Mechanism of the Conversion of Xanthine Dehydrogenase to ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 280, No. 26, Issue of July 1, pp. 24888 –24894, 2005 Printed in U.S.A.

Mechanism of the Conversion of Xanthine Dehydrogenase to Xanthine Oxidase IDENTIFICATION OF THE TWO CYSTEINE DISULFIDE BONDS AND CRYSTAL STRUCTURE OF A S NON-CONVERTIBLE RAT LIVER XANTHINE DEHYDROGENASE MUTANT*□ Received for publication, February 17, 2005, and in revised form, March 28, 2005 Published, JBC Papers in Press, May 4, 2005, DOI 10.1074/jbc.M501830200

Tomoko Nishino‡, Ken Okamoto‡, Yuko Kawaguchi‡, Hiroyuki Hori‡§, Tomohiro Matsumura‡, Bryan T. Eger¶, Emil F. Pai¶, and Takeshi Nishino‡储 From the ‡Department of Biochemistry and Molecular Biology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan and ¶Departments of Biochemistry, Medical Biophysics, and Molecular and Medical Genetics, Division of Molecular and Structural Biology, University of Toronto, Ontario Cancer Institute/University Health Network, Toronto, Ontario M5G 2M9, Canada

Xanthine oxidoreductase (XOR),1 xanthine dehydrogenase (XDH, EC 1.1.1.204), or xanthine oxidase (XO, EC 1.2.3.2) is a * This work was supported by Grants-in-aid 08249104 and 11169231 (to Takeshi Nishino) for Science Research on Priority Areas and a Grant-in-aid 09480167 (to Takeshi Nishino) for Science Research from the Ministry of Education, Science, Sports, and Culture of Japan and a grant from the Canadian Institutes for Health Research (to E. F. P.). 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. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental text and references, Tables S1 and S2, and Figs. S1–S4. The atomic coordinates and structure factors (code 1WYG) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). § Present address: Dept. of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama 790-8577, Japan. 储 To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan. Tel.: 81-3-3822-2131; Fax: 81-35685-3054; E-mail: [email protected]. 1 The abbreviations used are: XOR, xanthine oxidoreductase; XO, xanthine oxidase; XDH, xanthine dehydrogenase; DTT, dithiothreitol;

complex metalloflavoenzyme that catalyzes oxidation of hypoxanthine to xanthine and xanthine to uric acid with concomitant reduction of NAD⫹ or molecular oxygen. The enzyme is a homodimeric protein of Mr 300,000 and is composed of independent subunits; each subunit contains one molybdopterin, two non-identical iron sulfur centers ([2Fe-2S] clusters), and one FAD (1, 2, 3). The oxidative hydroxylation of xanthine to uric acid takes place at the molybdenum center, and reducing equivalents thus introduced are transferred rapidly via two iron sulfur centers to FAD, where physiological oxidation occurs (4). The mammalian enzymes exist in the NAD⫹-dependent form (xanthine dehydrogenase, XDH) in freshly prepared samples from organs under normal conditions, i.e. they exhibit low xanthine/O2 reductase activity but high xanthine/NAD⫹ reductase activity, even in the presence of O2 (5, 6). XDH can be converted reversibly to xanthine oxidase (XO) by oxidation of cysteine residues or irreversibly by limited proteolysis (5–13). XO has high reactivity toward O2 but negligible reactivity toward NAD⫹. As XO can reduce molecular oxygen to superoxide and hydrogen peroxide (1), XO is thought to be one of the key enzymes producing reactive oxygen species (14). The crystal structures of bovine milk XDH and proteolytically produced XO have been solved and showed large conformational differences around the FAD (15). Although the transition seems to occur in a similar way, whether caused by cysteine modification or proteolysis, the identification of the responsible cysteine residues is still a matter of controversy. It is not easy to identify the responsible residues, because the enzyme contains as many as 36 cysteine residues/monomer of rat XOR (12), and many residues are modified by common cysteine-modifying reagents, such as 5,5⬘-dithiobis(nitrobenzoic acid) or iodoacetamide (16). It was, however, reported that only four cysteine residues were modified during the conversion from rat liver XDH to XO by titration with 4,4⬘dithiodipyridine (4,4⬘-DTPY) (16). During the titration, two disulfide bonds were suggested to be formed by modification with 4,4⬘-DTPY, because the addition of 2 mol of 4,4⬘-DTPY stoichiometrically provides 4 mol of 4-thiopyridone. A similar observation was made by Hunt and Massey (11) with the bovine milk enzyme. Although only four cysteine residues were modified during conversion from XDH to XO by this reagent, it was still difficult to identify the residues, because the modifier was released from the residues involved as part FDNB; fluorodinitrobenzene; 4,4⬘-DTPY, 4,4⬘-dithiodipyridine; MB, methylene blue; FAD, flavin adenine dinucleotide.

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Mammalian xanthine dehydrogenase can be converted to xanthine oxidase by modification of cysteine residues or by proteolysis of the enzyme polypeptide chain. Here we present evidence that the Cys535 and Cys992 residues of rat liver enzyme are indeed involved in the rapid conversion from the dehydrogenase to the oxidase. The purified mutants C535A and/or C992R were significantly resistant to conversion by incubation with 4,4ⴕ-dithiodipyridine, whereas the recombinant wildtype enzyme converted readily to the oxidase type, indicating that these residues are responsible for the rapid conversion. The C535A/C992R mutant, however, converted very slowly during prolonged incubation with 4,4ⴕ-dithiodipyridine, and this slow conversion was blocked by the addition of NADH, suggesting that another cysteine couple located near the NADⴙ binding site is responsible for the slower conversion. On the other hand, the C535A/C992R/C1316S and C535A/C992R/ C1324S mutants were completely resistant to conversion, even on prolonged incubation with 4,4ⴕ-dithiodipyridine, indicating that Cys1316 and Cys1324 are responsible for the slow conversion. The crystal structure of the C535A/ C992R/C1324S mutant was determined in its demolybdo form, confirming its dehydrogenase conformation.

