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• W2084250862 abstract "Rhodobacter capsulatus xanthine dehydrogenase (XDH) is a cytoplasmic enzyme with an (αβ)2 heterodimeric structure that is highly identical to homodimeric eukaryotic xanthine oxidoreductases. The crystal structure revealed that the molybdenum cofactor (Moco) is deeply buried within the protein. A protein involved in Moco insertion and XDH maturation has been identified, which was designated XdhC. XdhC was shown to be essential for the production of active XDH but is not a subunit of the purified enzyme. Here we describe the purification of XdhC and the detailed characterization of its role for XDH maturation. We could show that XdhC binds Moco in stoichiometric amounts, which subsequently can be inserted into Moco-free apo-XDH. A specific interaction between XdhC and XdhB was identified. We show that XdhC is required for the stabilization of the sulfurated form of Moco present in enzymes of the xanthine oxidase family. Our findings imply that enzyme-specific proteins exist for the biogenesis of molybdoenzymes, coordinating Moco binding and insertion into their respective target proteins. So far, the requirement of such proteins for molybdoenzyme maturation has been described only for prokaryotes. Rhodobacter capsulatus xanthine dehydrogenase (XDH) is a cytoplasmic enzyme with an (αβ)2 heterodimeric structure that is highly identical to homodimeric eukaryotic xanthine oxidoreductases. The crystal structure revealed that the molybdenum cofactor (Moco) is deeply buried within the protein. A protein involved in Moco insertion and XDH maturation has been identified, which was designated XdhC. XdhC was shown to be essential for the production of active XDH but is not a subunit of the purified enzyme. Here we describe the purification of XdhC and the detailed characterization of its role for XDH maturation. We could show that XdhC binds Moco in stoichiometric amounts, which subsequently can be inserted into Moco-free apo-XDH. A specific interaction between XdhC and XdhB was identified. We show that XdhC is required for the stabilization of the sulfurated form of Moco present in enzymes of the xanthine oxidase family. Our findings imply that enzyme-specific proteins exist for the biogenesis of molybdoenzymes, coordinating Moco binding and insertion into their respective target proteins. So far, the requirement of such proteins for molybdoenzyme maturation has been described only for prokaryotes. Xanthine oxidoreductase is a complex metalloflavoprotein that catalyzes the hydroxylation of hypoxanthine and xanthine, the last two steps in the formation of urate, using a water molecule as the ultimate source of oxygen incorporated into the product (1Hille R. Sprecher H. J. Biol. Chem. 1987; 262: 10914-10917Abstract Full Text PDF PubMed Google Scholar). The enzyme exists in two forms; the xanthine dehydrogenase form (XDH; EC1.17.1.4) 2The abbreviations used are: XDH, xanthine dehydrogenase; MPT, molybdopterin;Moco, molybdenum cofactor; bis-MGD, bis-molybdopterin guanine dinucleotide cofactor; NTA, nitrilotriacetic acid;, hSO, human sulfite oxidase; HPLC, high pressure liquid chromatography. uses NAD+ as electron acceptor, whereas the xanthine oxidase (EC1.17.3.2) form uses O2 as electron acceptor (2Hille R. Chem. Rev. 1996; 96: 2757-2816Crossref PubMed Scopus (1480) Google Scholar). Xanthine oxidoreductases are found both in eukaryotes and prokaryotes, with the enzymes isolated from bovine milk and Rhodobacter capsulatus being functionally and structurally the best characterized (3Enroth C. Eger B.T. Okamoto K. Nishino T. Pai E.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10723-10728Crossref PubMed Scopus (592) Google Scholar, 4Truglio J.J. Theis K. Leimkuhler S. Rappa R. Rajagopalan K.V. Kisker C. Structure (Camb.). 