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J. Biol. Chem., Vol. 280, Issue 4, 2840-2846, January 28, 2005

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Staphylococcus aureus IsdG and IsdI, Heme-degrading Enzymes with Structural Similarity to Monooxygenases^*

Ruiying Wu, Eric Patrick Skaar¶||, Rongguang Zhang, Grazyna Joachimiak, Piotr Gornicki**, Olaf Schneewind¶, and Andrzej Joachimiak¶¶

From the Structural Biology Center and Midwest Center for Structural Genomics, Biosciences Division, Argonne National Laboratory, Argonne, Illinois 60439 and the Departments of ^¶Microbiology, ^**Molecular Genetics and Cell Biology, and Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637

Received for publication, August 18, 2004 , and in revised form, October 27, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Heme-degrading enzymes are involved in human diseases ranging from stroke, cancer, and multiple sclerosis to infectious diseases such as malaria, diphtheria, and meningitis. All mammalian and microbial enzymes identified to date are members of the heme oxygenase superfamily and assume similar monomeric structures with an all -helical fold. Here we describe the crystal structures of IsdG and IsdI, two heme-degrading enzymes from Staphylococcus aureus. The structures of both enzymes resemble the ferredoxin-like fold and form a -barrel at the dimer interface. Two large pockets found on the outside of the barrel contain the putative active sites. Sequence homologs of IsdG and IsdI were identified in multiple Gram-positive pathogens. Substitution of conserved IsdG amino acid residues either reduced or abolished heme degradation, suggesting a common catalytic mechanism. This mechanism of IsdG-mediated heme degradation may be similar to that of the structurally related monooxygenases, enzymes involved in the synthesis of antibiotics in Streptomyces. Our results imply the evolutionary adaptation of microbial enzymes to unique environments.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Staphylococcus aureus acquires iron, an essential nutrient required for infection, by binding host heme-carrying proteins and extracting and transporting heme across the bacterial cell wall and plasma membrane envelope (1-3). Once inside the staphylococcal cytoplasm, heme is either incorporated into bacterial heme proteins or degraded to release iron for subsequent incorporation into polypeptides and cofactors (4-6). One heme acquisition system, encoded by the isd (iron-regulated surface determinants) gene cluster, encodes the heme-degrading enzyme IsdG (1, 5). IsdI, a homolog of IsdG, is encoded elsewhere on the staphylococcal chromosome (5). IsdG and IsdI show no significant sequence similarity to known heme oxygenases (7-10) and do not contain the conserved N-terminal histidine or the GXXXG motif characteristic for these enzymes. Nonetheless, purified IsdG and IsdI cleave heme tetrapyrrole in the presence of suitable electron donors (2, 5). This activity can functionally substitute for the classical heme oxygenase activity and permits growth of Corynebacterium ulcerans (5) lacking the HmuO heme oxygenase on media with hemin as a sole source of iron (8). Here we address the question of whether IsdG and IsdI have structures and catalytic mechanisms similar to those of members of the heme oxygenase superfamily (11), enzymes degrading heme in the cytoplasm of eukaryotic cells and in some bacterial species such as Corynebacterium diphtheriae, Pseudomonas aeruginosa, and Neisseria spp (9).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Preparation of Proteins for Crystallization—The cells were grown at 37 °C in Luria-Bertani broth in the presence of 100 µg/ml ampicillin and 30 µg/ml kanamycin, respectively. Expression of His-tagged fusion proteins in Escherichia coli strain BL21(DE3) (12) was induced with 1 mM D-isopropyl--thiogalactoside when the optical density at 600 nm reached 0.6 and incubated at 20 °C overnight. The cells were harvested by centrifugation and suspended in five volumes of lysis buffer containing 50 mM HEPES, pH 8.0, 300 mM NaCl, 10 mM imidazole, 10 mM -mercaptoethanol, 5% glycerol, and inhibitors of proteases (P8849; Sigma-Aldrich). For IsdG cells obtained from 2 liters of culture (5.0 g) were incubated for 30 min on ice with 1 mg/ml lysozyme (Sigma-Aldrich) followed by sonication (6 x 30 s, on ice) (13). The sample was centrifuged at 30,000 x g (RC5C-Plus centrifuge; Sorvall) for 20 min, and supernatants were filtered through 0.4- and 0.22-µm in-line membranes (Gelman Sciences Inc., Ann Arbor, MI). His₆-tagged IsdG was purified by affinity chromatography using Ni-NTA¹ Superflow resin (Qiagen, Valencia, CA). Eluted IsdG (total yield, 119 mg) was treated with the His₇-tagged tobacco etch virus protease to remove the His₆ tag for 16-24 h at 4 °C following basic protocol (13). The cleavage was monitored by SDS-PAGE and Coomassie Brilliant Blue R (Amersham Biosciences) staining. After the cleavage, the reaction mixture was applied to a 1-ml Ni-NTA column, and the column was washed with 3 column volumes of buffer A. All of the chromatographic steps were performed at 22 °C. The column flow-through and wash were collected and then applied to a desalting column Sephadex G-25 fine XK 26/20 (Amersham Biosciences) and equilibrated with storage buffer containing 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 2 mM dithiothreitol. The protein was analyzed by SDS-PAGE stained with Coomassie Brilliant Blue R. The IsdG was concentrated with simultaneous buffer exchange using Centriplus-3 (Amicon Bioseparations, Bedford, MA) (3-kDa cutoff). The final IsdG yield was 90 mg.