Conversion of Xanthine Dehydrogenase to Oxidase

EXPERIMENTAL PROCEDURES

Materials—Spodoptera frugiperda (Sf9) cells and wild-type circular Autographa californica nuclear polyhedrosis virus (AcNPV) DNA for wild-type XOR were obtained from Invitrogen (20). Triple cut virus DNA for mutant cotransfection was obtained from Novagen (BacVectorTM 2000). The transfer vector pJVP10Z was kindly provided by Dr. Palmer Taylor (University of California, San Diego). The restriction and modification enzymes were from Takara Shuzo Co., Ltd. (Tokyo, Japan) or New England Biolabs, Inc. The culture media IPL41 or Sf-900II, fetal calf serum, and Grace medium were purchased from Invitrogen. The polyclonal antibody used in these experiments was raised in our laboratory against purified rat liver XOR, as described previously (12). DE52 was purchased from Whatman Ltd., hydroxylapatite resin BIOGEL HTP was from Bio-Rad, and NAD⫹ was from Sigma (grade V-C). All other chemicals were of reagent grade. DNA Manipulations and Site-directed Mutagenesis—A baculovirusinsect cell system (21) was employed for the expression of the wild-type and mutant XOR enzymes. The site-directed mutagenesis was done using a Muta-Gene Phagemid in vitro mutagenesis kit (Bio-Rad) and a QuikChange site-directed mutagenesis kit (Stratagene). All of the altered DNA sequences were analyzed using a Sequenase version 2.0 DNA sequencing kit (United States Biochemical). Escherichia coli strain CJ236 (Bio-Rad) was transformed with pUC118EP2 carrying the cDNA of rat liver XOR, and this transformant was infected in the mid-log phase with helper phage M13KO7 (Bio-Rad) in the presence of ampicillin, chloramphenicol, and kanamycin to prepare single-stranded DNA. The phage particles were collected by polyethylene glycol precipitation, and single-stranded DNA was extracted with phenol-chloroform and recovered by ethanol precipitation. The following 5⬘-phosphorylated DNA oligomer was utilized to construct the XOR variant: 5⬘-GGG GTC CAG TTT GCC GGC CAT ATC CTC-3⬘ for C535A (Cys535 codon substituted by Ala codon) and 5⬘-CCC TCT CTT TTT CCA TCG ATT TTC CCT GTT GAA TTT C-3⬘ for C992R (Cys992 codon substituted by Arg codon). Each oligonucleotide was hybridized to single-stranded pUC118MXL and introduced into E. coli strain HB101 (Takara Shuzo, Tokyo, Japan). The resultant double-stranded vectors were isolated and digested with SmaI and SpeI for the C535A mutant

and SmaI and PmaCI for the C992R mutant. Each obtained fragment was ligated with pRXD203 that had been digested with the same restriction enzymes. The double mutant C535A/C992R was constructed by insertion of the DNA fragment containing C535A at the restriction sites SmaI and SpeI into pRXD203C992R at the same sites. As regards the triple mutants C535A/C992R/C1324S and C535A/C992R/C1316S, the former mutant was constructed by using the plasmid containing full-length XOR DNA/C535A/C992R and the same plasmid containing XOR cDNA/C1324S, which was reported previously (13), by digestion and ligation at the SpeI and EcoRV restriction sites. The latter mutant was constructed with the 5⬘-phosphorylated DNA oligomer 5⬘-C CAG TTC ACC ACC CTG TCT GTC ACT GGA GTA CCA G-3⬘ for C1316S (Cys1316 codon substituted by Ser codon) using a QuikChange sitedirected mutagenesis kit and plasmid containing full-length cDNA of XOR/C535A/C992R. The DNA fragment encoding each mutant enzyme was excised with NheI and ligated into baculovirus transfer vector pJVP10Z. The direction of the cDNA was identified by DNA sequencing. The transfer vector pJVP10Z contains the ␤-galactosidase gene, and color selection can be done using 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (X-gal) as a substrate (20, 21, 22). Construction of Recombinant Viral Variant Types—The wild-type recombinant virus was prepared as described previously (20). Cotransfection with AcNPV DNA and constructed transfer vectors was conducted using a BacVectorTM 2000 (Novagen), and screening of the recombinant virus was carried out by plaque assay according to the manufacturer’s manual (20, 21). To identify recombination, direct Western blots were performed with infected cell extracts of the second virus amplification, and positive clones were selected. We usually prepared a virus stock with a titer of 1 ⫻ 107 plaque-forming units/ml. Overexpression of Rat XOR and Its Variants Using the BaculovirusInsect Cell System—The maintenance of Sf9 cell lines and expression procedures were the same as described previously (20). Purification Procedures of Recombinant Enzymes—Purification procedures were performed as described previously (20); the recombinant active XORs and demolybdo-dimeric XORs were separated by affinity column chromatography (23, 24). The enzymes were concentrated and incubated with 5 or 10 mM dithiothreitol (DTT) for 1 h at 25 °C to generate xanthine dehydrogenase forms of XOR, followed by gel filtration to remove excess DTT, as necessary. SDS-Polyacrylamide Gel Electrophoresis—SDS-PAGE was performed as described by Laemmli (25) using 10% polyacrylamide gel. Protein markers purchased from Bio-Rad consisted of a mixture of recombinant proteins (10 –250 kDa). Enzyme Assays—Enzyme assays were carried out at 25 °C in 50 mM potassium phosphate buffer (pH 7.8) containing 0.4 mM EDTA in a final volume of 3.0 ml. Xanthine oxidoreductase activities with various electron acceptors were determined by monitoring the absorbance changes as follows: O2 (air-saturated buffer; 295 nm) and NAD⫹ (500 ␮M; 340 or 295 nm). NADH-methylene blue (MB) activity was determined by monitoring the absorbance change at 340 nm with 50 ␮M NADH in the presence of 50 ␮M methylene blue. XOR concentration was determined from the absorbance at 450 nm using an extinction coefficient of 35.8 mM⫺1 cm⫺1 (26) for the native enzyme. XO was converted to XDH by incubation with 5 or 10 mM DTT at 25 °C for 1 h. The dehydrogenase to oxidase ratio (the D/O ratio) as defined by Waud and Rajagopalan (8) was determined as the ratio of the absorbance change at 295 nm under aerobic conditions in the presence of NAD⫹ to that in the absence of NAD⫹. Activity to flavin ratio (1) was obtained by dividing the change in absorbance/min at 295 nm in the presence of NAD⫹ under aerobic conditions by the absorbance at 450 nm of the enzyme used in the assay at 25 °C. Photometric experiments were performed with a Hitachi U-3210 photometer. Crystallization and Data Collection of the C535A/C992R/C1324S Mutant of XOR—The enzyme used for crystallization was further purified on a gel filtration column of TSKSW3000XL (Tosoh Co.) just before crystallization to remove aggregated enzyme after folate affinity chromatography. As described previously, the recombinant XOR was expressed in multiple forms (20), and the enzyme used for crystallization was the demolybdo-dimeric form. The enzyme was concentrated to 8 mg/ml in buffer A (20) and incubated with 5 mM DTT for 60 min at 25 °C. Crystals of the mutant XDH were grown by vapor diffusion, equilibrating a mixture of 1 ␮l of protein and 1 ␮l of reservoir solution containing 9 –11% polyethylene glycol 8000, 0.6 M Li2SO4, 5 mM DTT, 1 mM sodium salicylate, 0.4 mM EDTA, 15% glycerol, and 40 mM HEPES (pH 6.20), against 1.5 ml of reservoir solution. Crystals of the enzyme were flash-frozen with their mother liquor as a cryoprotectant and mounted in cryoloops. Diffraction data were collected at beamline BL38B1, SPring8, Harima Garden City,