2002; 10: 115-125Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). R. capsulatus XDH is a cytoplasmic enzyme with an (αβ)2 heterodimeric structure, with the two subunits encoded by the xdhA and xdhB genes, respectively (5Leimkühler S. Kern M. Solomon P.S. McEwan A.G. Schwarz G. Mendel R.R. Klipp W. Mol. Microbiol. 1998; 27: 853-869Crossref PubMed Scopus (90) Google Scholar). It was shown that XdhA binds the two [2Fe-2S] clusters and the FAD cofactor, whereas XdhB binds the molybdopterin (MPT) form of the molybdenum cofactor (Moco) (4Truglio J.J. Theis K. Leimkuhler S. Rappa R. Rajagopalan K.V. Kisker C. Structure (Camb.). 2002; 10: 115-125Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 5Leimkühler S. Kern M. Solomon P.S. McEwan A.G. Schwarz G. Mendel R.R. Klipp W. Mol. Microbiol. 1998; 27: 853-869Crossref PubMed Scopus (90) Google Scholar). DNA sequence analysis revealed that a third gene is co-transcribed with xdhAB, designated xdhC (6Leimkühler S. Klipp W. J. Bacteriol. 1999; 181: 2745-2751Crossref PubMed Google Scholar). It was shown by interposon mutagenesis that the xdhC gene product is required for XDH activity; however, XdhC was not identified as a subunit of active XDH (4Truglio J.J. Theis K. Leimkuhler S. Rappa R. Rajagopalan K.V. Kisker C. Structure (Camb.). 2002; 10: 115-125Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 6Leimkühler S. Klipp W. J. Bacteriol. 1999; 181: 2745-2751Crossref PubMed Google Scholar). Analysis of the Moco content of inactive XDH purified from an R. capsulatus xdhC– strain showed that in the absence of XdhC, Moco is not inserted into XDH, but XDH still consisted of an (αβ)2 heterodimer with a full complement of FeS clusters and FAD (6Leimkühler S. Klipp W. J. Bacteriol. 1999; 181: 2745-2751Crossref PubMed Google Scholar). An interaction of XdhC with apo-XDH was not analyzed; however, studies of the electrophoretic mobility revealed a different conformation of Moco-free XDH in xdhC– and Moco-deficient R. capsulatus mutant strains in comparison with mature XDH (6Leimkühler S. Klipp W. J. Bacteriol. 1999; 181: 2745-2751Crossref PubMed Google Scholar). It was assumed that during Moco biosynthesis, the Moco-free apo-XDH stays in a suitable “open” conformation for the insertion of Moco into XdhB. XdhC was proposed to act as an XDH-specific Moco carrier protein, a Moco insertase, or a chaperone involved in proper folding during or after the insertion of Moco into XDH. In previous work, we have reported a system for the heterologous expression of R. capsulatus XDH in Escherichia coli (7Leimkühler S. Hodson R. George G.N. Rajagopalan K.V. J. Biol. Chem. 2003; 278: 20802-20811Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Whereas preparations of bovine milk XDH/xanthine oxidase typically possess a large quantity of the desulfo form of the enzyme (8Massey V. Komai H. Palmer G. Elion G.B. Vitam. Horm. 1970; 28: 505-531Crossref PubMed Scopus (24) Google Scholar), R. capsulatus XDH can be purified from E. coli cells in a form with a full complement of the equatorial Mo=S ligand required for functionality (9Leimkühler S. Stockert A.L. Igarashi K. Nishino T. Hille R. J. Biol. Chem. 2004; 279: 40437-40444Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). This shows that E. coli contains a sulfurtransferase capable of adding the terminal sulfur ligand to the molybdenum site of Moco; however, nothing is yet known about the nature of this protein in E. coli. This report describes the detailed analysis for the requirement of XdhC to produce active XDH during heterologous expression in E. coli TP1000 cells. Analysis of XDH expressed under different aeration levels in the presence or absence of XdhC showed that, especially under aerobic conditions, XdhC is required to produce active XDH containing the terminal sulfur ligand of Moco. In addition, we purified and characterized XdhC after heterologous expression in E. coli. We could show that XdhC binds Moco/MPT in stoichiometric amounts and is able to insert bound Moco into Moco-free apo-XDH. In addition, a specific interaction between XdhC and XdhB was identified. We showed that XdhC acts as a Moco-binding protein, which protects the sulfurated form of Moco from oxidation. We propose that sulfurated Moco is inserted into apo-XDH by the aid of XdhC to produce active XDH. This is the first example of a system-specific protein involved in maturation of a molybdoenzyme for which Moco binding could be shown. Bacterial Strains, Plasmids, Media, and Growth Conditions—E. coli ER2566(DE3) cells and plasmid pTYB2 were obtained from New England Biolabs, and expression vector pET29b was purchased from Novagen. E. coli TP1000 cells (10Palmer T. Santini C.