odCPO/Hem13p structure Phillips, et al.

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Crystal structure of the oxygen-dependant coproporphyrinogenoxidase (odCPO/Hem13p) of Saccharomyces cerevisiae*

John D. Phillips1§¶, Frank G. Whitby2§, Christy A. Warby1, Pierre Labbe3, ChengYang4, James W. Pflugrath4, Joseph D. Ferrara4, Howard Robinson5, James P.Kushner1, Christopher P. Hill2¶

Departments of Medicine1 and Biochemistry2, University of Utah School of Medicine, Salt LakeCity, UT 84132, USA3 Laboratoire de Biochimie des Porphyrines, Institut Jacques Monod CNRS, Université Paris 7,2 place Jussieu, 75251 Paris Cedex 5 France4 Rigaku/Molecular Structure Corporation, 9009 New Trails Drive, The Woodlands, TX 77381,USA5 Biology Department, 463 Brookhaven National Laboratory, Upton, New York 11973-5000,USA

§ Contributed equally to this work.

¶ Corresponding authors.

* This work was supported by National Institutes of Health grants GM56775 and DK20503.Operations of the National Synchrotron Light Source are supported by the U.S. Department ofEnergy, Office of Basic Energy Sciences, and by the National Institutes of Health.

Running title: odCPO/Hem13p structure

Classification: Biochemistry

Keywords: heme biosynthesis, crystallography, protein structure, coproporphyrinogen oxidase

The atomic coordinates and structure factor amplitudes have been deposited in the proteindatabank with accession codes 1tkm, 1tk1, 1tkl, and 1tlb.

The abbreviations used are: odCPO, oxygen-dependent coproporphyrinogen oxidase; RMSD,root mean square deviation; URO-D, uroporphyrinogen decarboxylase; PDB, Protein Data Bank.

Christopher P. Hill John D. PhillipsDepartment of Biochemistry Department of MedicineUniversity of Utah School of Medicine University of Utah School of MedicineSalt Lake City, UT 84132 Salt Lake City, UT 84132 (801) 585-5536 voice (801) 581-6650 voice(801) 581-7959 fax (801) 585-5469 faxchris@biochem.utah.edu john.phillips@hsc.utah.edu

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Summary

Coproporphyrinogen oxidase (CPO) is an essential enzyme that catalyses the sixth step of

the heme biosynthetic pathway. Unusually for heme biosynthetic enzymes, CPO exists in

two evolutionarily and mechanistically distinct families, with eukaryotes and some

prokaryotes employing members of the highly conserved oxygen-dependent CPO (odCPO)

family. Here we report the crystal structure of the odCPO from Saccharomyces cerevisiae

(Hem13p), which was determined by optimized sulfur anomalous scattering and refined to

a resolution of 2.0 Å. The protein adopts a novel structure that is quite different from

predicted models, and features a central flat seven-stranded antiparallel sheet that is

flanked by helices. The dimeric assembly, which is seen in different crystal forms, is

formed by packing of helices and a short isolated strand that forms a beta ladder with its

counterpart in the partner subunit. The deep active site cleft is lined by conserved residues

and has been captured in open and closed conformations in two different crystal forms. A

substrate-sized cavity is completely buried in the closed conformation by the ~8 Å

movement of a helix that forms a lid over the active site. The structure therefore suggests

residues that likely play critical roles in catalysis, and explains the deleterious effect of

many of the mutations associated with the disease hereditary coproporphyria.

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Introduction

Heme is one of the most common prosthetic groups of proteins in both prokaryotes and

eukaryotes. This abundance reflects essential roles in energy metabolism, stress response,

oxygen transport, and signal transduction. Accordingly, heme is essential in all organisms

tested, heme biosynthetic enzymes have been highly conserved throughout evolution, and

mutations in these enzymes cause several human diseases.

The sixth step in the biosynthesis of heme, which is catalyzed by coproporphyrinogen oxidase

(CPO), is the oxidative decarboxylation of two propionate side chains of coproporphyrinogen III

to form vinyl groups in the product protoporphyrinogen IX (Fig. 1) (1,2). The enzyme from

Saccharomyces cerevisiae is called Hem13p (3). In plants, this step of the heme biosynthetic

pathway is also required for the production of chlorophyll.

