Comparison of the Heme-free and -bound Crystal Structures of
Human Heme Oxygenase-1*
Latesh
Lad
,
David J.
Schuller§,
Hideaki
Shimizu
¶,
Jonathan
Friedman
,
Huiying
Li
,
Paul R.
Ortiz de
Montellano
, and
Thomas L.
Poulos
**
From the
Department of Molecular Biology and
Biochemistry, Program in Macromolecular Structure, University of
California, Irvine, California 92697, the § Cornell
High Energy Synchrotron Source and Department of Molecular Biology and
Genetics, Cornell University, Ithaca, New York 14853, and the
Department of Pharmaceutical Chemistry, University of
California, San Francisco, California 94143-0446
Received for publication, November 10, 2002, and in revised form, December 23, 2002
 |
ABSTRACT |
Heme oxygenase (HO) catalyzes the degradation of
heme to biliverdin. The crystal structure of human HO-1 in complex
with heme reveals a novel helical structure with conserved
glycines in the distal helix, providing flexibility to accommodate
substrate binding and product release (Schuller, D. J., Wilks,
A., Ortiz de Montellano, P. R., and Poulos, T. L. (1999)
Nat. Struct. Biol. 6, 860-867). To structurally understand
the HO catalytic pathway in more detail, we have determined the crystal
structure of human apo-HO-1 at 2.1 Å and a higher resolution structure
of human HO-1 in complex with heme at 1.5 Å. Although the 1.5-Å
heme·HO-1 model confirms our initial analysis based on the 2.08-Å
model, the higher resolution structure has revealed important new
details such as a solvent H-bonded network in the active site that may
be important for catalysis. Because of the absence of the heme, the
distal and proximal helices that bracket the heme plane in the holo
structure move farther apart in the apo structure, thus increasing the
size of the active-site pocket. Nevertheless, the relative positioning and conformation of critical catalytic residues remain unchanged in the
apo structure compared with the holo structure, but an important
solvent H-bonded network is missing in the apoenzyme. It thus appears
that the binding of heme and a tightening of the structure around the
heme stabilize the solvent H-bonded network required for proper catalysis.
 |
INTRODUCTION |
Heme oxygenase (HO)1
catalyzes the oxidation of heme to biliverdin and is the key reaction
in the recycling of porphyrin and iron in mammals. Biliverdin is
reduced by biliverdin reductase to bilirubin, which is then conjugated
with glucuronic acid and excreted (1, 2). Excretion of bilirubin is
often deficient in newborn children, giving rise to neonatal jaundice
and the potential for neurological damage (3). CO, the other product of
the HO reaction, has been suggested to serve as a signaling molecule
through the guanylate cyclase system in a manner similar to nitric
oxide, although this remains a controversial topic (4, 5). Finally, the
iron released by HO is normally recycled and represents the major
source of this metal in heme homeostasis, whereas increased iron
release due to elevated HO activity can trigger enhanced lipid and
protein peroxidation (6, 7). There are two HO isoforms, denoted HO-1
and HO-2 (8-10). A third isoform discovered in rats has been described
(11), although the expressed protein is not an active HO, and
therefore, its significance is currently unclear.
Mammalian HO-1 is a membrane-bound protein. The expression of
truncated, water-soluble, fully active forms of human (12) and rat (13)
HO-1 missing the C-terminal 23-26 amino acid residues has facilitated
major advances in structure/function studies of HO, especially
mechanistic studies. The mechanism of HO resembles that of cytochrome
P450 in its ability to oxidize unactivated C-H bonds (14). However, in
contrast to cytochrome P450 and heme peroxidases, the action of HO does
not proceed through a ferryl intermediate (15, 16), but appears rather
to involve a peroxo ligand capable of attacking the
-meso
bridge of the heme (Fig. 1). Furthermore,
unlike true hemoproteins, the heme in HO serves as both cofactor and
substrate in a "substrate-assisted" reaction. The HO oxidation
consumes a total of three molecules of oxygen and 7 electrons (17) and
produces CO and free iron (Fe2+). For the human HO enzymes,
electrons are provided by NADPH-cytochrome P450 reductase (18), whereas
alternative electrons donors are employed in plants and bacteria (19,
20).