-L. Iobbi-Nivol C. Eaves D.J. Boxer D.H. Giordano G. Mol. Microbiol. 1996; 20: 875-884Crossref PubMed Scopus (146) Google Scholar) were used for expression of XDH from pSL207 (7Leimkühler S. Hodson R. George G.N. Rajagopalan K.V. J. Biol. Chem. 2003; 278: 20802-20811Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) and for expression of the Moco domain of human sulfite oxidase (hSO) from pTG818 (11Temple C.A. Rajagopalan K.V. Arch. Biochem. Biophys. 2000; 383: 281-287Crossref PubMed Scopus (108) Google Scholar). Moco-free apo-XDH was obtained after expression in E. coli RK5200 (chlA::Mu cts [moaA–]) (12Johnson M.E. Rajagopalan K.V. J. Bacteriol. 1987; 169: 117-125Crossref PubMed Google Scholar). Molybdenum-free hSO was obtained after expression in E. coli RK5202 (chlD202::Mu cts [modC–]) (13Miller J.B. Scott D.J. Amy N.K. J. Bacteriol. 1987; 169: 1853-1860Crossref PubMed Google Scholar). Expression of XDH from pSL181 was carried out in E. coli RK4353(DE3) cells (14Stewart V. MacGregor C.H. J. Bacteriol. 1982; 151: 788-799Crossref PubMed Google Scholar). His6-tagged CsdA was expressed in E. coli BL21(DE3) cells (Novagen) from plasmid pSL215 and purified as described previously (15Leimkühler S. Rajagopalan K.V. J. Biol. Chem. 2001; 276: 22024-22031Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Cell strains containing expression vectors were grown aerobically in LB medium at 30 °C in the presence of either 150 μg/ml ampicillin or 25 μg/ml kanamycin. Construction of Expression Vectors—For expression of xdhB and xdhC, primers were designed to allow cloning into the NdeI and KpnI sites of expression vector pTYB2, resulting in plasmid pAK22 and pAK20, respectively. For construction of pSL181, the NdeI-HindIII xdhABC fragment from pSL207 was cloned into pET29b, resulting in a C-terminal His6 fusion to XdhC. pMS3 was obtained from pSL207 by deletion of a 500-bp XhoI fragment in the coding sequence of xdhC. Expression and Purification of XdhB and XdhC—For the heterologous expression of R. capsulatus XdhC in E. coli, pAK22 and pAK20 were transformed into E. coli ER2566(DE3). The proteins were expressed and purified following the IMPACT instruction manual (New England Biolabs) as described previously (16Leimkühler S. Freuer A. Santamaria Araujo J.A. Rajagopalan K.V. Mendel R.R. J. Biol. Chem. 2003; 278: 26127-26134Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). XdhB was further purified by an additional gel filtration step. Immediately before use, XdhC was transferred into the required buffer by gel filtration using PD10 columns (GE Healthcare). Heterologous Expression of XDH in the Absence and Presence of XdhC under Different Oxygen Supply—XDH was expressed in E. coli TP1000 or DH5α cells (17Hanahan D. J. Mol. Biol. 1983; 166: 557-580Crossref PubMed Scopus (8210) Google Scholar) with the expression vectors pSL207 (xdhABC) and pMS3 (xdhAB) under different oxygen supply by using shaking rates of 50, 130, and 210 rpm. After expression, XDH was purified by Ni2+-NTA chromatography (Qiagen) and Q-Sepharose ion exchange chromatography (GE Healthcare). Co-purification of XDH with XdhC—For co-purification of XDH with XdhC, pSL181 was transformed using the E. coli RK4353(DE3) strain, and cells were grown in 1 liter of LB medium. For purification of the C-terminal His6-tagged XdhC, 0.5 ml of Ni2+-NTA matrix was used. The column was washed with 10 column volumes of 50 mm phosphate buffer, 