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The mechanism of assembly and cofactor insertion into Rhodobacter capsulatus xanthine dehydrogenase
Citation key ISI:000256497100037
Author Schumann, Silvia and Saggu, Miguel and Moeller, Nadine and Anker, Stefan D. and Lendzian, Friedhelm and Hildebrandt, Peter and Leimkuehler, Silke
Pages 16602-16611
Year 2008
ISSN 0021-9258
DOI 10.1074/jbc.M709894200
Address 9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3996 USA
Journal J. Biol. Chem.
Volume 283
Number 24
Month JUN 13
Publisher AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Abstract Rhodobacter capsulatus xanthine dehydrogenase (XDH) is a molybdo-flavoprotein that is highly homologous to the homodimeric mammalian xanthine oxidoreductase. However, the bacterial enzyme has an (alpha beta)(2) heterotetrameric structure, and the cofactors were identified to be located on two different polypeptides. We have analyzed the mechanism of cofactor insertion and subunit assembly of R. capsulatus XDH, using engineered subunits with appropriate substitutions in the interfaces. In an (alpha beta) heterodimeric XDH containing the XdhA and XdhB subunits, the molybdenum cofactor (Moco) was shown to be absent, indicating that dimerization of the (alpha beta) subunits has to precede Moco insertion. In an (alpha beta)(2) XDH heterotetramer variant, including only one active Moco-center, the active (alpha beta) site of the chimeric enzyme was shown to be fully active, revealing that the two subunits act independent without cooperativity. Amino acid substitutions at two cysteine residues coordinating FeSI of the two [2Fe-2S] clusters of the enzyme demonstrate that an incomplete assembly of FeSI impairs the formation of the XDH (alpha beta)(2) heterotetramer and, thus, insertion of Moco into the enzyme. The results reveal that the insertion of the different redox centers into R. capsulatus XDH takes place sequentially. Dimerization of two (alpha beta) dimers is necessary for insertion of sulfurated Moco into apo-XDH, the last step of XDH maturation.
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