For human HO-1, the expression of even shorter but still active
versions of HO-1 facilitated crystallization and structure determination of human HO-1 complexed with heme (21). The structure reveals a novel protein fold that consists primarily of
-helices, with the heme wedged between the distal and proximal helices. Conserved
glycines in the distal helix provide flexibility, causing the two HO
molecules in the crystallographic unit cell to differ. In one molecule,
the active-site pocket is more open, with relatively loose distal
helix-heme contact, whereas in the other molecule, contacts between the
heme and distal helix are tighter. This, together with the high
crystallographic thermal factors, suggests that flexibility of the
distal helix enables the heme pocket to be opened and closed to bind
the heme substrate and to permit dissociation of the biliverdin
product. The crystal structure of a truncated rat HO-1 isoform (22)
and, more recently, the structure of a bacterial HO from the
Gram-negative pathogenic bacterium Neisseria meningitidis
(23) reveal structural information very similar to that of human
HO-1.
Recently, the crystallization and three-dimensional structure of rat
apoheme oxygenase were published at 2.55 Å (24). Here we report the
human apo-HO-1 structure at 2.1 Å and a high-resolution structure of
human HO-1 complexed with heme refined to 1.5 Å. Both higher
resolution structures have enabled us to detect important structural
features that were not apparent at the lower resolution. We also report
the refined structure of the heme·HO-1 complex in the B crystal form
to 2.6 Å. This form was important for initial phasing of HO (21).
Although the B form structure is of considerably lower resolution than
the C form structure of the heme·HO-1 complex, it may be of interest
to investigators studying crystal packing.
 |
MATERIALS AND METHODS |
Bacterial Expression and Protein Purification--
Bacterial
fermentation of cells and purification of HO-1 were carried out
according to published procedures (12, 25). Enzyme purity was assessed
by examination of
A405/A280; in all cases,
A405/A280 > 2.1 for the
heme·HO-1 complex was considered pure. Enzyme purity was additionally
assessed by SDS-PAGE, and the preparations were judged to be
homogeneous by the observation of a single band on a Coomassie
Blue-stained reducing SDS-polyacrylamide gel.
Crystallization--
Heme·HO-1 complex crystals were grown as
previously described (25, 21) using the sitting-drop vapor diffusion
method with a well solution of 2.08 M ammonium sulfate, 100 mM HEPES (pH 7.5), and 0.9% 1,6-hexanediol. Drops
consisted of protein stock (5 µl) at 35 mg/ml in 20 mM
potassium phosphate (pH 7.4) mixed with well solution (5 µl) on
siliconized coverslips. Apo crystals were grown with a well solution of
2.25 M ammonium sulfate, 100 mM HEPES (pH 7.5),
and 0.9% 1,6-hexanediol. All crystals were grown at 28 °C, although
we have recently found that heme·HO-1 complex crystals tended
to grow as single plates rather than clusters of plates at room
temperature. For cryogenic data collection, heme·HO-1 crystals were
transferred to a solution of 2.32 M ammonium sulfate, 100 mM HEPES (pH 7.5), 0.9% 1,6-hexanediol, and 20% (v/v) glycerol. For apo-HO-1 crystals, D(+)-trehalose was used as
the cryoprotectant. Cryogenic data collection involved a seven-step transfer to artificial precipitant solution with an increased D(+)-trehalose concentration up to 35% (v/v). Apo- and
holo-HO-1 crystals all belong to the monoclinic space group
P21, with the cell dimensions listed in Table I.
Data Collection--
Apo-HO-1 data and the B form heme·HO-1
complex were collected using an in-house R-AXIS IV imaging plate
detector equipped with a rotating copper anode x-ray generator with
Osmic optics. Crystals were maintained at
160 °C in a steam of
nitrogen (Crystal Logic, Los Angeles). For both sets of data
collection, a 180° scan using 1° frames was collected. Data
collection for the high-resolution C crystal form heme·HO-1 complex
was carried out at Stanford Synchrotron Radiation Laboratory
beamline 7-1 with a Mar345 imaging plate. Optimization of data
collection was guided by the STRATEGY function of MOSFLM (26). All data
were reduced using DENZO and SCALEPACK (27), with rejections performed
using ENDHKL (Louis Sanchez, California Institute of Technology) in
conjunction with SCALEPACK.