100 mm NaCl, pH 8.0, containing 10 mm imidazole and 20 column volumes of the buffer containing 20 mm imidazole. The proteins were eluted with the same buffer containing 250 mm imidazole and immediately analyzed by SDS-PAGE. Moco was extracted from 1 ml of 390 μm hSO and added to the crude extract in conjunction with 80 μm Na2MoO4 prior to Ni2+-NTA chromatography. XDH showed no interactions with the Ni2+-NTA matrix under these conditions. Moco was quantified as Form A. Sulfuration of Moco Bound to XdhC by CsdA—240 μm CsdA was persulfurated by incubation with 10 mm l-cysteine for 15 min at 4 °C, and excess cysteine was removed by passage through a PD10 column. For in vitro sulfuration experiments, 3 μm XdhC was incubated in a total volume of 0.5 ml under anaerobic conditions with 32 μm Moco extracted from hSO, 60 μm CsdA, and 150 μm sodium dithionite for 30 min. CsdA was removed by Ni2+-NTA chromatography (0.5-ml bed volume) equilibrated in 50 mm phosphate buffer, 300 mm NaCl, pH 8.0, and a volume of 1 ml of the flow-through was collected and incubated with 0.75 μm apo-XDH for 2 h. Control experiments were carried out without inclusion of XdhC, CsdA, or sodium dithionite in the incubation mixtures. As positive controls, either 60 μm sodium sulfide was added to the incubation mixtures containing XdhC for chemical sulfuration of Moco or 32 μm Moco extracted from active XDH. Enzyme Assays—XDH assays were performed at room temperature using a Shimadzu UV-2401PC spectrophotometer. 1 ml of mixture contained 500 μm xanthine and 1 mm NAD+ in 50 mm Tris, 1 mm EDTA, pH 7.5. The specific enzyme activity (units/mg) is defined as the reduction of 1 μmol of NAD+/min/mg of enzyme. Metal and Moco/MPT Analysis—The molybdenum and iron contents of the purified proteins were quantified by inductively-coupled plasma optical emission spectrometry (ICP-OES) analysis with a PerkinElmer Life Sciences Optima 2100 DV ICP-OES. 500 μl of a 10 μm solution was mixed with 500 μl 65% nitric acid and incubated overnight at 100 °C before the addition of 4 ml of water. As reference, the multielement standard solution XVI (Merck) was used. Moco/MPT was quantified by conversion to Form A as described earlier (16Leimkühler S. Freuer A. Santamaria Araujo J.A. Rajagopalan K.V. Mendel R.R. J. Biol. Chem. 2003; 278: 26127-26134Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 18Johnson J.L. Hainline B.E. Rajagopalan K.V. Arison B.H. J. Biol. Chem. 1984; 259: 5414-5422Abstract Full Text PDF PubMed Google Scholar). Moco/MPT was extracted by the addition of 50 μl of acidic iodine. Following incubation at room temperature for 14 h, excess iodine was removed by the addition of 55 μl of 1% ascorbic acid, and the sample was adjusted with 1 m Tris to pH 8.3. The phosphate monoester of Form A was cleaved by the addition of 40 mm MgCl2 and 1 unit of calf intestine alkaline phosphatase. After the addition of 10 μl of acetic acid, Form A was identified and quantified by HPLC analysis with a C18 reversed phase HPLC column (4.6 × 250-mm ODS Hypersil; particle size 5 μm) with 5 mm ammonium acetate, 15% methanol at an isocratic flow rate of 1 ml/min. In-line fluorescence was monitored by an Agilent 1100 series detector with an excitation at 383 nm and emission at 450 nm. For quantification of Form A, the corresponding fractions were collected, and the concentration was determined as described earlier (18Johnson J.L. Hainline B.E. Rajagopalan K.V. Arison B.H. J. Biol. Chem. 1984; 259: 5414-5422Abstract Full Text PDF PubMed Google Scholar). Quantification of the Cyanolyzable Sulfur—250 μl of a 13 μm XDH in 100 mm Tris acetate, pH 8.6, was incubated with 27.5 μl of 1 m KCN overnight at 4 °C as originally described by Massey and Edmondson (19Massey V. Edmondson D. J. Biol. Chem. 1970; 245: 6595-6598Abstract Full Text PDF PubMed Google Scholar). The protein was separated with a 3000 molecular weight cut-off Centricon concentrator (Millipore), and 250 μl of the SCN–-containing flow-through was mixed with 250 μl of ferric nitrate reagent (10 g of Fe(NO3)3 × 9H2O and 20 ml of 65% HNO3 per 150 ml). The FeSCN– complex was quantified at 460 nm using a standard curve from 1–100 mm KSCN. Generation of Free Moco/MPT—The Moco domain of hSO was expressed from pTG818 in E. coli TP1000 and purified as described by Temple et al. (11Temple C.A. Rajagopalan K.V. Arch. Biochem. Biophys. 