©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Site-directed Mutagenesis of Recombinant Sulfite Oxidase
IDENTIFICATION OF CYSTEINE 207 AS A LIGAND OF MOLYBDENUM (*)

(Received for publication, November 3, 1995; and in revised form, December 19, 1995)

Robert M. Garrett (§) K. V. Rajagopalan (¶)

From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Each of the four cysteines in rat sulfite oxidase was altered by site-directed mutagenesis to serine, and the mutant proteins were expressed in Escherichia coli. Three of the replacements proved to be silent mutations, while a single cysteine, Cys-207, was found to be essential for enzyme activity. The C207S mutation was also generated in cloned human sulfite oxidase. The mutant human enzyme also displayed severely attenuated activity but was expressed at higher levels allowing purification and spectroscopic analysis. The absorption spectrum of the isolated molybdenum domain of the human C207S mutant displayed marked attenuation of the peak at 350 nm and a lesser decrease in absorbance from 450-600 nm as compared with the native human molybdenum domain. The molybdenum and molybdopterin contents of the two samples were comparable. These data suggest that the major features in the absorption spectrum of the native molybdenum domain arise from the binding of Cys-207 to the molybdenum and indicate that this residue functions as a ligand of the metal.


INTRODUCTION

Sulfite oxidase, located in the intermembrane space of animal mitochondria, catalyzes the oxidation of sulfite to sulfate, the terminal reaction in the oxidative degradation of the sulfur-containing amino acids, cysteine and methionine. The enzyme is a dimer of identical subunits of mass 52 kDa. The N-terminal domain of mass 10 kDa forms a b(5)-type cytochrome, and the C-terminal domain of mass 42 kDa anchors the molybdenum cofactor. The molybdenum cofactor in sulfite oxidase consists of molybdopterin (MPT), (^1)a 6-alkyl-dihydropterin containing a unique cis-dithiolene moiety coordinated to molybdenum (1) . EXAFS studies of rat liver sulfite oxidase have provided evidence for the presence of 2 Mo=O, 2 to 3 Mo-S, and 1 Mo-O(N) bonds at the molybdenum center(2, 3) .

The complete amino acid sequences of sulfite oxidase from chicken(4) , rat(5) , and human (6) sources have been reported. In addition, the amino acid sequences of a related enzyme nitrate reductase have been reported from a variety of fungal and plant sources (9, 10, 11, 12, 13, 14, 15, 16, 17) . (^2)(^3)Nitrate reductase catalyzes the reduction of nitrate to nitrite, a critical reaction in the nitrogen assimilation pathway in fungi and higher plants. The enzyme contains three prosthetic groups: the molybdenum cofactor, a b cytochrome, and FAD in binding domains encoded by distinct segments of the primary sequence. Unlike in sulfite oxidase, the molybdenum domain of nitrate reductase is at the N terminus, followed by the central heme domain and the C-terminal flavin domain. The amino acid sequences of the molybdenum domains of sulfite oxidase and nitrate reductase are approximately 37% identical, and a single cysteine residue, corresponding to Cys-207 of sulfite oxidase, is invariant in all of the sulfite oxidases and nitrate reductases sequenced to date. It has been postulated that this cysteine functions as a ligand to molybdenum(4, 6) . Recently it was shown that mutation of the corresponding cysteine residue in nitrate reductase leads to loss of activity(18) .

This report describes the site-directed mutagenesis of rat and human sulfite oxidase to generate cysteine to serine mutants for each of the four cysteines in sulfite oxidase. The molybdenum domain of the human sulfite oxidase C207S mutant has been purified, and spectroscopic data indicate that Cys-207 functions as a ligand of molybdenum.


MATERIALS AND METHODS

Chemicals and Reagents

All common reagents were from Sigma and of the highest grade available. Premixed cell culture media were obtained from Life Technologies, Inc. Restriction enzymes were purchased from Stratagene, Life Technologies, Inc., or Amersham Corp. Escherichia coli host strain JM109 was purchased from Stratagene. Expression vector pPROK1, E. coli host strain DH5-alpha, and reagents for site-directed mutagenesis were purchased from Clontech. Reagents for DNA sequencing were obtained from Amersham.

