Comparison of the Heme-free and -bound Crystal Structures of Human Heme Oxygenase-1*

Latesh LadDagger , David J. Schuller§, Hideaki ShimizuDagger , Jonathan FriedmanDagger , Huiying LiDagger , Paul R. Ortiz de Montellano||, and Thomas L. PoulosDagger **

From the Dagger  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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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).


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Fig. 1.   Overall reaction catalyzed by heme oxygenase.

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 alpha -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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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REFERENCES

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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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 3sigma level and weak 2Fo - Fc electron density at the 1sigma 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 alpha -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 1.5sigma . 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 alpha -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 alpha -meso-carbon.

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 1sigma 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 II
r.m.s. deviation of backbone atoms between the four molecules of the human apo structure

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-alpha 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).

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 alpha -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 beta -carbon of Leu141, the N-zeta atom of Lys179, the carboxylate side chain of Glu145, and the C-delta 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 beta -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-delta 1 atom of Leu138 is broken, causing the side chain of Leu138 to move away from the heme site, thus reorienting the C-delta 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.

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.

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 delta -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-epsilon 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-epsilon 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-epsilon atom of Gln38 in molecule D H-bonding to the C-delta 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 delta -meso edge, is exposed. The alpha -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 beta -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 beta -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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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-epsilon 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 alpha -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 beta -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.


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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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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