of the reaction. We then reported that relatively few cysteine residues were modified upon chemical reaction with FDNB (13). During the initial 10 min of reaction with FDNB, two specific cysteine residues, Cys535 and Cys992, were labeled, and these residues were suggested to be involved in the rapid conversion from XDH to XO. Recently, Rasmussen et al. (17) reported the sulfhydryl group modification of bovine milk XOR using radioactive iodoacetic acid, and their results are consistent with our assignment. On the other hand, this interpretation has subsequently been challenged in a report describing gel analyses of the proteolytically cleaved disulfide form of XO (18). The crystal structure of bovine XOR shows that Cys992 is situated on the surface of the molecule, but Cys535 seems to be located in the long linker peptide between the FAD and the molybdopterin domains, although the residue is not visible in the crystal structure most probably due to its flexibility. The proteolytic cleavage site is also on the linker peptide. Based on detailed analyses of crystal structures of reversible XDH and proteolytic XO, as well as site-directed mutagenesis, Kuwabara et al. (19) concluded that the unique amino acid cluster of Phe549, Arg335 (corresponding to rat 334), Trp336 (rat 335), and Arg427 (rat 426) in the bovine enzyme sits at the center of a relay system that transmits modifications of the linker peptide caused by cysteine oxidation or proteolytic cleavage to the active site loop (Gln423–Lys433). The movement of the active site loop is considered to be the direct cause of the change in chemical behavior between XDH and XO (15). In the present work, we describe two cysteine couples, one of which is responsible for rapid and the other for slow conversion from XDH to XO. We also present, at 2.6-Å resolution, the crystal structure of a non-convertible rat mutant XOR in the demolybdo-inactive form and show that its polypeptide chain adopts the XDH conformation.

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Japan, at a temperature of 100 K. Radiation of 1.0 Å wavelength and a Q4 area detector (Area Detector Systems Corporation) were used. RESULTS

FIG. 1. a, change in the activity of C535A/C992R and wild-type XDHs during purification and various treatments. Oxidase activity (xanthine oxidase activity) was determined by following the absorbance change at 295 nm in the absence of NAD⫹. Open and closed circles represent wild-type and mutant enzymes, respectively. A, change in oxidase activity of wild-type and C535A/C992R mutant during purification. B, purified XORs were treated with 5 mM DTT for 60 min at 25 °C. C, after removal of excess DTT by gel filtration, XOR (5 ␮M wild type or 2.4 ␮M C535A/C992R) was reacted with 100 ␮M 4,4⬘-DTPY in 0.1 M pyrophosphate buffer (pH 8.5) for 60 min at 25 °C. D, 4,4⬘-DTPY-treated XORs were again reacted with 10 mM DTT for 60 min under the same conditions as for C. b, SDS-PAGE of various purified rat liver XDHs. Lanes 1 and 7, marker proteins (Bio-Rad) having molecular masses of 250, 150, 100, 75, 50, 37, and 25 kDa. Lane 2, XDH purified from rat liver (2 ␮g); lane 3, recombinant wild type XDH (3 ␮g); lane 4, C535A XDH (3 ␮g); lane 5, C992R XDH (1 ␮g); lane 6, C535A/C992R XDH (1 ␮g); lane 8, C535A/C992R/C1316S XOR (1 ␮g); lane 9, C535A/C992R/C1324S XOR (1.3 ␮g). c, treatment of purified wild-type and various mutant XDHs with 4,4⬘-DTPY. The enzymes (2.4 –5 ␮M) were treated at 25 °C with 100 ␮M 4,4⬘-DTPY in 0.1 M pyrophosphate buffer (pH 8.5), and aliquots were withdrawn at various times to determine the activity of uric acid formation in the absence of NAD⫹ (oxidase activity) by following the absorbance increase at 295 nm. Open circles, recombinant wild type XOR; closed circles, C535A/C992R XOR; closed triangles, C535A XOR; closed diamonds, C992R. Closed squares represent the activity of urate formation in the presence of NAD⫹ during conversion of the C992R mutant. d, 4,4⬘-DTPY treatment of C535A/C992R mutant XDH in the presence and absence of NADH. A, the C535A/C992R mutant (2.3 ␮M) was treated with 100 ␮M 4,4⬘-DTPY in the absence of NADH in 0.1 M pyrophosphate buffer (pH 8.5) containing 0.4 mM EDTA, 50 ␮g/ml superoxide dismutase, and 20 ␮g/ml catalase. B, 4,4⬘-DTPY treatment in the presence of 193 ␮M NADH under the same conditions as in A. After incubation for various times, aliquots were withdrawn, and various activities were determined. Open circles, total urate formation calculated from the increase of absorbance at 295 nm in the standard assay mixture with xanthine and NAD⫹ as substrates; closed circles, XO activity calculated from the increase of absorbance at 295 nm without NAD⫹; closed triangles, XDH activity calculated from the increase of absorbance at 340 nm with NAD⫹. e, treatment of triple mutants with 4,4⬘-DTPY. C535A/C992R/C1316S (closed symbols) or C535A/C992R/C1324S mutant (open symbols) enzyme was treated with 100 ␮M 4,4⬘-DTPY in 0.1 M pyrophosphate buffer (pH 8.5) containing 0.4 mM EDTA. After incubation for various times, aliquots were withdrawn, and various activities were determined. The triangles show total urate formation calculated from the increase of absorbance at 295 nm with NAD⫹; circles, XO activity calculated from the increase of absorbance at 295 nm without NAD⫹; and squares, XDH activity calculated from the increase of absorbance at 340 nm with NAD⫹.

from 14 to 52% after 60 min at 25 °C. Resistance to conversion from XDH to XO was also observed in the single mutants C535A and C992R, although the double mutant C535A/C992R was the most resistant (Fig. 1c). These results indicate that, although Cys535 and Cys992 are involved in rapid conversion by treatment with 4,4⬘-DTPY, the conversion from XDH to XO involves not only Cys535 and Cys992, but also other residues. It