Molybdenum Quantitation

Molybdenum was measured using a Perkin-Elmer Zeeman/3030 atomic absorption spectrophotometer equipped with autosampler model AS-60. All buffers and standard solutions were made using double-deionized water. Samples were equilibrated in 50 mM Tris acetate, pH 8.0, mixed with an equal volume of concentrated HNO(3), wet ashed at 100 °C for 6 h, and diluted in 0.2% HNO(3) to an approximate molybdenum concentration of 10 ppb. A standard curve was obtained using solutions of 0, 5, and 15 ppb molybdenum in 0.2% HNO(3). All other conditions were as described by Johnson(19) .

Strains and Growth Conditions of E. coli

All plasmids were maintained in E. coli strain JM109 or DH5-alpha. Cultures were grown in LB medium (Life Technologies, Inc.) containing 100 µg/ml ampicillin at 37 °C. Cultures of E. coli strain DH5-alpha for the expression of recombinant sulfite oxidase were grown in 2 liters of LB medium containing 100 µg/ml carbenicillin for 16 h at 37 °C, subcultured into 30 liters of LB medium containing 100 µg/ml ampicillin, 50 µM sodium molybdate, 5 ml of Antifoam A, 0.4 mM isopropyl beta-D-thiogalactopyranoside and grown for 6 h at 37 °C.

Purification of Sulfite Oxidase

All buffers used in the purification procedure contained 0.5 mM EDTA and 0.1 mg/ml phenylmethylsulfonyl fluoride. Cells from a 30-liter culture were harvested by centrifugation and resuspended in 10 mM potassium phosphate, pH 7.8. The cell pellet was weighed and resuspended in 1 volume of 50 mM potassium phosphate, pH 7.8 (phosphate buffer), and the cells were disrupted by two passes through a French pressure cell. An additional 4 volumes of phosphate buffer were added, and the homogenate was centrifuged at 23,000 times g for 20 min. Streptomycin sulfate was added to the supernatant to a final concentration of 2%, and the sample was centrifuged at 8000 times g for 15 min. The resulting supernatant was made 45% saturated with ammonium sulfate, stirred slowly on ice for 10 min, and centrifuged at 8000 times g for 15 min. The pellet was resuspended in 5 volumes of phosphate buffer, mixed with 0.75 volume of ice-cold acetone, and the solution was centrifuged at 8000 times g for 5 min. The supernatant was immediately applied to a column of G-25 Sepharose pre-equilibrated with phosphate buffer. The fraction containing sulfite oxidase was collected and applied to a column of DE52 cellulose pre-equilibrated with phosphate buffer. The enzyme was eluted from the column using a gradient of 50-350 mM potassium phosphate, pH 7.8. Fractions exhibiting a ratio of absorbance at 413 nm/280 nm greater than 0.3 were pooled, brought to 20% saturation with ammonium sulfate, and applied to a column of phenyl-Sepharose (Pharmacia Biotech Inc.) equilibrated with 20% saturated ammonium sulfate in phosphate buffer. The enzyme was eluted with a gradient of 15% to 0% saturated ammonium sulfate in phosphate buffer. Fractions with an absorbance ratio of 0.75 or greater were pooled, concentrated, and stored in phosphate buffer containing 50% glycerol at -20 °C.