IsdI was purified using this same procedure (13). 6.8 g of cells obtained from 2 liters of culture were treated with lysozyme (Sigma) followed by sonication (6 x 30 s, on ice) (13). The sample was centrifuged at 30,000 x g for 20 min, and the supernatants were filtered through 0.4- and 0.22-µm in-line membranes (Gelman Sciences Inc.). His₆-tagged IsdI was purified by affinity chromatography using Ni-NTA resin. The total yield of IsdI after Ni-NTA column was 110 mg. The IsdI was also treated with the His₇-tagged tobacco etch virus protease to remove the His₆ tag. The cleavage was monitored by SDS-PAGE. After the cleavage, the reaction mixture was applied to a 1-ml Ni-NTA column, and the column was washed with 3 column volumes of buffer A. The column flow-through and wash were collected and then applied to a desalting column Sephadex G-25 fine XK 26/20 (Amersham Biosciences) equilibrated with storage buffer containing 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 2 mM dithiothreitol. The protein was analyzed by SDS-PAGE. The protein was concentrated with simultaneous buffer exchange using Centriplus-3 (Amicon Bioseparations, Bedford, MA) (3-kDa cut-off). The final IsdI yield was 78 mg. Selenomethionine-labeled IsdI and IsdG proteins were prepared using regular expression strains in M9 medium and the methionine biosynthesis inhibition method (14). The protein expression and final yield were very similar to those of native IsdG and IsdI proteins.

A 2 mM protein stock solution in 10 mM Tris-HCl, pH 7.4, 20 mM NaCl, and 1 mM dithiothreitol were used for crystallization. The best crystals of IsdI were obtained at 295 K using vapor diffusion and hanging droplets in VDX plates (Hampton Research) from sodium cacodylate buffer pH 6.5 and 250 mM NaCl using 25% polyethylene glycol 4000 as a precipitating agent. The IsdI crystals reach the data collection dimensions (0.3 x 0.2 x 0.2) in 2 days. The best crystals of IsdG were obtained at 295 K using vapor diffusion and hanging droplets in VDX plates from 100 mM Bis-Tris HCl buffer, pH 6.5, 200 mM NH₄SO₄ using 30% polyethylene glycol 4000 as a precipitating agent. For cryoprotection of crystals, the polyethylene glycol 4000 concentration was increased to 33%, and the crystals were flash frozen in liquid nitrogen. The IsdG crystals reach the data collection dimensions (0.2 x 0.2 x 0.1 mm) in 3 days.