2000; 383: 281-287Crossref PubMed Scopus (108) Google Scholar). To 200 μl of hSO (220 μm in 100 mm Tris, pH 7.2), 50 μl of 1 m Na2MoO4 was added prior to heat treatment at 95 °C for 5 min. After centrifugation at maximum speed for 5 min, the supernatant was used as a Moco source. For purification of molybdenum-free MPT, hSO was expressed from pTG818 in E. coli RK5202 (13Miller J.B. Scott D.J. Amy N.K. J. Bacteriol. 1987; 169: 1853-1860Crossref PubMed Google Scholar) without the addition of 1 mm Na2MoO4, and Na2MoO4 was omitted from the incubation mixtures. The sulfurated form of Moco was obtained from 80% active XDH using the same procedure. Reconstitution of Apo-XDH with Moco—To obtain Moco-free XDH, pSL207 was expressed in E. coli RK5200 (moaA–) cells, and apo-XDH was purified immediately before use by Ni2+-NTA affinity chromatography. Varying amounts of XdhC (0–15 μm) were incubated with an excess of purified Moco from hSO at 4 °C. Unbound Moco was removed from the incubation mixtures by gel filtration using Nick columns (GE Healthcare). The amount of Moco bound to XdhC was quantified by conversion to Form A, and the Form A concentration was determined using a Form A standard curve. 300 μl of Moco-XdhC was incubated with 100 μl of 4 μm apo-XDH for 90 min at 4 °C. After incubation, XDH was separated from XdhC by Ni2+-NTA chromatography. As control, different concentrations of free Moco (0–35 μm) were incubated with apo-XDH. The amount of Moco inserted into XDH was quantified by conversion to Form A. The same procedure was used to insert the sulfurated form of Moco (obtained from heat-treated XDH) into apo-XDH in the presence or absence of XdhC. 2 μm XdhC were preincubated with Moco for 5, 30, or 60 min at 4 °C before the addition of 0.8 μm apo-XDH. In parallel, extracted Moco was incubated without any additions before the addition of apo-XDH at the same time points. After an incubation time of 2 h at 4 °C, XDH activity was tested. Binding of Moco/MPT to XdhC—6 μm XdhC (in 100 mm Tris, pH 7.2) was incubated with free Moco/MPT (0–35 μm) for 15 min at 4 °C. The samples were transferred to Microcon concentrators (molecular weight cut-off 10,000; Millipore) and centrifuged at 14,000 × g for 5 min. As a control, free Moco/MPT was used in the absence of XdhC. The flow-through containing unbound Moco/MPT was converted to Form A and quantified. Fitting was based on the law of mass action for a Moco/MPT to XdhC ratio of n × m. KDapp=[Mocofree]n[XdhCfree]m[XdhCm−Mocon] (Eq. 1) Surface Plasmon Resonance Measurements—Binding experiments were performed with the surface plasmon resonance-based instrument Biacore™ 2000 and sensor chips CM5, using the control software 2.1 and evaluation software 3.0 (Biacore AB, Uppsala, Sweden). XDH, apo-XDH, XdhB, and bovine serum albumin were immobilized via amine coupling at 6,000–13,000 resonance units (RU) per flow cell. The running buffer was 20 mm phosphate, pH 7.5, 250 mm NaCl, 50 μm EDTA, 0.005% Tween 20. XdhC with concentrations of 0.2, 0.4, 0.8, 1.6, 3.2, and 6.4 μm was injected at 25 °C for 4.5 min at a flow rate of 20–40 μl/min followed by a 10–15-min dissociation using kinject and regeneration of the sensor surface with 10–20 mm HCl for 1 min. Binding curves were normalized by substraction of buffer injection curves for all flow cells and XdhC curves for the control flow cell. Heterologous Expression and Purification of R. capsulatus XdhC in E. coli—For purification of XdhC, a fusion protein was generated containing both a C-terminal intein tag and a chitin-binding domain for affinity purification. After binding to chitin