Site-directed Mutagenesis, Plasmids, and Strains

Mutations were introduced into pRG140 (5) and pRG118H (6) essentially as described by Deng and Nickoloff(20) . The RSO-C207S expression vector pRG140-C207S was constructed using primer C207SB (tcactctgcaatctgctggtaacc) to change guanosine at position 667 to cytosine, and two selection primers, 140-S1 (caggaaacagatatcatgccatgg) to change the EcoRI site in the multiple cloning site of pRG140 to EcoRV, and primer 140-0 (ttcccggaattgcatatcaagctt) to delete the EcoRI and EcoRV sites in the multiple cloning site of pRG140. All other plasmids expressing mutants of RSO were constructed using the identical selection primers, 140-S1, and 140-0. The RSO-C242S expression vector pRG140-C242S was constructed using primer C242S (gggccggggcgcggctctctgatgtctt) to change guanosine at position 774 to cytosine. The RSO-C260S expression vector pRG140-C207S was constructed using primer C260S (gaaaccgaagcccatgtctcttttgaag) to change guanosine at position 838 to cytosine. The RSO-C451S expression vector pRG140-C451S was constructed using primer C451S (ttgaacatcatttctaaagcggta) to change guanosine at position 1401 to cytosine. The HSO-C207S expression vector pRG118-C207S was constructed from pRG118H using selection primer RG118S (gacctgcagtctagattggctgttttggcg), which changes the unique HindIII site in the multiple cloning site of pRG118H to XbaI, and primer 118C207S (tctctctgcaatctgccggcaaccgacgct) to change guanosine 555 to adenosine, and guanosine 557 to cytosine, introducing the C207S mutation and deleting an internal PstI site to allow rapid screening of successful recombinants. The HSO-K108R expression vector pRG118-K108R was constructed using RG118S as the selection primer, and primer K108R (tcctgaagacagggtagcccccaccg) to change adenosine 1226 to guanosine, introducing the K108R mutation. The HSO-C207S-K108R expression vector was constructed from pRG118-C207S using selection primer RG118S2 (gacctgcagccaagcttggctgttttggct) to change the unique XbaI site of pRG118-C207S to a HindIII site, and primer K108R to introduce the K108R mutation.

Tryptic Cleavage of Sulfite Oxidase

For isolation of the molybdenum domain of HSO-K108R and HSO K108R-C207S, enzyme (7 mg/ml in 50 mM Tris-HCl, pH 8.1) was incubated at 6 °C for 18 h in the presence of trypsin at a ratio of 100 µg of trypsin/mg of sulfite oxidase. Trypsin inhibitor was then added at a ratio of 1.5 µg of trypsin inhibitor/µg of trypsin. The cleavage reaction mixture was chromatographed on a GF-250 gel filtration column (250 times 9 mm, DuPont) equilibrated with 100 mM potassium phosphate, pH 7.8, with 1 mM EDTA at a flow rate of 1 ml/min on a Hewlett-Packard HP-1090 high pressure liquid chromatograph equipped with a Hewlett-Packard HP-1040A diodearray detector.

Sulfite Oxidase Assays

Sulfite oxidase was assayed by monitoring reduction of cytochrome c. For assays of purified sulfite oxidase, cuvettes contained 5-10 µl of enzyme, 50 µl of 1 mM horse heart cytochrome c, and 0.1 M Tris-HCl, pH 8.5, with 0.1 mM EDTA in a final volume of 1 ml. The rate of increase in absorbance at 550 nm was measured before and after addition of 4 µl of 0.1 M sodium sulfite. One unit of sulfite oxidase activity is defined as the amount of enzyme required to yield an absorbance change of 1.0/min at 25 °C. Under these conditions one unit of sulfite oxidase activity corresponds to the reduction of 0.053 µmol of cytochrome c/min(21) . Activity of the molybdenum domain of sulfite oxidase was assayed by monitoring reduction of ferricyanide(22) . Sodium sulfite (4 µl of 0.1 M) was added to a cuvette containing the enzyme in 1 ml of 0.4 mM K(3)Fe(CN)(6), 0.1 M Tris-HCl, 0.1 mM EDTA, pH 8.5. The rate of decrease in absorbance at 420 nm was monitored. One unit of sulfite:ferricyanide activity is defined as the amount of enzyme required to yield an absorbance change of 1.0/min at 25 °C.