CPO is unusual among heme biosynthetic enzymes in that evolution has selected two very

different and unrelated enzymes to catalyze the same reaction (4). Some prokaryotes encode

oxygen-independent CPO (oiCPO), which, as shown by the crystal structure of E. coli oiCPO

(5), are radical SAM enzymes (6,7) that utilize both a 4Fe-4S cluster and S-adenosylmethionine

as cofactors. In contrast, the oxygen-dependent CPO (odCPO/Hem13p) enzymes encoded by

eukaryotes (and some prokaryotes) employ a very different mechanism. There are reports of

requirement for copper (8) and manganese (9), although other studies find no metal ion or other

cofactor dependence (except O2) for odCPO activity (10-12). Regardless of mechanistic details,

the first decarboxylation has been shown to be the rate limiting step for the overall reaction and

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transient formation of the 3-carboxyl intermediate, harderoporphyrinogen, has been

demonstrated (13,14).

The mature odCPO is a ~35 kDa protein that exists as a stable ~70 kDa dimer in solution

(3,9,11,12,15-17). It is located in the mitochondria of higher eukaryotes (1,12,15,18,19) but

resides in the cytosol of S. cerevisiae (16). Despite these different intracellular localizations, the

mature protein sequence has been highly conserved with the only significant differences being

the presence or absence of mitochondrial targeting sequences that are removed during import

(20). Mutations of odCPO cause the autosomal dominant disease hereditary coproporphyria,

with more than 20 different odCPO mutations identified in afflicted families (21).

In an effort to better understand the biochemical basis for catalytic activity and the deleterious

effect of clinically identified mutations, we have determined the crystal structure of yeast (S.

cerevisiae) odCPO/Hem13p in two different crystal forms at resolutions of 2.0 Å and 2.4 Å. The

enzyme adopts a unique fold that presents two independent active sites on the dimeric structure

that are revealed by deep clefts lined with evolutionarily conserved residues. In one crystal form

the cleft is open to bulk solvent, whereas in the other form movement of two helices closes the

cleft entrance to leave a cavity that is the size and shape of a substrate molecule. Finally,

mapping of the mutations associated with coproporphyria indicates that most of these changes

will destabilize the protein structure or distort the active site cavity.

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Materials and methods

Expression and purification of odCPO/Hem13p

A cDNA encoding S. cerevisiae Hem13p was amplified using PCR and cloned into the

expression vector pET16B. Protein was expressed in the E. coli strain BL21 (DE3)plysS

(Novagen, Madison, WI) grown in LB media at 37°C. Induction (500µM IPTG) at an OD600 of

0.5 was followed by growth for four hours before harvesting by centrifugation and storing of

pellets at –80°C. Cell pellets from 6L of culture were resuspended in 50 mL lysis buffer (100

mM NaCl, 50 mM Tris, pH 8.0) and incubated on ice for 1 hour with 1.2 mL of 10 mg/mL

lysozyme prior to sonication. The membrane fraction was removed by centrifugation at 20,000 x

g for 30 min. The supernatant was loaded onto a 1 mL Ni2+-NTA (Qiagen, Chatsworth, CA)

column at 4°C. The column was washed with 40 mL buffer A (300 mM NaCl, 50 mM NaPO4,

pH 7.0, 10% (v/v) glycerol, 2.5 mM -mercaptoethanol), followed by elution of the protein in 30

mL buffer B (300 mM NaCl, 50 mM NaPO4, pH 7.6, 150 mM imidazole, 2 mM -

mercaptoethanol). Fractions containing purified histidine-tagged odCPO/Hem13p were dialyzed

against 4L of 20 mM Tris, pH 7.5, 5% (v/v) glycerol, concentrated to 25 mg/mL using

Centriprep concentrators (Amicon, Beverly, MA), and used in the crystallization trials.

Crystallization

Form C crystals, named because the protein was cleaved during crystallization, grew with bi-

pyramidal morphology in sitting drops at 21°C. The reservoir solution (20% PEG3000, 0.1 M

HEPES pH 7.5, 0.2 M Na-acetate) was mixed with an equal volume of protein solution in the

drop. A washed form C crystal ran with an apparent molecular weight of 30 kDa on SDS-PAGE

(a weaker band was also seen at 10 kDa). N-terminal sequencing of protein from a form C

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crystal revealed that the first 5 residues in the protein were DPRNL, indicating that the

crystallized protein was cleaved at residue Asp6 and suggesting that ~60-70 of the C-terminal

residues had also been removed.