beads, cleavage with dithiothreitol resulted in purified XdhC in a soluble form with a yield of 0.2 mg of protein/liter of E. coli culture (Fig. 1). The purified protein displayed a single band on Coomassie Brilliant Blue-stained SDS-polyacrylamide gels with a monomeric mass of 33 kDa, which is in close correspondence to the calculated monomeric mass of 33.4 kDa. The observed elution position of native XdhC from a size exclusion chromatography suggested that XdhC exists as a dimer in solution (data not shown). Characterization of the Role of XdhC for the Maturation of R. capsulatus XDH in E. coli—As reported previously, XdhC was shown to be essential for the insertion of Moco into XDH when expressed in R. capsulatus under anaerobic conditions, but not for XDH heterologously expressed under aerobic conditions in E. coli TP1000 cells, containing a deletion in the mobAB genes (6Leimkühler S. Klipp W. J. Bacteriol. 1999; 181: 2745-2751Crossref PubMed Google Scholar, 7Leimkühler S. Hodson R. George G.N. Rajagopalan K.V. J. Biol. Chem. 2003; 278: 20802-20811Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). However, in both cases, XDH was found to be inactive. To determine whether different aeration levels during expression influence the levels of Moco insertion into XDH, R. capsulatus XDH was expressed in E. coli TP1000 cells in the presence or absence of XdhC under variation of shaking velocities (50, 130, and 210 rpm). XDH was purified (see “Experimental Procedures”), and XDH activity was determined, in addition to the content of molybdenum, iron, cyanolyzable sulfur, and MPT in the enzyme (Table 1). As shown in Table 1, XdhC was not required for the insertion of MPT into XDH; however, increasing aeration resulted in lower levels of MPT in the purified protein. Analysis of the molybdenum content of the proteins corresponded well with the MPT content, showing that mainly all MPT bound to the protein is present as Moco. In addition, all enzyme preparations contained the appropriate levels of FeS and FAD cofactors, as judged by their respective visible absorption spectra (data not shown) and by determination of the iron content (Table 1). However, XDH activity was drastically decreased in the absence of XdhC with increasing levels of culture aeration (Table 1). Whereas XDH expressed in the absence of XdhC at 50 rpm under semianaerobic conditions retained 30% of its activity, the protein expressed at 210 rpm was almost inactive. Analysis of the terminal Mo=S ligand required for XDH activity showed that loss of enzyme activity correlated well with a low concentration of the cyanolyzable sulfur present in Moco. Thus, during heterologous expression in E. coli TP1000, XdhC is not required for Moco insertion into XDH, but in particular under aerobic conditions XdhC is essential for the stabilization of the sulfurated form of Moco. We also investigated whether the presence of Moco in XDH in the absence of XdhC is due to the usage of E. coli TP1000 cells for expression, which accumulate Moco to unphysiological high levels due to an impaired conversion of Moco into bis-MGD. Our analyses showed that expression of xdhAB in DH5α cells under high culture aeration resulted in an inactive, Moco-free XDH (data not shown), consistent with the results obtained for XDH purified from R. capsulatus xdhC– cells. Thus, under physiological concentrations of Moco, XdhC is essential for Moco insertion into XDH both in R. capsulatus and E. coli.TABLE 1Effect of XdhC on activity and cofactor content of R. capsulatus XDH after heterologous expression in E. coli TP1000 cells under different culture aerationrpm Values, expression of genes on plasmid usedMolybdenumaMolybdenum (μm molybdenum/μm XDH) and iron (μm 2[2Fe2S]/μm XDH) were determined by ICP-OES (see “Experimental Procedures”) using a multielement standard. The molybdenum and iron concentrations determined in the purified proteins were compared to a XDH preparation with a 100% Moco and 2[2Fe2S] content.IronaMolybdenum (μm molybdenum/μm XDH) and iron (μm 2[2Fe2S]/μm XDH) were determined by ICP-OES (see “Experimental Procedures”) using a multielement standard. The molybdenum and iron concentrations determined in the purified proteins were compared to a XDH preparation with a 100% Moco and 2[2Fe2S] content.Cyanolyzable sulfurbThe concentration of the terminal sulfur ligand of Moco was determined spectrophotometrically as an iron-thiocyanate complex at 420 nm as described under “Experimental Procedures.” 