RESULTS AND DISCUSSION

The kinetic mechanism of sulfite oxidase consists of several steps, including oxidative hydroxylation of sulfite by Mo(VI) generating Mo(IV), stepwise 1 electron transfers from Mo(IV) to the heme domain, followed by electron transfer from the heme to the physiological electron acceptor cytochrome c. Thus, any mutation causing a loss of activity could be due to an effect on any of several steps in the reaction pathway and may or may not involve changes in molybdenum coordination. In order to demonstrate that a particular residue is a ligand of molybdenum, it is necessary to demonstrate that site-directed mutagenesis of that residue alters the absorption spectrum of the MPT-molybdenum chromophore. In the case of the majority of molybdoenzymes, including sulfite oxidase, it is virtually impossible to detect changes in the absorption properties of the molybdenum center owing to the presence of other much stronger chromophores. In particular, the cytochrome b(5)-type heme of sulfite oxidase completely masks the absorption spectrum of the molybdenum center. However, studies using sulfite oxidase purified from rat liver (19) showed that gentle treatment with trypsin followed by gel filtration allowed separation of the two chromophores and isolation of the molybdenum domain. Absorption spectra of the isolated molybdenum domain revealed certain features of the MPT-molybdenum chromophore, although total removal of heme was not achieved in those earlier studies. With the availability of recombinant sulfite oxidase and techniques for site-directed mutagenesis, it became possible to examine the effects of specific mutations on enzyme activity and then to further probe the effects of those mutations that impair activity by monitoring the absorption features of the molybdenum center. Changes in absorption properties would be indicative of disturbances in metal ligation and, along with the effects on enzyme activity, could be used to identify protein-derived molybdenum ligands.

Site-directed Mutagenesis of Sulfite Oxidase

RSO contains four cysteine residues, one of which, Cys-207, is conserved in sulfite oxidases and nitrate reductases from a variety of fungal, plant, and animal sources. Cys-207 lies in a region of the molybdenum domain highly conserved in all of the sulfite oxidases and nitrate reductases sequenced to date and thus is a likely candidate to serve as a ligand to molybdenum in these enzymes. In order to establish the role of the four cysteines in RSO, a set of cysteine to serine mutations was constructed to determine which, if any, of these residues are required for activity of the enzyme. Four RSO expression vectors, pRG140-C207S, pRG140-C242S, pRG140-C260S, and pRG140-C451S were constructed using site-directed mutagenesis as described under ``Materials and Methods.'' The mutant proteins were purified and assayed for sulfite:cytochrome c and sulfite:ferricyanide activity. A single cysteine, Cys-207, was found to be required for normal activity of the enzyme. The absorption spectrum of RSO-C207S is identical to that of the wild-type RSO, indicating the enzyme contains a normal heme chromophore(21) ; however, the enzyme has a specific activity at least 2000-fold lower than that of the wild-type enzyme.

Tryptic Cleavage of Sulfite Oxidase-Treatment of sulfite oxidase with trypsin has been shown to result in cleavage of the enzyme into two distinct domains, yielding the monomeric heme domain and the molybdenum domain, which remains a dimer(23) . Cleavage of sulfite oxidase results in loss of the sulfite:cytochrome c activity of the holoenzyme; however the molybdenum domain retains the ability to oxidize sulfite to sulfate using ferricyanide as the electron acceptor. Due to extremely low expression of the RSO cysteine mutants, tryptic cleavage of the rat enzyme into its constituent domains was not feasible. When HSO was subsequently cloned it was found to be expressed at a level approximately 5-fold higher than the rat enzyme, suitable for isolation and preparation of the molybdenum domain for spectroscopic characterization. However, HSO proved to be extremely insensitive to trypsin as compared with RSO (Fig. 1A). In order to characterize the molybdenum domain spectroscopically, it is necessary to completely cleave the enzyme, since even small amounts of residual heme domain will interfere with the spectrum of the isolated molybdenum domain. The wild-type human enzyme could be completely cleaved only under conditions which severely degraded the molybdenum domain. Examination of the amino acid sequences showed that HSO contains a lysine rather than an arginine at position 108, the site susceptible to trypsin cleavage. In order to generate an HSO mutant with increased sensitivity to trypsin the lysine at position 108 was altered to arginine by site-directed mutagenesis to create plasmid pRG118-K108R. The sulfite:cytochrome c and sulfite:ferricyanide activities of HSO-K108R were identical to those of wild-type HSO. The HSO-K108R mutant showed greatly enhanced sensitivity to trypsin, allowing cleavage of the holoenzyme under conditions which preserve the activity of the molybdenum domain (Fig. 1B). It is interesting to note that the human K108R mutant was less sensitive to trypsin than the wild-type rat enzyme, suggesting that the R108K substitution is not the sole factor responsible for the reduced trypsin sensitivity of the human enzyme. Plasmid pRG118-K108R-C207S was constructed to express the HSO-C207S mutant in a background that would allow tryptic cleavage of the enzyme and subsequent purification of the molybdenum domain.