The form I crystals grew with rod morphology in sitting drops at 4, 13, and 21°C. The reservoir

solution was 18% PEG8000, 0.1 M HEPES, pH 7.5, 2% isopropanol, 0.2 M Na-acetate. Drops

were equal parts protein and reservoir solutions. The form II crystals grew with plate

morphology in sitting drops at 21°C. The reservoir solution was 2.2 M ammonium sulfate, 0.1

M Tris, pH 8.5. The drops were a 2:1 mixture of protein and reservoir solutions.

X-ray data collection

All data were collected from crystals maintained at 100 K. Crystals were suspended in rayon

loops and plunged into liquid nitrogen. Form C crystals were transferred directly from the

crystallization drop to liquid nitrogen. Form I and II crystals were first transferred to a

cryoprotectant solution prior to cooling. Cryoprotectant for form I crystals was comprised of

10% (v/v) glycerol added to the reservoir solution, and for the form II crystals was 15% (v/v)

glycerol added to a 1:1 mixture of reservoir solution and 3.5 M aqueous ammonium sulfate.

Data were collected from a single form C crystal using a Rigaku R-AXIS IV imaging plate area

detector on a Rigaku RU-H3R rotating anode x-ray source with a chromium anode that generates

useable X-rays at 2.29 Å wavelength for increased sulfur anomalous signal. The custom-

designed diffraction experiment included Osmic confocal optics to focus the X-ray beam and a

helium path enclosure to minimize air absorption and scattering between the sample and detector

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(22). High-resolution data for refinement were collected from a second form C crystal and from

a form I crystal at the National Synchrotron Light Source (NSLS). Data were collected from a

single form II crystal using an imaging plate area detector on a rotating anode X-ray source with

a copper anode. Crystallographic statistics are given in Table I.

Structure determination and refinement

The form C crystal structure was determined by sulfur SAD phasing. SOLVE (23) was used to

locate seven sulfur positions using 3.3 Å data and to calculate phases based on this solution. The

M value was 0.27 and overall Z score was 19. Density modification was performed with

RESOLVE (24) to produce an electron density map that allowed building of several strands and

helices. Phases from this refined partial model were used to calculate an anomalous difference

Fourier map that confirmed the sulfur substructure and found an eighth site. SOLVE and

RESOLVE were rerun, fixing the eight sulfur positions, to give a slightly improved map (Fig. 2).

Refinement and rebuilding using 1.9 Å data collected at NSLS beamline X8C resulted in a model

of good stereochemistry and agreement with x-ray terms (Table I).

The form I and II crystal structures were determined by molecular replacement using the

program MOLREP (25) with the CCP4i interface (26). The refined form C model was used to

determine the form I structure, which has two molecules in the asymmetric unit. The refined

form I model was subsequently used to determine the form II structure, which has 6 molecules in

the asymmetric unit. In both cases, model building with O (27) and refinement with REFMAC5

(28) were straightforward (Table I).

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Results and discussion

Structure determination

Crystals of histidine-tagged yeast odCPO/Hem13p were grown in three different space groups.

One of these crystal forms is a truncated protein that underwent limited proteolysis during

crystallization to yield a protein that started at Asp6, as indicated by N-terminal sequencing of

protein from a washed crystal. SDS-PAGE analysis gave a molecular weight of approximately

30 kDa, suggesting that protein in these crystals was also cleaved at the C-terminus to remove

the last ~60-70 residues. We refer to these crystals as form C (cleaved). Protein from the other

two crystal forms (forms I and II) migrate similar to full length odCPO/Hem13p on SDS-PAGE.

N-terminal sequencing indicated that form I crystals have been processed at the N-terminus to

start at Ala3. This analysis was not performed on form II crystals.