Model Building and Refinement--
The apo-HO-1 structure was
determined by the method of molecular replacement using MOLREP (28). A
monomer of the human heme·HO-1 crystal structure (Protein Data Bank
code 1QQ8) (21) with the heme and waters removed was used as the
probe, with searches being carried out at 4.0 Å in space group
P21. The best cross-rotation and translation function
solution was rigid body-refined and fixed in place, followed by
searches for the remaining molecules in the asymmetric unit. A total of
four solutions were found, corresponding to the expected four
monomers/asymmetric unit. The final R factor was 48.9%,
with a correlation coefficient of 54.4%. The structure was further
refined in CNS (29). Protein atoms were initially refined by simulated
annealing, followed by a few cycles of conjugate gradient minimization
and water picking. Finally, temperature factors were refined. No
restraints for non-crystallographic symmetry were applied. The program
O (30) was used for further adjustment and modeling of protein atoms,
ligands, and water molecules. Backbone geometry was checked in PROCHECK
(31), and none of the residues were in the disallowed region. Data
collection and refinement statistics for all structures are summarized
in Table I.
The B form heme·HO-1 complex initial model was constructed by placing
the C crystal form molecules according to the NCS relationships uncovered in the initial molecular replacement (21). It was then
refined in non-crystallographic symmetry (CNS) (29). For the
high-resolution heme·HO-1 complex structure, the initial 2.08-Å structure was subjected to rigid-body refinement with the newly collected synchrotron data, followed by minimization and
B-factor refinement in CNS (29). When refinement proceeded
to the point where the use of multiple conformations and anisotropic
thermal factors was necessary and appropriate, SHELXL (32) was used. TOM was used for model building for both structures (33). To accommodate model building of the high-resolution structure with multiple conformations and anisotropic thermal factors, XtalView (34)
was used.
 |
RESULTS |
Structure Solution--
Apo-HO-1 crystallizes in two different
unit cells, the A and B forms (Table I).
The B form was produced by growing crystals in 10% glycerol and was
used to solve the initial structure of holo-HO-1 (21). Refinement of
the B form apo-HO-1 model was never successful because of inadequate
data quality, which led us to further investigate the A form. An
empirical approach to molecular replacement was taken with the A
crystal form. Rather than using one method, several programs, including
AmoRe (35), EPMR (36), BRUTE (37), CNS (29), and MOLREP (28), were used. Of these, only MOLREP was successful.
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Table I
Data collection and refinement statistics
Data collection statistics for the B crystal form were previously
reported (21) and are reproduced here for convenience. r.m.s.d., r.m.s.
deviation.
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Heme·HO-1 Structure--
The higher resolution 1.5-Å structure
of heme·HO-1 is basically the same as in our earlier report (21).
However, new details on solvent structure in the active site possibly
relevant to the catalytic mechanism are now visible. The relevant
region is shown in Fig. 2. As noted
earlier, the two molecules in the asymmetric unit adopt slightly
different conformations: one with the active site more "closed" in
molecule A and the other more "open" in molecule B. As shown in
Fig. 2, Asp140 in the closed molecule participates in an
H-bonded network involving well ordered solvent molecules. The
"peanut" shape electron density indicated by the arrows
in Fig. 2A could be due to two water molecules only 2.3 Å apart or, more likely, one water occupying two different positions,
each at fractional occupancy. In the open molecule, the positions of
these waters give only a small lobe of Fo
Fc electron density at the 3
level and weak
2Fo
Fc electron density at the
1
level, indicating a much less well ordered solvent structure in
the open molecule. Mutagenesis studies have shown that
Asp140 is essential for proper HO-1 function (38, 39) and
that changing Asp140 to Ala converts HO-1 into a peroxidase
(39). The reason is that, in the mutant, the peroxide O-O bond is
cleaved, giving a traditional peroxidase Fe(IV)-O oxyferryl
center (39). In the normal HO-1 reaction, the distal
peroxide atom (Fig. 1) undergoes electrophilic attack on the heme
-meso-carbon. One factor that controls which process
dominates, normal heme oxidation or cleavage of the O-O bond, is
undoubtedly a proper proton delivery mechanism to the iron-linked
oxygen. We earlier postulated that the proton donor must be water
because there are no active-site residues close enough to serve as a
direct proton donor (21). The Asp140-water H-bonded
network provides a potential proton delivery system. That the water
network is not well defined in the open molecule suggests that the
closed molecule is much closer to the active form of HO-1.