Figure 1: A, sulfite:cytochrome c activity of rat (circle) and human (bullet) sulfite oxidase during the course of tryptic cleavage. Trypsin treatment of wild-type recombinant RSO and HSO was essentially as described by Johnson and Rajagopalan(23) . Sulfite oxidase, 1 mg/ml in 50 mM Tris-HCl, pH 8.1, was incubated at room temperature for 30 min with 15 µg trypsin in a volume of 0.5 ml. Aliquots taken for activity assays were mixed with an equal volume of 50 mM Tris-HCl, pH 8.1, containing 1.5 µg of trypsin inhibitor/µg of trypsin. B, sulfite:ferricyanide () and sulfite:cytochrome c (box) activity of HSO-K108R during the course of tryptic cleavage (B). HSO-K108R was treated with trypsin at room temperature using 100 µg of trypsin/mg of sulfite oxidase in 50 mM Tris-HCl, pH 8.1. Aliquots taken for activity assays were mixed with an equal volume of 50 mM Tris-HCl, pH 8.1, containing 1.5 µg of trypsin inhibitor/µg of trypsin.



Spectroscopic Analysis of Sulfite Oxidase C207S

Purified HSO-K108R and HSO-K108R-C207S were cleaved with trypsin as described under ``Materials and Methods.'' The molybdenum domain was purified by HPLC with on-line diode array detection (Fig. 2). The proteins showed identical elution profiles with clear separation of the molybdenum and heme domains eluting at 8.8 and 10.6 min, respectively. As seen in Fig. 2, absorption spectra obtained from the wild-type HSO and HSO-C207S molybdenum domains are quite different. The wild-type molybdenum domain exhibits a distinct peak at 350 nm and a broad shoulder extending from 400 to 500 nm, identical to that reported for the isolated rat molybdenum domain(23) . In contrast, the spectrum of the HSO-C207S molybdenum domain exhibits a small shoulder at 350 nm. The most distinct feature of the spectrum is a peak at 404 nm which falls off rapidly to the base line at 500 nm. Denaturing polyacrylamide gel electrophoresis of the wild-type HSO and HSO-C207S molybdenum domains indicated that the proteins had been purified to near homogeneity. Both samples were assayed for molybdenum content by atomic absorption spectroscopy to assure that the cleavage reaction did not result in the loss of molybdenum from either enzyme. The HSO molybdenum domain was found to have a 0.76:1 molar ratio of molybdenum:protein monomer, while the HSO-C207S molybdenum domain displayed a ratio of 0.84:1. Sulfite oxidase holoenzyme routinely yields molybdenum:protein molar ratios of 0.8-0.9:1. The molybdopterin content of the enzyme was also measured. Samples of the molybdenum domains were acidified to pH 2.5 in the presence of excess iodine, which converts MPT to Form A, a stable oxidized derivative. Form A was assayed using fluorescence, with excitation at 380 nm and emission at 455 nm. The wild-type HSO molybdenum domain yielded 240 fluorescent units/mg of protein, compared with 215 fluorescent units/mg of protein for the HSO-C207S molybdenum domain. These data indicate that the cleavage reaction and subsequent purification of the molybdenum domain did not disrupt the molybdenum center of the enzymes, confirming that the spectral differences can be attributed to the loss of a cysteine ligand to molybdenum in the HSO-C207S mutant. The finding that the C242S, C260S, and C451S mutants showed no loss of activity makes it very unlikely that those three cysteines serve as molybdenum ligands. These findings are in accord with the proposal that the two dithiolene sulfur atoms and Cys-207 are the sources of the 3 Mo-S bonds observed in EXAFS. A model of the sulfite oxidase molybdenum center supported by all of the available data is shown in Fig. 3.


Figure 2: HPLC chromatograms of trypsin-treated HSO-K108R (A) and HSO-K108R-C207S (B). Absorbance at 280 nm is represented by the solid line; absorbance at 413 nm is represented by the dotted line. C shows the absorbtion spectrum of the molybdenum domain of HSO (solid line) and HSO-C207S (dashed line). The samples were matched in absorbance at 280 nm.