We were unable to prepare crystals of selenomethionine-substituted odCPO/Hem13p that

diffracted with sufficient strength to allow reliable phase determination. Structure determination

was therefore approached using intrinsic anomalous scattering from the eight sulfur atoms in the

truncated molecule of form C crystals. This crystal form offered the advantage of a relatively

high fraction of sulfur atoms. Data were collected using a rotating anode generator fitted with a

chromium anode, appropriate optics, and helium x-ray path (22). The SAD/solvent flattened

map (Fig. 2) was of sufficient clarity to allow building of an initial model that was subsequently

refined to an Rfactor/Rfree of 22.8/23.1% using 1.9 Å data collected from another form C crystal

at a synchrotron (Table I). This refined model starts at Arg8 and ends at Arg265. A total of 219

ordered residues are included in the model, and three flexible loops and the C-terminal 63

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residues have been omitted; the ordered residues are Arg8-Ala41, Gly51-Lys91, Asp107-

Lys191, and Gly207-Arg265.

The anomalous scattering of sulfur atoms was used in the classic structure determination of

crambin (29). Since then, sulfur anomalous scattering has contributed to a number of structure

determinations (30), although only a few of the new structures have resulted from sulfur

anomalous scattering data collected on a rotating anode source rather than at a synchrotron (31-

34). The primary limitation of this approach is the small anomalous signal obtained from sulfur

with conventional copper anode targets. In view of this, it has been suggested that use of a

chromium anode, which more than doubles the f” of sulfur, might allow a general approach to

crystal structure determination without the need to prepare heavy atom derivatives, modified

protein, or synchrotron radiation (22). To the best of our knowledge, this is the first report of a

new protein structure determined using sulfur anomalous scattering data collected on a

chromium target rotating anode generator. Our experience shows that this approach can work

well for a 30 kDa protein that contains eight ordered sulfur atoms and diffracts to 2.5 Å

resolution on a rotating anode source.

The structures of crystal forms I and II, both of which contain full-length protein, were

subsequently determined using molecular replacement starting with the form C structure. These

models were refined at resolutions of 2.0 Å (form I) and 2.4 Å (form II). The Rfactors/R free

values are 20.7%/25.4% (form I) and 20.8%/28.2% (form II), and the other statistics also

indicate that the models have been refined appropriately (Table I).

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Structure description

The crystallographic refinements resulted in models for the form I and II structures that start

from the first ordered residue (Pro4 form I; Ala3 form II) and extend to the C-terminus.

odCPO/Hem13p forms a seven-stranded anti-parallel beta sheet that is relatively flat and is

covered on both sides by helices (Fig. 3). This structure is essentially identical in all of the

crystal forms. Consequently, the form C structure will not be discussed further because, while

this form was important for structure determination of the full-length protein, it lacks more than

half of the residues that are visible in the form I and II crystals and does not form the

physiologically relevant dimer (discussed below). The two monomers in form I crystals overlap

with an RMSD of 0.6 Å on all pairs of C atoms, and the six monomers in form II crystals

overlap on each other with RMSD of 0.7-1.0 Å over all C . Form I and II monomers overlap

with RMSD values of ~1.5 Å on all pairs of C atoms except those before Arg13 and between

Gln77-Lys110, inclusive. The 12 N-terminal residues project in very different directions in the

two crystal forms, apparently because of different lattice constraints. The Gln77-Lys110 loop

appears to move significantly between the two crystal forms, in part because, as discussed below,

H2 serves as a lid that moves to cover the active site cavity in the form II structure.

odCPO/Hem13p crystallizes as a dimer in which the beta sheets face each other and project

above their surrounding helices (Fig. 4). This is consistent with multiple reports that the enzyme

is dimeric in solution (3,9,15,16). The dimeric interface buries a total of 2,638 Å2 of accessible

solvent surface area and includes 11 direct hydrogen bonding interactions between protein

atoms. There are also five water molecules buried at the interface, although most of the contact

surface is hydrophobic. The 28 residues that lose accessible surface area upon dimerization are

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derived from helices H5, H7, H8 and H9, strands S1, S2, S3, and S8, and connecting segments.

At the center of the interface, S8 forms an antiparallel beta ladder with its two-fold related

partner. The relevance of the crystallographic dimer is indicated by its extensive surface area,

the location of conserved residues at the interface (Fig. 4C), and the observation that the same

dimer is formed by the two monomers in the form I asymmetric unit and the six monomers in the

asymmetric unit of form II crystals. The dimeric arrangement appears to be important for

structure at the active site, since many residues that stabilize dimer formation are close to

residues that line the active site cleft, and failure to dimerize would likely destabilize the active

site conformation.