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Fig. 2.
Stereo diagrams showing the
Asp140-solvent H-bonded network in the distal pocket.
A, 2Fo Fc map
contoured at 1.5 . The arrows indicate the location of the
water that very likely occupies two positions at fractional occupancy.
B, the same as in A, showing the H-bonded network
that very likely serves to deliver protons to the iron-linked oxygen
required for catalysis. Note that one of these waters is 4.4 Å from
the heme -meso-carbon. An iron-liked oxygen/peroxide
would very likely displace this water as well as the water liganded to
the iron, thus enabling the distal peroxide oxygen atom to directly
contact the -meso-carbon.
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Apo Structure--
The structure of human apo-HO-1 has been
refined in the A crystal form to an R-factor of 0.217 and a
free R-factor of 0.262 at 2.1-Å resolution. There are four
molecules of apo-HO-1 in the asymmetric unit (designated A-D), in
comparison with two molecules in the heme·HO-1 structure. The final
structure contains a total of 7065 protein atoms, 450 water atoms, and
one non-water solvent, assigned as Cl
. The first 9-10
and last 10 residues of the 233-residue protein are not ordered in
electron density maps for molecules B and D. For molecule A, residues
10-224 are resolved, whereas for molecule C, we observed, for the
first time, the N terminus (Fig.
3A), although the last 10 residues of the C terminus are disordered. The overall r.m.s. deviation
of backbone atoms between the four molecules ranges from 0.38 to
0.55 Å (Table II). However, there are regions between the molecules where larger deviations (>0.8 Å)
exist and are localized between residues 156-159 and 188-195.

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Fig. 3.
Comparison of the human apo-HO-1 and
heme·HO-1 structures. A, diagram of the
2Fo Fc composite-omit electron
density map contoured at 1 showing the N-terminal region of apo-HO-1
molecule C; B, least-squares superimposition of the closed
conformation of heme·HO-1 and molecule A of apo-HO-1, with the
black line representing the apo molecule and the gray
line representing the holo structure; C, least-squares
superimposition of the open conformation of heme· HO-1 and molecule
A of apo-HO-1, with the black line representing the apo
molecule and the gray line representing the holo
structure.
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Table III lists the overall r.m.s.
deviation of backbone atoms between the four apo-HO-1 molecules and the
two heme·HO-1 molecules. At first glance, the r.m.s. deviation
between the two sets of structures is also low, suggesting very little
structural change. However, closer analysis revealed that one of the
heme·HO-1 molecules (molecule A/closed molecule) has a higher r.m.s.
deviation than the other (molecule B/open molecule) with respect to the
four apo-HO-1 molecules. The plot of the r.m.s. deviation for C-
atoms between the two holo-HO-1 molecules and the four apo-HO-1 forms (Fig. 4B) shows regions of
large deviation in the proximal helix, but only for the closed molecule
in the heme·HO-1 structure are there large deviations in the distal
helix and following loop. Fig. 3 (B and C) shows
the superimposition of an apo-HO-1 molecule with the open and closed
molecules of human heme·HO-1. Therefore, as might be expected, the
apo-HO-1 active site more closely matches the open form of
heme·HO-1.
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Table III
r.m.s. deviation of backbone atoms between the two holo molecules and
four apo molecules of the human HO-1 structures
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Fig. 4.
A, plot of the r.m.s. deviation
of backbone atoms between the four apo molecules in the asymmetric
unit; B, plot of the r.m.s. deviation of backbone atoms
between apo-HO-1 and the two conformations of heme·HO-1: closed
(dotted line) and open (solid line).