Data Collection—Diffraction data were collected at 100 K at the 19ID beam line of the Structural Biology Center at the Advanced Photon Source, Argonne National Laboratory. For IsdI, the three-wavelength inverse beam multi-wavelength anomalous diffraction (MAD) data set (peak, 12.6603 keV (0.9794 Å); inflection point, 12.6620 keV (0.9793 Å); high energy remote, 13.0000 keV (0.9538 Å)) was collected from a selenomethionine-labeled protein crystal. One crystal (0.3 x 0.2 x 0.2 mm) was used to collect all MAD data to 1.5 Å resolution, with 5 s of exposure/1°/frame using a 200-mm crystal to detector distance. The total oscillation range was 180 degrees as predicted using strategy module of the HKL2000 suite (15). The space group was P2₁ with cell dimensions of a = 45.23 Å, b = 38.01 Å, c = 61.55 Å, = = 90°, = 93.9°. All of the data were processed and scaled with HKL2000 (Tables I and II). For phase extension and model refinement using CNS, peak data were used.

Expression and Purification of IsdG for Biochemical Analysis— E. coli BL21(DE3) strains carrying pET15BisdG were grown overnight at 37 °C in Luria-Bertani broth containing 100 µg/ml ampicilin. The cells were subcultured into fresh medium and grown at 37 °C to mid-log phase. At this time, the expression of the vectors was induced using 1 mM isopropyl--D-thiogalactopyranoside. Cell growth was continued for 3 h at 30 °C, and the cells were harvested by centrifugation (10,000 x g for 15 min). The cells were lysed at 14,000 p.s.i. using a French press after suspension in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl containing 100 µM phenylmethylsulfonyl fluoride. The cell suspension was then centrifuged at 100,000 x g for 60 min. After centrifugation, the soluble supernatant was applied to the Ni-NTA column, equilibrated with 50 mM Tris-HCl, pH 7.5, 150 mM NaCl. The column was washed with 20 volumes of 50 mM Tris-HCl, pH 7.5, 150 mM NaCl followed by a second washing with 30 volumes of 50 mM Tris-HCl, pH 7.5, 150 mM NaCl containing 10% glycerol and 10 mM imidazole. The protein was then eluted in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl containing 500 mM imidazole, and the fractions were dialyzed against 50 mM Tris-HCl, pH 7.5, 150 mM NaCl. The purified proteins were stored at -20 °C. The protein yields were similar to those described previously for IsdG and IsdI (5).

Generation of Mutant IsdG Proteins—IsdG proteins containing amino acid substitutions were created using the Pfu mutagenesis technique (Stratagene, La Jolla, CA). Briefly, complimentary oligonucleotides containing the desired alanine mutation were synthesized (IDT Technologies, Coralville, IA; oligonucleotide sequence available upon request) and used in polymerase chain reactions with pET15BisdG as template. PCR conditions followed recommendations by the manufacturer (Stratagene) including 1x reaction buffer, 10 ng of template DNA, 22 nM oligonucleotide primers, 200 µM equivalently mixed dNTPs, and water to a final volume of 50 µl. The cycling parameters included 16 cycles comprised of 30 s at 94 °C, 1 min at 55 °C, and 12 min at 68 °C. Following PCR, 1 µl of the restriction enzyme DpnI was added directly to each amplification reaction to digest methylated DNA, and the reactions were incubated overnight at 37 °C. Following digestion, DNA was transformed into DH5, and successful transformants were selected on ampicillin agar. Successful incorporation of single amino acid changes were verified by DNA sequencing, and mutant proteins were purified as described above.

Hemin Degradation Assay—All of the absorption spectra were obtained using a Cary 50BIO (Varian, Walnut Creek, CA). Ascorbic acid-dependent degradation of hemin was monitored spectrophotometrically as previously described (7) with the listed changes. IsdG-hemin (10 µM) in 50 mM Tris-HCl, pH 8.0, was incubated at 37 °C with ascorbic acid at a final concentration of 10 mM. The spectral changes between 300 and 800 nm were recorded every 2 min for the duration of the experiment.