Expression and Purification of Three Mutants of Rat Liver Recombinant XOR—We reported previously that two cysteine residues, Cys535 and Cys992, were involved in the conversion from XDH to XO, based on the results of chemical modification with FDNB. To confirm the role of these residues, we constructed three mutants of rat XOR, C535A, C992R, and C535A/ C992R. In the chicken enzyme, which cannot be converted to XO by any treatment, the residue Cys992 is replaced by an arginine, whereas the residue Cys535 is conserved (27). The soluble form of recombinant rat liver XOR was expressed in the baculovirus/Sf9 cell system (20). As described previously, the expressed enzyme consisted of multiple forms; it contained inactive demolybdo-monomeric, inactive demolybdo-dimeric, and active molybdo forms (20). In this study, we used the active enzyme fraction eluted from the first folate affinity chromatography, which removes the demolybdo form, for functional analyses of both wild and mutant enzymes, except in NADH reduction experiments, where the demolybdo form does not influence the results (20). After purification, the activity to flavin ratio value of active XDH at 25 °C was ⬃130, which is comparable with that of native XOR purified from rat liver without the second affinity chromatography step (23). All of the enzymes were expressed in their XDH form and could be identified as such in crude extracts. Although the wild-type XOR retained negligible XDH activity only for 1 day after the start of purification, the mutant enzymes retained significant XDH activity even seven days after purification; they exhibited 25–50% XDH activity. All of the enzymes, including wild type, could be reconverted to XDH. Each of these purified recombinant XDHs showed mostly a single band of molecular mass at 150 kDa on SDS-PAGE, although there was a minor amount of proteolytic nicking in the C535A mutant (Fig. 1b). The values of the activity to flavin ratio of these purified enzymes were between 117 and 160, corresponding to a 59 – 80% active form (the activity to flavin ratio for fully active enzyme is 200 (24)), presumably because of the presence of desulfoenzyme, as in the case of the natural enzyme (10). The NADH-MB activities of these various DTT-treated XDHs were almost the same, at 732–742 mol/min/mol FAD (data not shown), suggesting that the FAD binding site is intact. Thus, the purified wild type and mutants were extremely similar in various aspects, other than conversion property. Treatment of Wild-type and C535A/C992R, C535A, and C992R Mutant XORs with 4,4⬘-DTPY—The purified recombinant wild-type and mutant XOR enzymes were treated with 5 mM DTT to convert them to the XDH type, followed by gel filtration to remove DTT, and the obtained XDHs were treated with 4,4⬘-DTPY. A typical example is shown in Fig. 1a comparing the recombinant wild-type XOR and C535A/C992R double mutant XOR. The single mutant C535A and C992R XORs also showed similar profiles. Fig. 1a, A, shows the auto-oxidation process of cysteine residues during purification. Both enzymes were converted readily to XDH by 5 mM DTT during 60 min of incubation (Fig. 1a, B). In the case of recombinant wild-type XDH, the XO activity was increased from 20 to 100% within 15 min by treatment with 0.1 mM 4,4⬘-DTPY, as shown in Fig. 1a, C. The urate formation activity determined in the presence of NAD⫹ remained at almost the initial level during the reaction. On the other hand, the conversion process from XDH to XO of the mutant was significantly slower than that of wild-type with 4,4⬘-DTPY, although even the double mutant was still converted slowly to the oxidase form (Fig. 1a, C) during prolonged incubation; the oxidase activity of C535A/C992R increased


should be noted that the oxidase activities after reaction with 4,4⬘-DTPY were reconverted to the XDH type by incubation with 10 mM DTT, indicating that the conversion is due to modification of sulfhydryl residues (Fig. 1a, D). Reduction of C535A/C992R Mutants Having Various Levels of Oxidase Activity with NADH and Protection of Slower Conversion by the Presence of NADH—The NADH-methylene blue activity decreased concomitantly with increase of XO activity in a similar way to the xanthine-NAD⫹ activity, suggesting that the conversion is related to pyridine nucleotide binding (Fig. 2a). To assess the binding ability of the C535A/C992R mutants with NADH, double-mutated enzymes with three different activities of NADH-MB were prepared, and reduction experiments were performed by mixing them with NADH under anaerobic conditions. As shown in Fig. 2a, the level of absorbance bleaching by NADH was approximately parallel to NADH-MB activity, suggesting that other cysteine residues may exist near the NAD⫹ binding site. To confirm this, XDHtype or DTT-treated C535A/C992R mutant was incubated with 100 ␮M 4,4⬘-DTPY in the presence of NADH. As shown in Fig. 1d, the conversion from XDH to XO was completely blocked, and the mutant retained XDH-form activity even after 80 min (Fig. 1d). This suggests strongly that the other cysteine couple exists near the NAD⫹ binding site and can be protected from modification by the binding of pyridine nucleotide. In the crys-

tal structure of bovine XDH (but not XO (15)), the C-terminal peptide is inserted into the neighboring molecule pointing toward the NAD⫹ binding pocket. Two cysteine residues that could form a disulfide bond are located near the C terminus. To test the potential involvement of these residues, we constructed two further mutants, C535A/C992R/C1316S and C535A/ C992R/C1324S. In the chicken enzyme, the residue Cys1324 is replaced by phenylalanine, whereas the residue Cys1316 is conserved (27). C535A/C992R/C1316S and C535A/C992R/C1324S XOR Mutants—As described above, the double mutant C535A/C992R XOR was expressed in its XDH form, but it gradually converted to the level of 50 – 60% XO activity during the 7–10-day purification period. On the other hand, triple mutants C535A/ C992R/C1316S and C535A/C992R/C1324S were also expressed as XDH enzymes, but their XDH/XO activity ratio was not changed over a period of 10 days. When exposed to air, they were not converted to XO enzymes even after a month. Fig. 2b shows the spectra of purified C535A/C992R/C1316S and C535A/C992R/C1324S XORs without DTT treatment before and immediately after reduction with NADH under anaerobic conditions. The reduction level after 20 min (not shown) was the same as in the case of DTT-treated native rat liver XDH. Although we performed the same experiments with DTTtreated C535A/C992R/C1316S or C535A/C992R/C1324S XOR, the spectra were indistinguishable from each other, e.g. they showed the same levels of absorbance bleaching and semiquinone formation at ⬃600 nm that are typical of the XDH form. Fig. 1e displays the reaction time courses of the mutants C535A/C992R/C1316S and C535A/C992R/C1324S with 4,4⬘DTPY. Both mutants showed no activity change at all from XDH to XO; they retained the initial dehydrogenase activity during the whole 1 h reaction period. These results are consistent with the fact that these triple mutants are not converted to XO by sulfhydryl oxidation and support the view that Cys1316 and Cys1324 are involved in the slower conversion from XDH to XO by sulfhydryl oxidation. Crystal Structure of the C535A/C992R/C1324S Mutant of Rat Liver XOR—The C535A/C992R/C1324S mutant of rat liver XOR in the demolybdo form was successfully crystallized, and diffraction data were collected to 2.6 Å resolution. The structure was determined by molecular replacement techniques and refined using molecular dynamics refinement. The asymmetric unit contains one subunit, in contrast to the previously structurally characterized bovine milk XDH, which contains one homodimer. Overall, however, the two structures are very similar (Fig. 3a). In both cases, the monomer consists of three major domains, the N-terminal, iron-sulfur center-binding domain, the FAD-containing intermediate domain, and the largest, the C-terminal domain (12, 15). The latter corresponds to the molybdopterin-binding domain in bovine milk XDH, although the molybdopterin cofactor was absent in this mutant, as already predicted from the results of chemical analysis (20). It is intriguing that the overall structure of the molybdopterin domain of this demolybdo-form mutant is not greatly different from that of the molybdo form of bovine milk XDH, and the amino acid residues surrounding the molybdopterin cofactor are situated at very similar positions and in similar orientations, leaving the space for the molybdopterin cofactor vacant. Although the electron density of the interdomain linkers was not fully visible in the bovine milk XDH structure (Protein Data Bank code 1FO4), probably because of flexibility, the electron density linking the FAD and the molybdopterin domains was well defined in this mutant even though the B factors of residues Arg528–Gly536 were relatively high (70 – 84 Å2) compared with the mean B value of 37.3 Å2 for whole