The active site cleft/cavity

The odCPO sequences have been highly conserved throughout evolution; 76 (23%) of the

residues are invariant between ten highly diverged species that range from cyanobacteria to man

(Fig. 3B). The invariant residues are mostly buried in the hydrophobic core or at the dimer

interface, while the surface exposed invariant side chains are primarily centered about a deep

cleft of the form I crystal structure that appears to house the enzyme active site (Fig. 5). The

cleft is sandwiched between one face of the beta sheet (strands S3-S7) and helices H7-H9. These

secondary structural elements pack against each other at the base of the cleft. Residues from

helix H4 also contribute to the base and to one side of the cleft, and helix H2 is positioned above

the cleft in the form I structure, rather like a raised lid.

Remarkably, the active site cleft is not open to bulk solvent in the form II crystal structure (Fig.

5B). Instead, the top of the cleft has been closed by helix H2, for which equivalent C atoms

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move by 6-9 Å between form I and form II structures (Fig. 5D). Helix H8 also moves by ~4 Å,

and these two helices pack tightly against each other in the form II conformation to close the

active site cleft. As a consequence of this conformational change, the form II active site is

completely sequestered from bulk solvent and encloses a cavity that approximates the size and

shape expected for the substrate molecule. The 30 residues that expose accessible surface area to

the cavity are indicated with green dots in figure 3B, and the importance of the cavity for

enzymatic function is further supported by the observation that 18 of these residues are invariant.

The completely buried active site explains why substitutions throughout the tetrapyrrole

macrocycle limit catalytic turnover (35-37).

Because of the striking match between the dimensions of the enclosed cavity of the form II

structure and a substrate molecule, we have modeled coproporphyrinogen into this space. It

appears that the few close contacts that result in the model might be relieved by relatively minor

changes such as adjustment of side chain rotamer angles. The substrate molecule modeled in this

way would also occupy an unimpeded position in the form I structure, and it is included in figure

5 for illustrative purposes. We are cautious about proposing specific contacts based upon this

crude modeling exercise, although the model does suggest some tentative conclusions about

substrate binding. The bound tetrapyrrole appears constrained to lie with the macrocycle plane

oriented approximately as shown in the figure. The specific locations of propionate side chains

are not obvious, although candidate binding partners include the two invariant positively charged

side chains exposed to the cavity, Arg135 and Arg275. Other candidate propionate ligands

include the invariant polar side chains of Ser72, Ser117, His131, and Asn133, as well as a

number of main chain amide groups. We speculate that the central NH groups of the substrate’s

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four pyrrole rings are coordinated by the invariant side chain of Asp274, which is the only

invariant carboxylate side chain exposed to the cavity and is located near the middle of one face.

This possibility is attractive since uroporphyrinogen decarboxylase (URO-D), which catalyzes

the preceding step in heme biosynthesis and provides the best model for cyclic tetrapyrrole

binding by an enzyme, uses an invariant aspartate side chain to coordinate all four pyrrole NH

groups of its substrate (38). Precisely determining the substrate binding geometry and locating

the specific sites of oxygen binding and the decarboxylation reaction, which are not currently

apparent, will be a priority for our future experiments.

Comparison with other structures

As expected, the structure is very different from that of the unrelated oxygen-independent

oiCPO, which is a monomeric two-domain protein whose most prominent feature is a curved

parallel beta sheet (5). It was predicted, based upon sequence analysis, that odCPO/Hem13p

would fold as two T-fold domains (39); T-folds are comprised of four sequential antiparallel beta

strands and a pair of antiparallel helices between the second and third strands (40). The observed

odCPO/Hem13p structure, however, is quite different from this prediction. Furthermore, T-fold

proteins have a distinctive mode of assembly in which 3-5 protein subunits form rings of 12-20

beta strands, and two of these rings pack face to face (40). This results in assembly around a

distinctive central tunnel (T-fold; Tunnel-fold). Thus, odCPO/Hem13p does not possess

significant similarity with the T-fold proteins in the structure of the monomer or in higher order

assembly.