Coordinates—The coordinates and the structures factors have been deposited in the Protein Data Bank (accession codes 1SQE [PDB] and 1XBW).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Crystal Structure of IsdG and IsdI—The high resolution crystal structures of S. aureus IsdG and IsdI in their native forms were determined at 1.9 and 1.5 Å resolution, respectively (Tables I, II, III, IV). Crystallographic phases were determined by MAD from selenomethionine-containing enzymes. IsdG and IsdI crystallized as homodimers in the monoclinic space group but in two different crystal packing environments: IsdI with one dimer in asymmetric unit and IsdG with two dimers in asymmetric unit. Both proteins assume a virtually identical ferredoxin-like fold consistent with their 64% sequence identity (78% similarity) (Fig. 1). The IsdI and IsdG structures can be superimposed with root mean square deviation 1.03 Å over 100 residues. The distance matrix alignment analysis placed these two proteins in the + sandwich superfold family, defined by the ferredoxin-like + sandwich with an anti-parallel -sheet. Each monomer is assembled into a very tight dimer (Fig. 2) similar to the ActVA-Orf6 monooxygenase from Streptomyces coelicolor (Protein Data Bank entry 1LQ9 [PDB] ) (21) (Fig. 3), a protein with little sequence similarity to IsdG and IsdI. The structures can be superimposed with root mean square deviation 1.92 Å over 112 residues. IsdG and IsdI also show structural similarity to several other proteins in the Protein Data Bank. The closest structural homolog is TT1380, a hypothetical protein from Thermus thermophilus (root mean square deviation 1.89 Å, 102 residues; Protein Data Bank entry 1IUJ [PDB] ). Other structural homologs include proteins with ferredoxin-like folds such as the hypothetical protein YjcS from Bacillus subtilis (Protein Data Bank entry 1Q8B [PDB] ). IsdG amino acid numbering is used throughout the text to describe the structures and results of functional analysis.

View larger version (79K): [in this window] [in a new window]   FIG. 1. The crystal structure of IsdG and IsdI. Residues fully conserved in all members of the IsdG family are shown in green, and other strongly conserved residues are shown in gray. A and B, IsdG dimer with subunit A is labeled in shades of blue, and subunit B is labeled in shades of orange.In B, the homodimer of IsdG is presented with the 2-fold axis of symmetry perpendicular to the plane of the picture in A. C and D, IsdI dimer with subunit A is labeled in shades of aqua, and subunit B is labeled in shades of pink. In D, the homodimer of IsdI is presented with the 2-fold axis of symmetry perpendicular to the plane of the picture in C. E, IsdG homodimer in the same orientation as B. F, IsdI homodimer in the same orientation as D, both showing predicted heme binding pockets. C (light gray) and N (dark gray) termini are shown along with the predicted binding sites of the heme substrate (red).

View larger version (25K): [in this window] [in a new window]   FIG. 2. Stereo image of the IsdI dimer. Both subunits are shown with the secondary structure elements. The N and C termini are labeled.

View larger version (60K): [in this window] [in a new window]   FIG. 3. Comparative structural analysis of the IsdG and ActVA families of monooxygenases. A, ActVA-Orf6 homodimer with subunits shaded in blue and red. B, IsdG homodimer with subunits shaded in orange and green. C, IsdI homodimer with subunits shaded in aqua and pink. D, residues predicted to be involved in binding and oxidation of the substrate in ActVA-Orf6 (blue) that are conserved in IsdG (green) and IsdI (pink). E, residues shown to be required for oxidation of heme in IsdG (green) and the equivalent residues in IsdI (pink). ActVA-Orf6 residues that occupy a similar space as the catalytic residues of IsdG are shown in blue.

View larger version (48K): [in this window] [in a new window]   FIG. 4. Multiple sequence alignment of IsdG, IsdI, and members of the ActVA family of monooxygenases. Residues involved in substrate binding and catalysis in the IsdG family are shown in red. Residues involved in substrate binding and catalysis in the ActVA family are shown in blue. Regions of strong sequence similarity are shown in gray.