100% was set to a fully sulfurated control XDH.ActivitycSpecific enzyme activity (units/mg) is defined as the reduction of μmol of NAD/min/mg of enzyme under assay conditions.MPT/MocodMPT/Moco was quantified after conversion to the stable oxidized fluorescent degradation product Form A, as described under “Experimental Procedures.” 100% was set to a control XDH with a full complement of Moco.%%%units/mg%50 rpm, xdhABC (pSL207)71.5 ± 6.7105.2 ± 5.063.5 ± 0.637.8 ± 0.772.7 ± 5.950 rpm, xdhAB (pMS3)71.0 ± 5.7101.7 ± 1.425.5 ± 2.111.7 ± 0.667.7 ± 2.4130 rpm, xdhABC (pSL207)71.5 ± 0.397.6 ± 3.166.9 ± 1.845.3 ± 0.771.8 ± 4.1130 rpm, xdhAB (pMS3)71.0 ± 0.793.6 ± 0.42.5 ± 0.90.95 ± 0.0365.2 ± 4.9210 rpm, xdhABC (pSL207)31.3 ± 1.1106.0 ± 4.831.1 ± 0.635.1 ± 0.350.8 ± 4.4210 rpm, xdhAB (pMS3)32.6 ± 3.3101.1 ± 3.90.85 ± 0.90.19 ± 0.0249.4 ± 2.8Moco-free control XDH (xdhABC, pSL207) expressed in E. coli RK5200 at 130 rpmNDeND, not detectable.102.8 ± 1.2NDNDNDa Molybdenum (μm molybdenum/μm XDH) and iron (μm 2[2Fe2S]/μm XDH) were determined by ICP-OES (see “Experimental Procedures”) using a multielement standard. The molybdenum and iron concentrations determined in the purified proteins were compared to a XDH preparation with a 100% Moco and 2[2Fe2S] content.b The concentration of the terminal sulfur ligand of Moco was determined spectrophotometrically as an iron-thiocyanate complex at 420 nm as described under “Experimental Procedures.” 100% was set to a fully sulfurated control XDH.c Specific enzyme activity (units/mg) is defined as the reduction of μmol of NAD/min/mg of enzyme under assay conditions.d MPT/Moco was quantified after conversion to the stable oxidized fluorescent degradation product Form A, as described under “Experimental Procedures.” 100% was set to a control XDH with a full complement of Moco.e ND, not detectable. Open table in a new tab Analysis of Moco/MPT Binding to XdhC—From the previous results, the question arose whether XdhC is able to bind Moco and might act as a Moco insertase for XDH, inserting the sulfurated form of Moco and protecting it from oxidation. Since hSO contains the MPT form of Moco and can be purified in large quantities (11Temple C.A. Rajagopalan K.V. Arch. Biochem. Biophys. 2000; 383: 281-287Crossref PubMed Scopus (108) Google Scholar), recombinant, heat-denatured hSO was chosen as a source of Moco/MPT for binding studies. To compare differences in binding of MPT or Moco to XdhC, Moco was obtained from the Moco domain of hSO expressed in TP1000 cells in the presence of molybdate, whereas molybdenum-free MPT was obtained from inactive hSO expressed in RK5202(modC–) cells in the absence of molybdate. To determine the dissociation constants of Moco/MPT to XdhC, purified XdhC was incubated with extracted Moco/MPT for 15 min before unbound Moco/MPT were separated by ultrafiltration using a membrane with a molecular weight cut-off of 10,000. The Moco/MPT concentration in the flow-through was quantified after conversion to the stable, fluorescent oxidation product Form A (see “Experimental Procedures”). Quantification of Moco/MPT after ultrafiltration in the presence and absence of XdhC allowed the determination of KD values of Moco/MPT binding to XdhC (Fig. 2). The amount of Moco/MPT bound to XdhC and free XdhC concentrations were calculated according to the determined free and total Moco concentrations and the given total XdhC concentration. Fitting revealed a function according to the law of mass action for a Moco/MPT-XdhC-ratio