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A search with DALI (41) found just one structure, LMAJ006828, with significant similarity

(Z=39) to odCPO/Hem13p. LMAJ006828 is a hypothetical protein from Leishmania major that

was determined by the Structural Genomics of Pathogenic Protazoa program

(http://depts.washington.edu/sgpp) at 1.4 Å resolution and recently deposited in the Protein Data

Bank (PDB) with entry code 1VJU. There is no corresponding publication currently available

for this structure and the PDB header describes it as having unknown function. LMAJ006828

has the same topology and forms the same dimer as odCPO/Hem13p; 263/291 C atoms

superimpose with an RMSD of 1.0 Å for the monomer, and 526/582 C atoms superimpose with

an RMSD of 1.5 Å for the dimer. Note that this dimer is not the same as implied by the

asymmetric unit defined in the 1VJU PDB entry, although it is revealed by application of

crystallographic symmetry operators and has a much larger interface than the other lattice

contacts of the deposited structure. The high level of structural similarity is reflected in the

amino acid sequence; 37% of the residues are identical to their structural counterparts in yeast

odCPO/Hem13p, including all but four of the 76 invariant residues indicated in figure 3B. This

high level of structural and sequence similarity strongly implies that LMAJ006828 is a

coproporphyrinogen oxidase. LMAJ006828 residues are disordered between strands S2 and S3,

indicating that this structure is in an open conformation. Indeed, it seems likely that the

disordered conformation for the H2 loop corresponds to the fully open state, whereas the form I

structure seen for yeast odCPO/Hem13p, in which the opening to the active site cleft is only ~8

Å between atom centers, is better viewed as midway between the fully open and fully closed

conformations.

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Clinically identified mutations

A number of mutations have been identified that encode full-length odCPO protein yet appear to

cause hereditary coproporphyria. Because of the high degree of sequence identity (52%)

between yeast Hem13p and human odCPO, we have mapped these 19 mutations onto the

Hem13p structure (Table II, Fig. 6). The deleterious effect of most of these mutations can be

explained as likely caused by a decrease in stability. These “destabilizing” residues are scattered

over the structure, including at the dimer interface. We are unable to explain the deleterious

effect of mutation at the surface-exposed positions of 169, 182, and 321 (yeast Hem13p

numbering), although it is impossible to rule out an effect on stability or perhaps mediation of an

as yet unidentified protein-protein contact. In contrast, the mutations at residue Ser72 and

Arg275 are especially suggestive. These residues are both invariant, and although these

substitution seem unlikely to greatly perturb stability, they would alter the size, shape, and

polarity of the active site cavity.

In summary, the structure explains how many of the mutations give rise to coproporphyria and

supports the model that the enzymatic reaction proceeds in an isolated cavity that is formed by

conformational change upon binding substrate. If the reaction intermediate is able to reposition

within the active site cavity, this architecture would explain why the first decarboxylation, on the

pyrrole A ring, is rate limiting and rapidly followed by decarboxylation of the pyrrole B ring

propionate (13,14). One potential advantage of the substrate-induced conformational change is

that it might generate a specific pathway and binding site for the molecular oxygen cofactor,

such as seen for cholesterol oxidase (42,43). This is an attractive possibility, since it would

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provide a mechanism to protect the highly oxygen sensitive substrate and product from

inappropriate oxidation.

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Table I. Crystallographic data and refinement

COP-OX Crystal Form C C I II

CrystalSpace group P41212 P41212 P43212 C2Cell dimensions (Å) a=71.8, c=118.1 a=70.9, c=114.5 a=86.8, c=207.8 a=233.7, b=65.4,

c=166.5, =108.1°# mols/AU 1 1 2 6

Crystallographic DataWavelength (Å) 2.29 1.10 1.10 1.5418Data collectiona RAXIS-Cr NSLS-X8C NSLS-X25 RAXIS-CuData processingb d*TREK DENZO DENZO DENZOResolution (Å) 38.5-2.50 30.0–1.90 30.0–2.00 30.0–2.40

(2.59-2.50) (1.97–1.90) (2.07–2.00) (2.55–2.40)# Refs Measured 222,966 661,152 486,505 478,664# Unique Reflns 20,453 23,022 53,840 87,606Complete (%) 99.9 (99.3) 97.0 (95.6) 98.4 (97.5) 93.9 (90.7)<I/ I> 24.3 (4.2) >20 (6.0) 19 (3.2) 15 (2.5)Mosaicity (o) 0.40 0.58 0.47 0.83Rsymc (%) 0.057 (0.383) 0.072 (0.580) 0.057 (0.583) 0.077 (0.309)