Putative Active Site of IsdG and IsdI—Four short -helices of each subunit decorate the outside of the IsdG and IsdI -barrel. Two large, symmetry-related cavities are formed between these -helices and the -sheets of the -barrel. All of the -helices contribute residues to the cavity. The cavity includes the -sheet residues 2-12 (1) and residues 71-79 from -helix 3 (Figs. 1, E and F, and 3D). A similar cavity was shown to nest the ActVA-Orf6 monooxygenase active site (21). In IsdG and IsdI, the surface of this cavity is lined mainly with hydrophobic side chains. The edges of the cavity are hydrophilic with several positively and negatively charged residues resulting in a positively charged surface near the cavities and negatively charged surface further away. It appears that IsdG and IsdI dimers may present two separate cavities for heme binding and oxygenation. Interestingly, there is no obvious site for metal cofactor binding. IsdI and IsdG seem to carry out oxygenation of the heme without the assistance of any of the prosthetic groups or cofactors normally associated with activation of molecular oxygen. However, enzyme cofactors in the form of reductant or reductase are required for the reaction to proceed.

The majority of residues lining the putative active site cavities are similar in IsdG and IsdI; however, conformation of some side chains in the cavity is different (for example Trp⁶⁷ and His⁷⁷), suggesting some flexibility and structural adjustments of the active site upon substrate (heme) binding. In fact, a loop located near the active site is disordered in both structures (residues 81-88 in IsdG and residues 80-87 in IsdI). The majority of the amino acid substitutions between IsdG and IsdI occur on the protein surface, whereas the electrostatic potential near the active site cavities is conserved.

Conserved Residues in the Putative Active Site of IsdG Are Required for Heme Degradation—The dimeric -barrel type structure of IsdG and IsdI is in stark contrast with the monomeric -helical folding pattern of the heme oxygenase family, suggesting that they represent a novel family of heme-degrading enzymes. IsdG and IsdI homologs can be identified in numerous Gram-positive pathogens including Bacillus anthracis, the causative agent of anthrax, and Listeria monocytogenes, an important food-borne pathogen (Fig. 5A). Amino acid residues in the putative heme binding cavity, as well as at the interface between the monomers are highly conserved among members of the family (Figs. 1 and 4). The precise location of the active site could not be determined directly from the structures of free enzymes (without bound heme).

View larger version (75K): [in this window] [in a new window]   FIG. 5. Conserved residues required for IsdG family mediated heme degradation. A, multiple sequence alignment of the IsdG family of proteins and their secondary structure. Green and gray side chains represent absolutely or strongly conserved residues, respectively. B-I, UV spectral analysis of reactions between heme and ascorbic acid, the electron donor, catalyzed by IsdG mutants. The Soret peak at 412 nM is indicative of the intact porphyrin ring of heme, and a decrease in absorbance at this wavelength indicates heme degradation. Spectra at time 0 are shown in red, and spectra at 30 min are shown in blue, with intermediate measurements shown in gray. B, wild-type IsdG; C, N7A; D, K17A; E, F23A; F, M38A; G, W67A; H, S70A; I, H77A.