FIG. 2. a, anaerobic reduction of C535A/C992R mutants with various levels of NADH-methylene blue activity. Enzymes (4.1 ␮M) with different activities were mixed with NADH (final 63 ␮M) in 50 mM potassium phosphate buffer, pH 7.8, containing 0.4 mM EDTA under anaerobic conditions, and the spectra were recorded immediately after mixing. Solid line, before mixing NADH; dash-dotted line, the enzyme with NADH-MB activity of 192 mol/min/FAD; dotted line, the enzyme with activity of 388 mol/min/FAD; dashed line, the enzyme with activity of 588 mol/min/FAD. Initial spectra of these mutant XORs were adjusted to the solid spectrum by calculation. Inset, relationship of the absorbance bleaching at 450 nm versus NADH-MB activity. b, immediate spectral changes of C535A/C992R/C1316S and C535A/C992R/C1324S mutants by addition with NADH under anaerobic conditions. Dashed line, initial spectrum of C535A/C992R/C1316S; dotted line, spectrum after mixing of C535A/C992R/C1316S with 83 ␮M NADH; closed circle, initial spectrum of C535A/C992R/C1324S XDH; open circle, spectrum after mixing of C535A/C992R/C1324S XDH with 83 ␮M NADH. The enzyme concentration of C535A/C992R/C1316S or C1324S mutant used was 4.8 or 5.5 ␮M, respectively.

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enzyme peptide. This indicates a much reduced, although still recognizable, mobility in this part of the polypeptide chain of the rat enzyme when compared with the bovine one (19). The C992R residue is located at the end of a helix of the molybdopterin domain, and C535A is part of the linker connecting the FAD to the molybdopterin domain (Fig. 3b). The distance between the ␣ carbon atom of residue 992 and that of residue 535 is ⬃18 Å. As we described previously, there is a unique cluster of amino acids that plays a dual role by forming the core of the relay system for the XDH/XO transition and by gating a solvent channel leading toward the FAD ring (19). The crystal structure of the present rat liver XDH mutant also contains this amino acid cluster, and the loop (Gln422–Lys432) takes the same conformation as in bovine milk XDH. These findings are consistent with the fact that the mutant is in the XDH form. Although the C-terminal stretch of amino acids was inserted into the FAD active site

of the neighboring molecule in the bovine crystal structure (15), that C-terminal peptide interacts with the NAD⫹ binding pocket of the same molecule in the crystal structure of the rat enzyme (Fig. 3c). The peptide makes contact with a loop (Leu493–Met503), which seems to support the binding of NAD⫹ to the XDH form. Although electron density for the five amino acid residues between 1319 –1323 was missing, the density corresponding to Cys1316 and Ser1324, mutated from Cys1324, was clearly observed in the present structure. The separation of 20.5 Å between Cys1316 and Cys1324 is rather long, and disulfide formation requires a large rearrangement of the flexible 9-amino-acid loop. Thus, it is very likely that the formation of a disulfide bridge between Cys1316 and Cys1324 will result in conformational changes of the C-terminal peptide, interfering with the insertion of this peptide, which seems to be important for pyridine nucleotide binding at the flavin active site.


FIG. 3. a, ribbon diagram of overall dimeric structure of rat XDH C535A/C992R/C1324S mutant. The interdomain loop (Ala529–Ala581) is shown in magenta. The C-terminal loop (Leu1315–Ile1331) is shown in orange. FAD and salicylate are shown as stick models. Fe-S centers are shown as space-filling models. The figures were generated with MOLSCRIPT (28) and RASTER3D (29) software. b, secondary structure elements around the residues that are involved in rapid XDH/XO conversion. The mutated residues Ala535 and Arg992 are shown as stick models. The interdomain loop region (Ala529–Ala581) is shown in magenta, as in a. FAD is shown as a stick model. Fe-S centers are shown as space-filling models. c, the C-terminal region of rat XDH in the NAD⫹ binding cavity. The C-terminal loop (Leu1315–Ile1331) is shown in orange. Cys1316 and Ser1324 are shown as stick models. The loop (Leu493–Met503) next to the C-terminal region is shown in yellow. The active site loop (Gln423–Lys433) is shown in blue. FAD is shown as a stick model. An Fe-S center is shown as a space-filling model. Pyridine moiety of NADⴙ cofactor binds between the space of the loop (Leu493–Met503) and FAD cofactor.


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FIG. 4. Schematic representation of the mechanism of conversion from xanthine dehydrogenase to xanthine oxidase. MOP, molybdopterin; SH, sulfhydryl group.

2 B. T. Eger, E. F. Pai, K. Okamoto, T. Nishino, unpublished observations.

are ⬃18 Å apart, so a large movement of the linker peptide would be required for disulfide bond formation between Cys992 and the mobile Cys535. It should be noted that the distance between the two residues would become feasible for disulfide bond formation if the flexible peptide loop 527–535 were extended to some extent. As summarized in Fig. 4, recent studies of bovine milk XOR by site-directed mutagenesis and more detailed analyses of the crystal structures of bovine milk XDH and XO forms have revealed the presence of a unique amino acid cluster acting as a switch and a solvent gate during the transition between the dehydrogenase and oxidase forms of XOR (19). Tight interactions between the amino acid residues of the cluster are crucial for stabilization of the XDH form of the enzyme. The cluster residues sit at the center of a relay system that transmits modifications of the linker peptide, caused by cysteine oxidation or proteolytic cleavage, to the active site loop (rat enzyme Gln422–Lys432), resulting in a dramatic change in the conformation of the latter. Both modifications lead to the removal of Phe549 from the cluster, probably the trigger for the subsequent rearrangements. This can be caused either by a change in conformation induced by disulfide formation between Cys535 and Cys992 or by proteolysis within the linker peptide. In the Rhodobacter capsulatus XDH, which cannot be converted to an oxidase form, the linker peptide is absent, because the flavin and molybdenum domains reside in separate subunits. The conformation of the R. capsulatus enzyme seems to be stabilized in the XDH form by additional steric factors (31, 32). Although the C535A/C992R mutant did not display the rapid XDH to XO conversion seen with wild-type enzyme, it was