Refinement StatisticsResolution (Å) 30.0–1.90 30.0–2.00 30.0–2.40

(1.97–1.90) (2.05–2.00) (2.46–2.40)Rcrystd (%) 0.228 (0.251) 0.207 (0.258) 0.208 (0.248)Rfreee(%) 0.231 (0.290) 0.254 (0.301) 0.282 (0.350)Protein residues 8-265 f 4-328 g 3-328 g

# solvent molecules 96 418 639h

RMSD bonds (Å) 0.011 0.016 0.021RMSD angles (°) 1.441 1.507 1.832

/ anglesi

Most favored (%) 91.3 94.0 90.1 Additional allowed(%) 7.1 6.0 9.6<B>protein (Å2) 29.8 42.7 47.2<B>main-chain (Å2) 29.2 42.4 47.1<B>water (Å2) 40.3 43.4 38.9

Values in parentheses refer to the high-resolution shell.a Data were collected on Rigaku RAXIS-IV detectors mounted on rotating anode sources withchromium (RAXIS-Cr) or copper (RAXIS-Cu) targets. Other data were collected on ADSCQuantum-4 CCD area detectors at beamlines X8C or X25 at Brookhaven National LaboratoryNational Synchrotron Light Source (NSLS).b Data processing was performed using DENZO/SCALEPACK (44) or d*TREK (45).c Rsym = |I-<I>|/ I where I is the intensity of an individual measurement and <I> is thecorresponding mean value.

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d Rcryst = ||Fo|-|Fc||/ |Fo|, where |Fo| is the observed and |Fc| the calculated structure factoramplitude.e Rfree is the same as Rcryst, calculated with a randomly selected test set of reflections (5% oftoal) that were never used in refinement calculations.f 219 residues were modeled. Disordered regions of the protein that could not be modeled are:42-50, 92-106, 192-206.g The full-length protein has been modeled as a single chain in all molecules in the asymmetricunit. For form II, residues 91-110 have only diffuse density and were assigned zero occupancies.h Includes one sulfate ion.i For non-Gly and non-Pro residues only.

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Table II. Human mutations in full length odCPO associated with coproporphyria

Yeast Human HumanRes # Res # Mut.(Ref) Comment on StructureE26 Q162 P (46) Destabilizing. Residue in helix H1. Incompatible with Pro.D35 D171 N (46) Destabilizing. Buried side chain. O 1 and O 2 H-bond m-c NH.G53 G189 S (47) Destabilizing. Buried residue. No room for Ser side chain.G61 G197 W (48) Destabilizing. Phi angle +ve. No room for Trp side chain.E65 E201 K (49) Destabilizing. Lose H-bond to Arg263.G67 A203 T (46) Destabilizing. Buried residue. Restricted environment for Thr.S72 S208 F (50) Active site. Projects into cavity.P122 P249 S (49) Destabilizing. Buried side chain making extensive contacts.G154 G280 R (51) Destabilizing. +ve phi and no room for a side chain.G167 A293 T (46) Destabilizing. Buried residue. Restricted environment for Thr.L169 H295 D (52) Surface exposed residue. May destabilize H3-H6 packing.D182 G308 V (46) Surface exposed residue. May destabilizes loop structure.R202 R328 C (50) Destabilizing. Buried. H-bonds E268, and E289 of dimer partner.T205 R331 W (53) Destabilizing. In surface pocket unable to accommodate Trp.R265 R391 W (46) Destabilizing. Buried residue making extensive contacts. R275 R401 W (46) Active site. Trp would restrict cavity.Q278 K404 E (54) Destabilizing. H-bonds CO groups at C-terminus of helix H7.W301 W427 R (48) Destabilizing. Buried at dimer interface.T321 R447 C (46) Surface exposed. No explanation.

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Figure legends

FIG. 1. Schematic of reaction catalyzed by odCPO/Hem13p. Propionate side chains on the

A and B pyrrole rings are decarboxylated to form vinyl groups and two molecules of carbon

dioxide. Molecular oxygen is converted to hydrogen peroxide, presumably via abstraction of a

hydrogen atom from each of the propionate/vinyl C atoms.