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N-terminal Region--
Of the four apo-HO-1 molecules in the
asymmetric unit, only in molecule C is the entire N-terminal region
seen (Fig. 3A). In molecule C, the N terminus curls away
from the molecule, whereas the location of the corresponding N-terminal
region in the heme·HO-1 structure (Fig. 3, B and
C) and other HO structures (rat HO-1 and bacterial N. meningitidis HO complexed with heme) (22, 23) appears to be
different. For the other HO structures, the N-terminal region bends
into the molecule, pushing down toward the proximal helix.
Distal Helix--
One of the most intriguing features of the HO
structures to date is the distortion in the distal helix that covers
part of the distal heme surface as well as forms an integral part of
the oxygen-binding pocket (21-23). Of the three heme·HO
structures now known, only in human HO-1 has the active site been
trapped in the open and closed conformation. In both rat HO-1 and
N. meningitidis HO, the distal helix is clearly in a
closed conformation (22, 23).
In the apo-HO-1 structure, the distal helix in all four molecules is
clearly in an open conformation (Figs. 5
and 6). The backbone atoms of Gly143, which, in the closed
molecule of the heme·HO-1 structure, directly contact the heme, have
shifted away in the apo-HO-1 structure. The amide group of
Gly143 is directed away from where the heme would be in all
four apo molecules, whereas movement of Gly139 is moderate
in comparison with the shift observed in Gly143. Only in
molecules A, B, and D is the
-carbon of Gly139 observed
shifting away from the heme site, with the carbonyl oxygen only
marginally moving away in molecules A and D, but still retaining the
same orientation as that in the closed molecule of heme·HO-1 (Fig.
5). These changes are important because the Gly139 carbonyl
oxygen can H-bond with the heme distal water ligand and hence might
also interact with oxygen in the oxyheme complex. Interestingly, the
catalytically important Asp140 discussed above is
relatively unchanged in the apo structure, with the exception of the
carboxylate side chain, which moves closer to the heme site because of
the small upward shift of Gly139. Several residues in the
distal helix differ between the apo and holo structures. These include
Ser142, which stabilizes the distal helix distortion
through H-bonds with the peptide backbones of Gly143 and
Leu141, the
-carbon of Leu141, the
N-
atom of Lys179, the carboxylate side chain of
Glu145, and the C-
1 and carbonyl oxygen atoms of
Leu138. Despite the change in these residues, the kink in
the distal helix is retained in the apo structure. For instance, the
redirection of the Gly143 amide bond stretches contact
between itself and the Ser142
-carbon from 2.91 Å in
the closed molecule of the heme·HO-1 structure to 3.23 Å in molecule
A, 3.15 Å in molecule B, 3.24 Å in molecule C, and 3.08 Å in
molecule D. However, the interaction between the Gly143 and
Ser142 amide bonds is enhanced from 3.02 Å in the closed
molecule of heme·HO-1 to 2.87 Å in molecule A, 2.90 Å in molecule
B, 2.84 Å in molecule C, and 2.86 Å in molecule D. Furthermore,
although the H-bond between the hydroxyl side chain of
Ser142 and the carbonyl oxygen of Leu138
remains intact, the non-bonded contact with the C-
1 atom of Leu138 is broken, causing the side chain of
Leu138 to move away from the heme site, thus reorienting
the C-
1 atom by ~100° away from the heme site in all four apo
molecules. The carbonyl oxygen of Ser142 that H-bonds to
the carboxylate side chain of Glu145 is also broken in all
four apo molecules. This causes the Glu145 side chain to
rotate and point in the opposite direction in all four apo molecules.
In addition, residues following Glu145, Val146,
and Leu147 have also shifted away from the heme, but
maintain a conformation similar to that of the closed molecule in
heme·HO-1.

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Fig. 5.
Diagram comparing the distal and proximal
heme regions between the closed conformation of the heme·HO-1
structure (gray) and four molecules in the asymmetric
unit of the apo-HO-1 structure (black).
A, molecule A; B, molecule B; C,
molecule C; D, molecule D.