View larger version (18K): [in this window] [in a new window]   FIG. 6. The putative heme binding cavity of IsdG and IsdI. A, IsdG and B, IsdI. Heme (red) is shown in the model proposed based on the enzymatic data of the IsdG mutants. Carbon (gray), oxygen (purple), nitrogen (blue), and sulfur (yellow) atoms are shown. The IsdG residues Asn7, Trp67, and His77 are required for heme degradation, and Met38 is required for maintaining the integrity of heme binding.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Pfam analysis assigns IsdG and IsdI to the ActVA family of monooxygenases responsible for the oxidation of aromatic polyketides (5). This enzyme family includes the ActVA-ORF6 from Streptomyces coelicolor (21), ElmH from Streptomyces olivaceus (22), and TcmH from Streptomyces glaucescens (23). The substrates of TcmH/ElmH (a tetracenomycin precursor) (22, 23), ActVA-Orf6 (6-deoxydihydrokalafungin (6-DDHK)) (21), and IsdG/IsdI (heme) exhibit similar five- or six-member aromatic ring architecture (Fig. 7). In the absence of any significant amino acid conservation (Fig. 4), the structural conservation of ActVA-Orf6 (21), IsdG, and IsdI (Fig. 3) is striking. These representatives of both monooxygenases subfamilies crystallized as homodimers with two substrate binding cavities. Trp⁶⁶ of ActVA-Orf6, Trp⁵⁸ of IsdG, and Trp⁵⁷ of IsdI occupy a structurally equivalent position in their respective substrate-binding sites. This residue is present in all members of the IsdG and ActVA family of monooxygenases. The Trp⁶⁶ residue was proposed to stabilize ActVA-Orf6-6-DDHK complex through H-bonding (21). Additionally, the carboxyl group of 6-DDHK extends from the active site pocket of ActVA-Orf6 and is exposed to solvent. This finding is consistent with our model of heme binding shown in Fig. 6.

View larger version (16K): [in this window] [in a new window]   FIG. 7. The substrates of the ActVA and IsdG family of enzymes. A, tetracenomycin precursor. B, 6-DDHK. C, protoporphyrin IX (heme).

We propose, based on earlier models for ActVA-Orf6 (21) and heme oxygenase-mediated catalysis (20), that hydroxylation of the heme porphyrin ring by IsdG and IsdI occurs via substrate self-hydroxylation. We predict that binding of heme in the cavity shown in Fig. 6 is followed by its reduction from the ferric to the ferrous form, thus allowing Fe^II-O₂ binding. A subsequent electron transfer to heme may form an activated peroxide intermediate capable of hydroxylating the porphyrin ring.

It seems plausible, that TT1380/1IUJ, a structural homolog of IsdG and IsdI from T. thermophilus, functions as a heme monooxygenase, suggesting that this bacterium is capable of degrading heme. The amino acid sequence of TT1380 is 27% identical to IsdG and 33% identical to IsdI and contains key residues of the IsdG/IsdI active site (Asn⁷, Trp⁶⁷, and His⁷⁷).

The abundance of heme-iron provides a rich source of iron for bacterial pathogens of vertebrates. The convergent evolution of the heme oxygenase family and the IsdG heme monooxygenase family provides an opportunity to compare two distinct enzyme families involved in similar processes. Divergent evolution of the monooxygenase substrate specificity under selective pressure to acquire iron from the host seems to have lead to a new heme-degrading enzyme family.

	FOOTNOTES

 
The atomic coordinates and structure factors (code 1SQE and 1XBW) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by National Institutes of Health Grant GM62414 (to A. J.) and Grants AI38897 and AI52474 (to O. S.), by a University of Chicago-Argonne National Laboratory joint research grant under H.28 of the prime contract, and by the United States Department of Energy, Office of Biological and Environmental Research, under Contract W-31-109-Eng-38. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors made equal contributions to this project.

^|| Supported by a Greater Midwest Affiliate Postdoctoral Fellowship of the American Heart Association.

To whom correspondence may be addressed: Dept. of Microbiology, The University of Chicago, 920 E. 58th St., Chicago, IL 60637. Tel.: 773-834-9060; Fax: 773-834-8150; E-mail:oschnee{at}bsd.uchicago.edu. ¶¶ To whom correspondence may be addressed: Structural Biology Center, Biosciences Division, Argonne National Laboratory, 9700 South Cass Ave., Bldg. 202, Argonne, IL 60439. Tel.: 630-252-3926; Fax: 630-252-6126; E-mail: andrzejj{at}anl.gov.

¹ The abbreviations used are: Ni-NTA, nickel-nitrilotriacetic acid; MAD, multiple-wavelength anomalous diffraction; 6-DDHK, 6-deoxydihydrokalafungin.

	ACKNOWLEDGMENTS

 
We thank all of the members of the Structural Biology Center at Argonne National Laboratory for help in conducting experiments. We also thank Dominique M. Missiakas (University of Chicago) for critical comments on this manuscript.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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