DISCUSSION

The results of the mutation analysis clearly point to an involvement of both Cys535 and Cys992 in the rapid conversion from xanthine dehydrogenase to oxidase. Both mutants C992R and C535A significantly resisted spontaneous conversion of XDH to XO by auto-oxidative disulfide formation or by the sulfhydryl modifier 4,4⬘-DTDY. As expected, the double mutant C535A/C992R was most resistant to the rapid conversion. Modification with FDNB also results in resistance to conversion, but on prolonged incubation of the mutants, the total activity decreases (data not shown) due to lysine modification near the xanthine binding site (13, 30). The fact that both single mutants resisted conversion suggests that these residues undergo disufide formation. McManaman and Bain (18) questioned the involvement of Cys992 and Cys535 in the XDH to XO conversion after they failed to detect peptides representative of the S-Slinked FAD and molybdopterin domains after proteolysis of oxidatively generated bovine XO. The gel chromatographic analysis that they applied, however, is prone to allow S-S exchange during the experiment when cysteine residues are incompletely alkylated. Involvement of the Cys992 residue is also indicated by the x-ray analysis of a bovine XO crystal, which was successfully obtained under special conditions, showing the electron density of a disulfide bond associated with Cys992.2 In the crystal structure of the rat liver XDH mutant elucidated in this study, the ␣ carbon atoms of residues 992 and 535

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Conversion of Xanthine Dehydrogenase to Oxidase ance, e.g. formation of a disulfide bridge, will result in the loss of NAD⫹ binding. Such an interpretation of the role of the C-terminal peptide is not only based on our structural studies but also is supported by our findings that the slow XDH to XO conversion reaction of C535A/C992R with 4,4⬘-DTPY was completely blocked in the presence of NADH (Fig. 1d) and that the XO form, which was generated slowly, probably by disulfide formation between the two C-terminal cysteine residues, cannot be reduced by NADH (Fig. 2a). The fact that the electron density of five amino acid residues between 1319 and 1323 was not observed in the crystal structure suggests that this region of the loop is highly disordered and may be able to move to allow disulfide bond formation between Cys1316 and Cys1324. Efforts are under way to grow well diffracting crystals of the fully oxidized form of XO, a form that also contains the Cys1316–Cys1324 disulfide, to obtain a clearer picture of which conformational changes near the flavin cofactor are caused by this oxidative modification. REFERENCES 1. Bray, R. C., (1975) in The Enzymes, (Boyer, P. D., ed) 3rd Ed., Vol. XII, pp. 299 – 419, Academic Press, New York 2. Hille, R., and Nishino, T. (1995) FASEB J. 9, 995–1003 3. Hille, R. (1996) Chem. Rev. 96, 2757–2816 4. Olson, J. S., Ballou, D. P., Palmer, G., and Massey, V. (1974) J. Biol. Chem. 249, 4363– 4382 5. Della Corte, E., and Stripe, F. (1968) Biochem. J. 108, 349 –351 6. Stripe, F., and Della Corte, E. (1969) J. Biol. Chem. 244, 3855–3863 7. Della Corte, E., and Stripe, F. (1972) Biochem. J. 126, 739 –745 8. Waud, W. R., and Rajagopalan, K. V., (1976) Arch. Biochem. Biophys. 172, 354 –364 9. Nakamura, M., and Yamazaki, I. (1982) J. Biochem. 92, 1279 –1286 10. Saito, T., and Nishino, T. (1989) J. Biol. Chem. 264, 10015–10022 11. Hunt, J., and Massey, V. (1992) J. Biol. Chem. 267, 21476 –21485 12. Amaya, Y., Yamazaki, K., Sato, M., Noda, K., Nishino, T., and Nishino, T. (1990) J. Biol. Chem. 265, 14170 –14175 13. Nishino, T., and Nishino, T. (1997) J. Biol. Chem. 272, 29859 –29864 14. McCord, J. M. (1985) N. Engl. J. Med. 312, 159 –163 15. Enroth, T., Eger, B. T., Okamoto, K., Nishino, T., Nishino, T., and Pai, E. F (2000) Proc. Natl. Acad. Sci. U. S. A. 10723–10728 16. Saito, T. (1987) Yokohama Med. Bull. 38, 151–168 17. Rasmussen, J. T., Rasmussenn, M. S., and Petersen, T. E. (2000) J. Dairy Sci. 83, 499 –506 18. McManaman, J. L., and Bain, D. L. (2002) J. Biol. Chem. 277, 21261–21268 19. Kuwabara, Y., Nishino, T., Okamoto, K., Matsumura, T., Eger, B. T., Pai, E. F., and Nishino, T. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 8170 – 8175 20. Nishino, T., Amaya, Y., Kawamoto, S., Kashima, Y., Okamoto, K., and Nishino, T. (2002) J. Biochem. 132, 597– 606 21. Summers, M. D., and Smith, G. E. (1987) A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experiment Station Bulletin No. 1555, College Station, Texas 22. Virlard, J., Lamuriere, M., Vernet, T., Briedis, D., Alkhatib, G., Henning D., Levin, D., and Richardson, C. (1990) J. Virol. 64, 37–50 23. Nishino, T., Nishino, T., and Tsushima, K. (1981) FEBS Lett. 131, 369 –372 24. Ikegami, T., and Nishino, T. (1986)) Arch. Biochem. Biophys. 247, 254 –260 25. Laemmli, U. K. (1970) Nature 227, 680 – 685 26. Jonson, J. L., Waud, W. R., Cohen, H. J., and Rajagopalan, K. V. (1974) J. Biol. Chem. 249, 5056 –5061 27. Sato, A., Nishino, T., Noda, K., Amaya, Y., and Nishino, T. (1995) J. Biol. Chem. 270, 2818 –2826 28. Kraulis, P. (1991) J. Appl. Crystallogr. 24, 946 –950 29. Merritt, E. A., and Bacon, D. J. (1997) Methods Enzymol. 277, 505–524 30. Nishino, T., Tsushima, K., Hille, R., and Massey, V. (1982) J. Biol. Chem. 257, 7348 –7353 31. Leimkuhler, S., Kern, M., Solomon, P. S., McEwan, A. G., Schwarz, G., Mendel, R. R., and Klipp, W. (1998) Mol. Microbiol. 27, 853– 869 32. Truglio, J. J., Theis, K., Leimkuhler, S., Rappa, R., Rajagopalan, K. V., and Kisker, C. (2002) Structure (Camb.) 10, 115–125