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Not surprisingly, the position of the distal helix and the sequence of
residues within this stretch (residues 139-147) in the apo-HO-1
structure adopt a conformation similar to that of the heme·HO-1 open
molecule (Fig. 6). However, the distal
helix in the apo-HO-1 molecules is more open, indicating that, although the distal helix is flexible, the open/closed conformation observed in
the heme·HO-1 structure is most likely a transient state captured in
the crystal lattice. This is in agreement with a recent NMR study on
human HO-1 that confirmed that parts of the distal helix are probably
mobile and that also found a closer approach of the distal helix in
human HO-1 than in the crystal structure (40).

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Fig. 6.
Diagram comparing the distal and proximal
heme regions between the open conformation of the heme·HO-1 structure
(gray) and four molecules in the asymmetric unit of
the apo-HO-1 structure (black). A,
molecule A; B, molecule B; C, molecule C;
D, molecule D.
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Proximal (His Ligand) Side--
A comparison between the two
structures in the region surrounding the heme His25 ligand
is shown in Figs. 5 and 6. The proximal helix in the apo structure is
relaxed and moves downwards away from the heme site, accompanied by
several conformational changes in the side chains of several residues.
In the apo structure, the position of the proximal histidine ligand
(His25) moves away from the heme pocket, with the ring
adopting random conformations. In the heme·HO-1 structure,
Glu29, underneath pyrrole A of the heme, is close enough to
form an H-bond with His25 (2.67 Å in the closed molecule
and 3.05 Å in the open molecule). However, in the apo molecules, the
movement of His25 away from Glu29 drastically
increases the distance between these groups (5.50 Å in molecule A,
4.32 Å in molecule B, 5.30 Å in molecule C, and 5.83 Å in molecule
D), thus eliminating an H-bonding interaction. Interestingly,
Gln38, which is H-bonded at a distance of 2.82 Å to the
carbonyl oxygen of Glu29 in the closed molecule of the
heme·HO-1 structure, is located near pyrrole A, in the vicinity of
the
-meso-carbon. However, the orientation and position
of the Glu38 side chain are uniquely different in the apo
structure. For the open molecule in the holo structure,
Gln38 is no longer H-bonded to Glu29 (the
distance between the N-
atom of Gln38 and
the oxygen atom of Glu29 is 6.40 Å) and has shifted
outwards away from the heme site, with the NH2 group
pointing in the same direction as in the closed molecule of the holo
structure. In all four apo molecules, Gln38 moves closer to
the heme position, thus preventing H-bonding with Glu29
because the distance between the N-
atom of Gln38 and
the oxygen atom of Glu29 increases to 5.74 Å in
molecule A, 5.42 Å in molecule B, 3.82 Å in molecule C, and 6.47 Å in molecule D. Furthermore, the orientation of the Gln38
side chain fluctuates between the apo molecules. In molecules A and C,
the side chain points down toward the proximal helix, whereas in
molecule B and, in particular, molecule D, the amide group is directed
up, with the N-
atom of Gln38 in molecule D H-bonding to
the C-
2 atom of Leu147 (Figs. 5 and 6).
Heme-Protein Interactions--
In the heme·HO-1 structure, the
heme meso edges are buried in the protein, whereas one, the
-meso edge, is exposed. The
-meso edge of
the heme, which is selectively targeted for hydroxylation in the HO
reaction, faces a wall of hydrophobic residues in the protein interior
consisting of Phe214, Met34, and
Phe37 (Figs. 7 and 8). In the
apo structure, these residues remain relatively unchanged. On the
opposite side of the heme, a number of ionic/H-bonding interactions
between the heme propionates and nearby side chains are important for
orientating the heme in the active site. Basic residues
Arg183, Lys18, and Lys22 surround
the propionates and hold the heme in place in the heme·HO-1 structure
(Figs. 7 and 8). In the apo structure,
Lys179 is unchanged in all molecules; the NH and
NH2 nitrogens of Arg183 point away from the
heme site in molecules A and D, whereas, because of the movement of the
proximal helix, Lys18 and Lys22 have markedly
shifted away from the heme pocket in all four molecules, with the side
chain of Lys22 bent farther away in molecule D than in
molecules A-C. The
-edge of the heme is in contact with
Tyr134 and Thr135 in the heme·HO-1 structure.
