and ~1 h for hHO
Phe166 NH (22). Moreover, the
Tyr53/Tyr58 OH groups, which exhibit saturation
transfer due to exchange, exhibit a much smaller saturation factor
(~10%) in HmuO than in hHO (~40%), dictating a much slower
exchange rate in HmuO. The ~700 factor decrease in the
Phe160/Phe166 NH exchange rate indicates that
the dynamic stability near fragment IV is ~4 kcal greater in HmuO
than in hHO (46). Although the extreme low-field shifts for the labile
protons of the H-bond networks are very similar in the two HO complexes
(Table III), indicating that the individual H-bonds are comparably
strong, other factors that contribute to the stability of the folding in the environment of the network are clearly much weaker in hHO than
in HmuO.
The 1H NMR data provide other indicators for a dynamically
more stable (and hence, less flexible) HmuO than hHO. On the one hand,
two new and strong H-bonds are observed, one of which could be uniquely
attributed to Tyr161 OH (which substitutes
for Phe167 in hHO). The likely acceptor, Gln46,
is suggested by the HmuO crystal structure,2 although the
strong low-field bias due to H-bonding suggests a stronger or more than
one acceptor. In fact, the crystal structure2 of
HmuO·PH·H2O places a water molecule within 3 Å of this
OH. The sequence origin of the other strong H-bond (peak a
at 12.4 ppm in Fig. 4A) is not identified, but has no
homolog in hHO. Nevertheless, these two strong H-bonds in HmuO are
incremental over those conserved relative to hHO (21, 22). The second observation is that the Tyr161 ring, unlike the
Phe167 ring, exhibits slow ring reorientation
about the C
-C
bond, as evidenced by the
resolution of two C
H peaks at low temperature, whereas
an averaged C
H peak is observed for Phe166
at all temperatures for hHO complexes (20, 21). The decreased mobility
of Tyr161 in HmuO relative to the Phe167 ring
in hHO (21) in 2-fold reorientation supports a tighter and more
constrained distal environment in HmuO than in hHO.
Role of the H-bond Network-- The conservation of the H-bond network/aromatic cluster/ordered water molecules in HmuO relative to hHO argues for important functional roles. The existence of a network of water molecules that includes water molecules near the catalytically critical distal helix Asp136/Asp140 (12, 14) supports the notion that water provides the stabilizing H-bond to the novel hydroperoxy intermediate. Interaction of Asp140 with the distal ligand via two water molecules has been recently characterized in the crystal structure of the rat HO·PH·N3 complex (23). The presence of a water/H-bond network that extends from the distal pocket through the enzyme to its surface on the opposite side from the substrate-binding pocket (22)2 suggests that the channel may funnel the required nine protons to the active site in a controlled manner.
The greater dynamic stability of the pocket near the catalytically
important Asp136/Asp140 is also apparent in two
other observations. The greater dynamic stability of the distal pocket
in HmuO relative to hHO, witnessed in both slower labile proton
exchange and aromatic ring reorientation, may be responsible for the
~4 factor slower turnover rate in HmuO (30) than in mammalian HOs (2,
11). More extensive NMR studies of both the dynamic properties of the
distal side and the distribution of oriented water molecules in HmuO
and other HO complexes are in progress.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants GM62380 (to G. N. L.) and GM57272 (to M. I.-S.) and Grants-in-aid from the Ministry of Education, Culture and Sports, 12157201 and 14380300 (to M. I.-S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains Figs. 1S-4S.
§ Present address: Amgen, Inc., Thousand Oaks, CA 91320.
To whom correspondence should be addressed: Dept. of
Chemistry, University of California, One Shields Ave., Davis, CA 95616. Tel.: 530-752-0958; Fax: 530-752-8995; E-mail:
lamar@indigo.ucdavis.edu.
Published, JBC Papers in Press, December 11, 2002, DOI 10.1074/jbc.M211249200
2 S. Hirotsu, G. C. Chu, D.-S. Lee, M. Unno, T. Yoshida, S.-Y. Park, Y. Shiro, and M. Ikeda-Saito, manuscript in preparation (Protein Data Bank code 1WI0).
3 Y. Li, R. T. Syvitski, G. C. Chu, M. Ikeda-Saito, and G. N. La Mar, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: HO, heme oxygenase; hHO, human heme oxygenase; PH, protohemin; DMDH, 2,4-dimethyldeuterohemin; DSS, 2,2-dimethyl-2-silapentane-5-sulfonate; NOE, nuclear Overhauser effect; NOESY, two-dimensional nuclear Overhauser effect spectroscopy; TOCSY, two-dimensional total correlation spectroscopy.
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