of 1:1, with a Moco saturation of 0.97 ± 0.06 (Fig. 2A) and a MPT saturation of 0.95 ± 0.11 (Fig. 2B). A KD value of 3.6 ± 0.1 μm was obtained for Moco binding, and a KD value of 3.5 ± 0.3 μm was obtained for MPT binding to XdhC at 4 °C, showing that XdhC is able to bind both molecules with the same efficiency in stoichiometric amounts. Analysis of the Ability of XdhC to Insert Moco into Apo-XDH—Further, it was of interest to determine whether XdhC is also able to transfer the bound Moco into purified Moco-free apo-XDH. Moco-containing XdhC was incubated with freshly purified apo-XDH for 90 min at 4 °C, before XdhC was removed from the incubation mixture by affinity chromatography (see “Experimental Procedures”). Since the Moco used for this experiment was obtained from hSO containing the dioxo form of Moco lacking the terminal sulfur ligand required for XDH activity, the reconstitution of XDH with Moco could not be determined by regaining XDH activity. Thus, to determine the amount of Moco present in reconstituted XDH, the protein was subjected to iodine oxidation to produce the fluorescent Form A derivative of the cofactor, which can be quantified by HPLC analysis. As shown in Fig. 3A, XdhC was able to insert bound Moco into Moco-free apo-XDH. The control experiment using free Moco showed the same level of Moco insertion into XDH. A half-maximal Moco insertion at 4 °C was observed at a Moco/XDH ratio of about 2:1. The achievable saturation level of XDH with Moco depended on the preparation of XDH and, as shown in Fig. 3A, was in the range of about 15%. A control experiment using bovine serum albumin instead of XDH showed that Moco insertion was specific to apo-XDH (data not shown). This reconstitution efficiency is less than the in vitro insertion of Moco described for hSO, where about 5" @default.
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• W2084250862 title "Rhodobacter capsulatus XdhC Is Involved in Molybdenum Cofactor Binding and Insertion into Xanthine Dehydrogenase" @default.
• W2084250862 cites W1555122393 @default.
• W2084250862 cites W1578121743 @default.
• W2084250862 cites W1584018467 @default.
• W2084250862 cites W1588405578 @default.
• W2084250862 cites W1615113694 @default.
• W2084250862 cites W1631792726 @default.
• W2084250862 cites W1719008040 @default.
• W2084250862 cites W1963703138 @default.
• W2084250862 cites W1972681803 @default.
• W2084250862 cites W1978663690 @default.
• W2084250862 cites W1988788131 @default.
• W2084250862 cites W1998514973 @default.
• W2084250862 cites W2001822890 @default.
• W2084250862 cites W2002207630 @default.
• W2084250862 cites W2006879511 @default.
• W2084250862 cites W2018046696 @default.
• W2084250862 cites W2018058209 @default.
• W2084250862 cites W2024110365 @default.
• W2084250862 cites W2028049009 @default.
• W2084250862 cites W2029572109 @default.
• W2084250862 cites W2039798321 @default.
• W2084250862 cites W2047876516 @default.
• W2084250862 cites W2049249409 @default.
• W2084250862 cites W2053360749 @default.
• W2084250862 cites W2059831206 @default.
• W2084250862 cites W2069852588 @default.
• W2084250862 cites W2070586798 @default.
• W2084250862 cites W2073070966 @default.
• W2084250862 cites W2078793942 @default.
• W2084250862 cites W2090221836 @default.
• W2084250862 cites W2095086526 @default.
• W2084250862 cites W2101943125 @default.
• W2084250862 cites W2102626746 @default.
• W2084250862 cites W2104594638 @default.
• W2084250862 cites W2106671813 @default.
• W2084250862 cites W2119073376 @default.
• W2084250862 cites W2128110461 @default.
• W2084250862 cites W2132417094 @default.
• W2084250862 cites W2136277512 @default.
• W2084250862 cites W2136310944 @default.
• W2084250862 cites W2156981649 @default.
• W2084250862 cites W2157433322 @default.
• W2084250862 cites W2169246184 @default.
• W2084250862 cites W4245399940 @default.
• W2084250862 doi "https://doi.org/10.1074/jbc.m601617200" @default.
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