The conformations of these residues are distinctly different in the apo
structure. The aromatic ring clearly leans away from the heme site and
tilts in the direction of the heme propionates. This change correlates
with movement of Thr135 in all four apo-HO-1 molecules,
flipping the side chain by ~150° such that it points toward the
position of the heme
-meso-carbon of the heme·HO-1
structure.

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Fig. 7.
Diagram comparing the heme pocket regions
between the closed conformation of the heme·HO-1 structure
(gray) and four molecules in the asymmetric unit of
the apo-HO-1 structure (black). A,
molecule A; B, molecule B; C, molecule C;
D, molecule D.
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Fig. 8.
Diagram comparing the heme pocket regions
between the open conformation of the heme·HO-1 structure
(gray) and four molecules in the asymmetric unit of
the apo-HO-1 structure (black). A,
molecule A; B, molecule B; C, molecule C;
D, molecule D.
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DISCUSSION |
The human apo-HO-1 structure supports our initial findings of a
dynamic active-site pocket (21) and is structurally similar to the
recently reported rat apo-HO-1 crystal structure (22). In the
comparisons between the apo-HO-1 and heme·HO-1 structures that
follow, we include the homologous rat HO-1 structures.
Differences in the Distal Pocket--
Differences between the
apo-HO-1 and heme·HO-1 structures are confined to the heme-binding
pocket. Interactions of backbone atoms of Gly139 and
Gly143 in the distal helix that contact the distal water
ligand of the heme in the holo-HO-1 structure are no longer present in
the apo-HO-1 structure, thus freeing the distal helix to move away from
the heme site in all four molecules, closely resembling the open
conformation (molecule B) in the human heme·HO-1 structure. The
absence of a bond between His25 and the heme iron in the
apo structure enables a relaxation of the proximal helix and, combined
with the upward movement of the distal helix, increases the overall
size of the apo active-site pocket. The larger pocket in the apo-HO-1
structure is reflected by the solvent-accessible volume of 57.9 A3, whereas the corresponding volume for the human
heme·HO-1 structure is 43.6 A3 (21). The rat apo-HO-1 and
heme·HO-1 structures reveal very similar changes (24). Not
surprisingly, because the sequence homology between the two HO-1
proteins is >80% (5), a comparison of human and rat apo-HO-1 reveals
that their overall structures are very similar, excluding the loop
region between Leu141 and Glu162
(Fig. 9A). The r.m.s.
deviation between molecules A of the two sets of apo structures is 1.11 Å.

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Fig. 9.
Structural comparison between human
(black) and rat (gray) apo-HO-1.
A, least-squares superimposition of apo-HO-1 molecules A of
rat and human; B, diagram comparing the distal and proximal
heme regions; C, diagram comparing the heme pocket
regions.
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Differences in the Proximal Pocket--
In the rat apo-HO-1
structure, a large portion of the proximal helix (residues 13-29),
which includes the proximal heme ligand (His25), is not
visible (24). In the human apo-HO-1 structure, the same region is
clearly visible, with the proximal histidine pulled away from the heme
pocket. In both the rat and human structures, the H-bonding interaction
between the N-
atom of Gln38 and the carbonyl oxygen of
the backbone of Glu29 observed in the holo structures is
lost in the apo form, allowing Gln38 to adopt a different
conformation in the apo structures. Gln38 appears to be
quite flexible because the side chain adopts different conformations in
all four molecules in the human apo-HO-1 structure. The differences in
the proximal helix between the human and rat HO-1 structures, which
might be due to differences in crystallization conditions or packing
constraints, give another good indication of the flexibility of the
enzyme in the apo state.