converted slowly by an Src homology modifier, suggesting the existence of another cysteine couple responsible for the slower conversion. Reduction experiments with excess NADH of the C535A/C992R mutants, which had various dehydrogenase activities as a result of prolonged auto-oxidation, suggested that the other cysteine couple might be close to the NADH binding site (Fig. 2). The fact that the slow conversion reaction of (C535A/C992R) XDH to XO with 4,4⬘-DTPY was completely blocked in the presence of NADH also supports such an interpretation. The crystal structure of the bovine milk enzyme suggested that Cys1316 and Cys1324 (rat numbering) were the most likely candidates for such a couple. The previous results of chemical modification with radioactive FDNB showed that 0.39 mol/FAD of labeled dinitrophenol was incorporated into Cys1324. We also produced the single mutant C1324S in a baculovirus-insect cell system. Although the protein was expressed in its XDH form, it had been converted almost entirely to its oxidase form by the second day of purification. Further, the DTT-generated XDH form of the single mutant C1324S was easily converted to the XO enzyme with 4,4⬘-DTPY under the same conditions as native XDH. However, it is not surprising that the single mutant C1324S still has the potential to convert, because the protective effect of cysteine modification at the C terminus can be superseded by the rapid conversion because of disulfide bond formation between Cys535 and Cys992. In this study, two triple mutants, C535A/C992R/C1316S and C535A/C992R/C1324S, were expressed completely in their XDH forms in crude extracts, and they stayed in this form throughout the purification. Incubating these two mutants with 4,4⬘-DTPY for 80 min did not cause conversion, and the sum and the ratio of XDH and XO activities were unchanged (Fig. 1e). In the crystal structure, the Cys1316 and Ser1324 residues are located close to the C terminus, and the distance between their ␣ carbon atoms is 20.5 Å (Fig. 3c). The C-terminal peptide is inserted into the same subunit, pointing in the direction of the NAD⫹ binding pocket, and makes contact with the loop (Leu493–Met503) that seems to be important for binding the NAD⫹ cofactor, an observation supported by the crystal structure of the NADH complex of the bovine enzyme.2 The surprising differences in location displayed by this C-terminal peptide (freely floating in bovine XO, binding to a neighboring molecule in the crystal lattice in bovine XDH (15) and entering the equivalent binding site of the same subunit in rat XDH) are well supported by the various electron density maps. In the case of bovine XO, no corresponding density can be observed, whereas in the bovine XDH crystals, it extends toward the neighboring molecule, although with elevated B-factors, and in rat XDH, the binding site on the next molecule is ⬎30 Å away. These results are consistent with a large degree of motional freedom of this C-terminal peptide, which lets it move in and out of the binding cavity. Given its location, it is easy to imagine that the C-terminal part of the XOR chain plays a role in pyridine nucleotide binding, possibly contributing to the generation of the actual binding site. Subsequently, any disturb-

SUPPLEMENTAL DATA CHARACTERIZATION OF MUTANTS

Steady-state kinetics of various mutants treated with or without DTT: The steady-state kinetic parameters of various DTT-treated or untreated mutants are summarized in Table S1. It was previously reported that the steady-state kinetics of native rat liver XOR afforded parallel Lineweaver-Burk plots when one substrate concentration was varied at a series of fixed concentrations of the other substrate. In the present study, Lineweaver-Burk plots of various mutants showed parallel lines for a series of fixed concentrations of the other substrate in all cases studied. All the parameters of xanthine-NAD+ activity, including Km for xanthine and NAD+ and Vmax, are very similar between DTTtreated wild-XDH, DTT-treated double mutant, and DTT-untreated triple mutants. These enzymes show rather higher Km values for oxygen and lower Vmax values than those of the wild-type XO form when xanthine oxygen activities were determined, consistent with the conclusion that DTT-untreated triple mutants (C535A+C992R+C1316S and C535A+C992R+C1324S) are in their XDH forms. Table S1. Steady-state kinetic parameters for rat liver and mutants enzymes Protein

DTT treat

D/O ratio

xanthine-NAD+ activity Km for X

Km for NAD

µM b

xanthine-O2 activity +

Vmaxa /min

µM

Km forX

Km for O2

Vmaxa

µM

µM

/min

1.8

46

1030

2.8

260

270

Rat liver XO

(-)

1

Not detected

XDH

(+)

7.1

1.3

(-)

1.9

2.0 ± 0.1

14.2 ± 0.8

433 ± 14

2.96 ± 0.2

47.7 ± 6.5

585±6

(+)

5.9

1.5 ± 0.2

10.7 ± 0.5

751 ± 15

2.8 ± 0.1

153.0 ± 0.6

267±6

(-)

5.8

1.7 ± 0.1

12.9 ± 0.4

730 ± 5

2.8 ± 0.1

150.0 ± 12

250±11

(-)

8.2

2.0 ± 0.1

13.4 ± 0.6

782 ± 10

2.9 ± 0.2

205.0 ± 14

275±5.5

C535A+C992R

C525A+C992R

8.5

810

+C1316S C535A+C992R +C1324S

a, Vmax was corrected for the measured value of AFR assuming that the AFR of fully active native rat liver enzyme is 200. Vmax for xanthine-O2 activity, urate mol/min/FAD; Vmax for xanthine-NAD activity, NADH mol/min/FAD. b, data from reference (S1). The enzyme concentrations used for the kinetic experiments were 6.6 nM for xanthine-O2 activity and 1.65 nM for xanthine-NAD activity. Xanthine-NAD+ Activity - The initial velocity of formation of NADH was measured at 25˚C by following the fluorescence emission at 460 nm with the excitation wavelength of 340 nm using a Nippon Bunko FP777 fluorescence spectrophotometer equipped with a temperature-controlled cell unit. The reaction mixture contained 50 mM KPB (pH 7.8), 0.4 mM EDTA, 1.6~1.74 nM XDH enzyme and various concentrations of ß-NAD+ (9.9, 15.8, 25, 50, 211 µM) and xanthine (0.91, 1.47, 3.05, 6 µM) in a volume of 3 ml. Xanthine-O2 activity - The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.8), 0.4 mM EDTA, various concentrations of xanthine (6, 7.5, 9, 12 µM) and 6.7 nM XDH or XO enzyme in a volume of 2 ml. Before addition of the enzyme, the incubation mixture was bubbled with different concentrations of oxygen gas for 5 min at 25˚C in an anaerobic cell equipped with an enzyme injector. The initial concentrations of oxygen were 69, 131, 258, 655 and 1290 µM. After addition of the enzyme, the absorbance at 295 nm was followed at 25˚C with a Hitachi 3210 double-beam spectrophotometer to obtain the initial velocity. Structure determination of the C535A+C992R+C1324S mutant of XOR:

1

Table S2. Data Collection and Refinement Statistics Space group Unit cell axes (Å), Unit cell angle (degree)

Resolution range (Å) Number of unique reflections (used for R-free calculation) Rsym# I/sigma I Completeness (%) Rcryst*(Rfree) Rmsd bond length (Å) Rmsd bond angles (degree) Number of nonhydrogen atoms Ramachandran plot (%)

I4122 a=b=134.3, c=523.3, alpha=beta= gamma=90 20-2.63 65,543 (1629) 6.5 (30.7) 35.0 (7.5) 100.0 (100.0) 0.247(0.283) 0.009 1.4 10,332 81.9, 16.5, 1.2, 0.4

#Rsym=∑hkl∑I|II - |/∑hkl∑I where II is the ith measurement and is the weighted mean of all measurements of I. *Rcryst=∑hkl |Fobs-Fcalc|/Fobs, where Fobs and Fcalc are the observed and calculated structure factors, respectively, and the summation is over the reflections used for model refinement. Rfree was the same as Rcryst for 2.5% of the data randomly omitted from the total data. Values in parentheses refer to the highest resolution shell (2.68 – 2.63 Å). Ramachandran statistics indicate the fraction of residues in the most favored, additionally allowed, generously allowed, and disallowed regions of the Ramachandran diagram, as defined by the program PROCHECK (S2). Data were reduced with the help of the program package HKL2000 and scaled using SCALEPACK (S3). The program package EPMR (S4) established the correct solutions of the respective molecular replacement function (20.0 to 4.0 Å resolution range). Bovine milk XDH (Protein Data Bank code 1FO4) without its cofactors was employed as a search model. The molecular models were built with the help of the program package O (S5). Subsequent refinement, including rigid body, simulated annealing, grouped B factors, and least-squares minimization were carried out with CNS, version 1.0 (S6).

2

Spectral properties of mutants: Visible absorption spectra of the purified recombinant XDHs of the wild type and each mutant are shown in Fig. S1. The spectra of all mutant enzymes were almost identical with that of the wild-type enzyme, having an extinction coefficient of 35.7 mM-1 (calculated from the FAD content), which is similar to the value of 35.8 mM-1 of native rat liver XDH (S7). The ratio of absorbance at 450 nm to 550 nm is 3.0, which is also similar to that of the native enzyme, indicating that the two iron sulfur centers are intact. Fig. S1. Absorption spectra of various purified rat liver XDH enzymes. The spectra were recorded after incubation of XORs with DTT followed by gel filtration on Sephadex G-25 equilibrated with potassium phosphate buffer, pH 7.8, containing 0.4 mM EDTA. Solid line, rat liver XDH purified from liver; closed circle, recombinant wild-type XDH; closed triangle, C535A mutant XDH; open circle, C992R; open square, C535A+C992R; closed square, C535A+C992R+C1316S; open triangle, C535A+C992R+C1324S.

The crystal structure of the mutant: Fig. S2. The difference in crystal structure between the molydbo-form of bovine milk XDH and the demolybdo-form of rat liver mutant XDH. The only electron density in the rat enzyme (green) found where the molybdopterin cofactor (red) sits in the bovine milk enzyme (yellow) corresponds to a phosphate ion from the buffer solution. It overlaps with the position of the cofactor phosphate in the bovine holoenzyme. Salicylate, which was added to the enzyme solution for stabilization, is found somewhat deeper in the active site pocket. It is surprising to see how little the positions of the surrounding side chains are influenced by the presence or absence of the cofactor. Figures were generated with MOLSCRIPT (S8) and RASTER3D (S9). Fig. S3: Electron density surrounding the residues in the linker peptide between flavin and molybdopterin domains. The 2Fo-Fc electron density map is contoured at 1.3 σ cutoff. The positions of alpha carbons of rat XDH are traced with a green line. The residues (Glu532-Gly536) that were not observed in the crystal structures of bovine milk XDH are shown in magenta. Fig. S4. Space-filling representation of the amino acid cluster and the loop that is important for XDH/XO conversion. The crystal structure of the rat liver XDH mutant contains the amino acid cluster stabilized by -cation interaction. Arg334 and Arg426 are shown in blue, Trp335 in green, Phe549 in magenta and FAD in yellow. The active site loop (Gln422-Lys432) is shown in red. It assumes the same conformation as in bovine milk XDH. Figures were generated with MOLSCRIPT (S8) and RASTER3D (S9).

REFERENCES FOR SUPPLEMENTAL MATERIALS S1. Saito, T., and Nishino, T. (1989) J. Biol. Chem. 264, 10015-10022 S2. Laskowski, R.A., MacArthur, M.W., Moss, D.S., and Thornton, J.M. (1993). J. Appl. Crystallogr. 26, 283-291 S3. Otwinoski, Z. and Minor, W. (1997) Methods Enzymol. 276, 307-326 S4. Kissinger, C. R., Gehlhaar, D. K. and Fogel, D. B. (1999) Acta Crystallogr. D Biol. Crystallogr. 55, 484-491 . S5. Jones, T. A., Zou, J. Y., Cowan, S. W. and Kjeldgaard, M. (1991) Acta Crystallogr. A, 47, 110-119. S6. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse- Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T. and Warren, G. L. (1998) Acta Crystallogr. D Biol. Crystallogr. 54, 905-921 S7. Jonson, J. L., Waud, W.R., Cohen, H. J., and Rajagopalan, K. V. (1974) J. Biol. Chem. 249, 5056-5061 S8. Kraulis, P. (1991) J. Appl. Crystallogr. 24, 946-950 S9. Merritt, E. A. and Bacon, D. J. (1997) Methods Enzymol. 277, 505-524

3

Enzyme Catalysis and Regulation: Mechanism of the Conversion of Xanthine Dehydrogenase to Xanthine Oxidase: IDENTIFICATION OF THE TWO CYSTEINE DISULFIDE BONDS AND CRYSTAL STRUCTURE OF A NON-CONVERTIBLE RAT LIVER XANTHINE DEHYDROGENASE MUTANT

J. Biol. Chem. 2005, 280:24888-24894. doi: 10.1074/jbc.M501830200 originally published online May 4, 2005

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Tomoko Nishino, Ken Okamoto, Yuko Kawaguchi, Hiroyuki Hori, Tomohiro Matsumura, Bryan T. Eger, Emil F. Pai and Takeshi Nishino