Figure 3: The proposed structure of the molybdenum center of sulfite oxidase showing the molybdenum atom coordinated to two terminal oxo groups, a third oxygen or nitrogen, the two dithiolene sulfurs of molybdopterin, and the side chain of Cys-207.



The region of the sulfite oxidase and nitrate reductase proteins immediately surrounding Cys-207 is highly conserved, and a consensus sequence TL(Q/V)CAGNRR(S/K)E can be identified containing Cys-207. The molybdenum coordination site consensus sequence was searched for in the sequences of two other human molybdopterin-containing enzymes, xanthine dehydrogenase (24) and aldehyde oxidase(25) . A stretch of similar amino acids was found in xanthine dehydrogenase (TLVSRGTRRTV). This sequence is identical to the consensus sequence in 7 out of 11 amino acids and similar in 9 out 11 positions. In the xanthine dehydrogenase sequence a serine replaces the conserved cysteine. It is possible that serine functions as a ligand to molybdenum in this enzyme and may explain the observed differences in the molybdenum centers of sulfite oxidase and xanthine dehydrogenase. However, no direct evidence for this assumption is available. Aldehyde oxidase, which is 50% identical to xanthine dehydrogenase, shows even less sequence similarity in this specific region. It would seem that the sequences of aldehyde oxidase and xanthine dehydrogenase are too divergent from those of sulfite oxidase and nitrate reductase to allow identification of possible molybdenum coordination sites based on sequence similarities

Recently the x-ray crystallographic structures of two proteins containing molybdopterin cofactors have been reported. The bis(MPT)tungsten-containing aldehyde ferredoxin oxidoreductase from Pyrococcus furiosus(7) and the (molybdopterin cytosine dinucleotide) molybdenum-containing aldehyde oxidoreductase from Desulfovibrio gigas(8) . The tungsten atom in the aldehyde ferredoxin oxidoreductase is coordinated to four dithiolene sulfur atoms provided by two MPT molecules. In the D. gigas enzyme the molybdenum atom is coordinated to the dithiolene of a single molybdopterin cytosine dinucleotide. Neither protein contains any protein derived ligands to the metal. The data presented in this paper thus represent the first established instance of the coordination of a protein side chain residue to molybdenum or tungsten in enzymes containing pterin-metal cofactors.

The results of these studies do not provide evidence for or against the possible coordination of Ser-207 to the metal in the C207S mutant. Ongoing EXAFS analysis of the mutant sulfite oxidase should provide the answer to that question. Comparison of the oxidation-reduction potentials for the Mo(VI) Mo(IV) and Mo(V) >Mo(IV) transitions of the mutant molybdenum center to those of the wild-type enzyme, in conjunction with stopped-flow spectroscopic studies to determine the effect of the mutation on the reductive and oxidative half-reactions should provide an understanding of the basis for the highly attenuated catalytic activity of the mutant. The successful molecular surgery for separation of the molybdenum center from the heme domain has made it feasible to apply spectroscopic techniques such as stopped-flow, magnetic circular dichroism, and resonance Raman to the detailed analysis of the C207S or any other mutant sulfite oxidase.


FOOTNOTES

*
This work was supported in part by Grants GM00091 and GM44283 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported in part by Grant T32 ES07031 from the National Institutes of Health.

To whom correspondence should be addressed: Dept. of Biochemistry, Duke University Medical Center, Durham, NC 27710. Tel.: 919-681-8845; Fax: 919-684-8919.

(^1)
The abbreviations used are: MPT, molybdopterin; EXAFS, extended x-ray absorption fine structure; RSO, rat sulfite oxidase; HSO, human sulfite oxidase; HPLC, high pressure liquid chromatography.

(^2)
P. E. Jensen, T. Hoff, B. M. Stummann, and K. W. Hennigsen, Genbank(TM) accession number U01029[GenBank].

(^3)
S. Wu, S. Lu, A. L. Kriz, and J. E. Harper, GenBank(TM) accession number L23854[GenBank].


ACKNOWLEDGEMENTS

We are grateful to Ralph Wiley for his expert assistance in the purification of recombinant sulfite oxidase. We also thank Dr. Jean Johnson for her valuable advice regarding the tryptic cleavage of sulfite oxidase and her critical reading of this manuscript.


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