Heme-Protein Interactions--
In the absence of bound heme, basic
residues near the propionate groups (Lys179,
Arg183, Lys18, and Lys22), which
play a fundamental role in correctly orienting the heme within the heme
pocket, have clearly repelled one another in the apo-HO-1 structures
(Fig. 9C). For example, Lys18 and
Lys22 are not visible in the rat apo structure, but are
clearly seen to shift away from the heme pocket in the human apo
structure. On the other hand, Lys179 and Arg183
are visible in both the rat and human apo structures, but vary significantly in conformation. In the human enzyme, Lys179
in all four apo-HO-1 molecules is unchanged from the heme·HO-1 structure, whereas the side chain of the same residue in molecules A
and B of rat apo-HO-1 approaches the position at which the propionate group of the heme was originally present in rat heme·HO-1. Similarly, Arg183 in the human apo structure has approximately the
same conformation as in the holo structure. In the rat apo structure,
Arg183 is flipped in molecules A and B. As a result, the
side chain of Arg183 approaches more closely to the
position at which the propionate group of the heme was originally
present in the rat heme·HO-1 structure (Fig. 9C).
Replacement of Arg183 with Glu or Asp, but not other
residues, including Gln and Asn, reportedly leads to changes in
regioselectivity (41). On the opposite side of the heme pocket,
Met34, Phe37, and Phe214 form a
hydrophobic wall opposite the heme
-meso edge. In the human apo-HO-1 structure, these residues are unchanged from those in
the heme·HO-1 structure, whereas in the rat apo-HO-1 structure, Met34 and Phe37 approach closer to where the
heme would be in the heme pocket (24). In addition, the side chain of
Phe207 (not shown in Fig. 9B), present under the
-meso edge of the heme, is flipped and points down toward
the position of the proximal helix in the rat apo structure, whereas
this residue is unchanged in the human apo-HO-1 structure.
Implications for Catalysis--
Unlike the globins and
peroxidases, the distal HO-1 active site does not have a histidine or
other obvious polar residues positioned to directly stabilize
iron-bound ligands. The only polar moieties that are close enough to
directly H-bond to the distal water ligand are the amide nitrogen of
Gly143 and the carbonyl oxygen of Gly139. In
addition, Asp140 could indirectly interact with an
iron-linked peroxide via H-bonded water molecules. Although the distal
and proximal helices must move to clamp down on the heme and to
form the His-Fe bond, and basic side chains reorient to interact with
the heme propionates, Asp140 remains essentially fixed in
the same place in both the apo-HO-1 and heme·HO-1 structures.
Asp140 is held in place through an array of H-bonding
interactions similar to those found in peroxidases. In peroxidases, the
conserved, catalytically essential, distal histidine is H-bonded to an
adjacent asparagine, fixing the imidazole ring at an optimum
orientation, allowing rapid and effective proton abstraction from the
bound peroxide (42-46). In human HO-1, Asp140 is H-bonded
and locked into a fixed space by an H-bonding network involving
Asn210, Arg136, and a second tier of residues
that includes Tyr58 and Try114. This network
remains the same in the apo-HO-1 and heme·HO-1 structures (Fig.
10). Nevertheless, the solvent
structure changes. The open and closed heme·HO-1 structures show that
the Asp140-solvent H-bonded network becomes ordered in the
closed form. It thus appears that, when heme binds and the active site
closes, critical waters become trapped in the distal cavity, where they form part of an essential Asp140-solvent H-bonded network
that may constitute the proton shuttle machinery required for oxygen
activation.

View larger version (22K):
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|
Fig. 10.
Structural comparison of the conserved
H-bonding interaction surrounding Asp140 between apo-HO-1
(black) and the two conformations of heme·HO-1
(gray): closed (A) and open
(B).
|
|
 |
ACKNOWLEDGEMENTS |
Dr. B. Bhaskar and Debrah Makino are
gratefully acknowledged for helpful discussions.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants GM33688 (to T. L. P.) and DK30297 (P. R. O. d. M.).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 atomic coordinates and the structure factors (code 1NI6 and 1N3U) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
¶
Present address: Molecular Neuropathology Group, RIKEN Brain
Science Inst., 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.
**
To whom correspondences should be addressed. Tel.: 949-824-7020;
Fax: 949-824-3280; E-mail: poulos@uci.edu.
Published, JBC Papers in Press, December 24, 2002, DOI 10.1074/jbc.M211450200
 |
ABBREVIATIONS |
The abbreviations used are:
HO, heme oxygenase;
r.m.s., root mean square.
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