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FIG. 2. Map computed from sulfur anomalous scattering. The solvent flattened SAD map

(cyan; 1.3 x RMSD) was computed at 3.3 Å resolution using data collected on a rotating

chromium anode. An anomalous difference map (red; 4 x RMSD) computed using phases

derived from the refined model shows the positions of Met and Cys residues. The final refined

model (yellow) is shown as a C trace. Top – adjacent helices. Bottom – a region of sheet.

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FIG. 3. Structure of odCPO/Hem13p

(A) Ribbon diagram. The chain is color ramped from blue (N-terminus) to red (C-terminus).

Secondary structural elements are labeled. Stereoview of odCPO/Hem13p with secondary

structural elements and N/C termini labeled. Figures 3A, 4, 5, and 6 were made using PyMOL

(55).

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(B) Amino acid sequence. The sequences of Saccharomyces cerevisiae Hem13p and human

odCPO are shown with secondary structural elements of the Hem13p crystal structure above.

Residues invariant across an alignment of 10 diverse odCPO sequences after alignment with

Clustal W (56) are shown on a magenta background. Sequences used in the analysis were from

Saccharomyces cerevisiae, Drosophila melanogaster, Homo sapiens, Aplysia californica, Mus

musculus, Nicotiana tabacum, Synechocystis sp. PCC 6803, Escherichia coli, Ralstonia

solanacearum, and Agrobacterium tumefaciens str. C58. Residues that expose at least 10 Å2 of

accessible surface area to the active site cavity of the form II structure are indicated with a green

dot. Residues that lose surface area upon dimer formation are indicated with a blue square.

Black diamonds indicate positions of mutations identified in cases of coproporphyria (Table II).

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FIG. 4. The odCPO/Hem13p dimer.

(A) Ribbon diagram. Top view, looking along the non-crystallographic two-fold axis. Form I

structure.

(B) Side view, two-fold axis vertical.

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(C) Stereoview of residues at the dimer interface. Residues that lose surface area upon dimer

formation (blue squares of figure 3B) are shown explicitly and colored magenta if invariant. A

modeled substrate molecule (white) indicates proximity of the dimer interface to the active site

cavity.

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FIG 5. Active site cleft/cavity.

(A) Form I crystal structure. Molecular surface of the odCPO/Hem13p dimer. Monomers are

colored gray and slate. Invariant residues are colored magenta. Substrate modeled into active

site, white. The view direction is similar to that of figure 4B, but with the two-fold axis tilted

from the vertical to show the cleft.

(B) Same as panel A but for the form II crystal structure. The modeled substrate is completely

sequestered in the active site cavity and hidden from view. As seen, the projections at the top of

this figure (loop between H2 and S4) have been built in very different conformations in form I

and II structures. To some extent, this results from displacement of H2 upon closing the active

site cavity (see text), although these residues are highly flexible and have been assigned zero

occupancy in the refined models.

(A) (B)

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(C) Stereoview of the form I active site cleft from panel A. A semitransparent surface is

displayed with helices H2 (cyan) and H8 (yellow) below. These helices close together to seal the

active site cavity in the form II structure.

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(D) Stereoview ribbon diagram showing open conformation (Form I; orange) and closed

conformation (Form II; green). The helices that move to enclose the active site cavity (H2 and

H8) are labeled.

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FIG. 6. Location of mutations identified in coproporphyria patients.

Worm representation stereoview of form II (closed) structure in same orientation as Figure 3A.

Sites of mutations identified in patients are shown as spheres. Substitutions expected to

destabilize the folded protein structure (gray). Mutations whose presumed deleterious effect is

not easily explained by the structure (blue). Mutations at the active site cleft (magenta). The

modeled substrate molecule (white) indicates the approximate location of the active site cavity.

Acknowledgments – We thank Heidi Schubert for critical comments on the manuscript. Data

collection at the NSLS was funded by the National Center for Research Resources.

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Pflugrath, Joseph D. Ferrara, Howard Robinson, James P. Kushner and Christopher P. HillJohn D. Phillips, Frank G. Whitby, Christy A. Warby, Pierre Labbe, Cheng Yang, James W.

(odCPO/Hem13p) of Saccharomyces cerevisiaeCrystal structure of the oxygen-dependent coproporphyrinogen oxidase

published online June 12, 2004J. Biol. Chem. 

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