The mechanism of assembly and cofactor insertion into Rhodobacter capsulatus xanthine dehydrogenase.
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 (alphabeta)(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 (alphabeta) heterodimeric XDH containing the XdhA and XdhB subunits, the molybdenum cofactor (Moco) was shown to be absent, indicating that dimerization of the (alphabeta) subunits has to precede Moco insertion. In an (alphabeta)(2) XDH heterotetramer variant, including only one active Moco-center, the active (alphabeta) 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 (alphabeta)(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 (alphabeta) dimers is necessary for insertion of sulfurated Moco into apo-XDH, the last step of XDH maturation.
Schumann
Silvia
S
Universität Potsdam, Institut für Biochemie and Biologie, Karl-Liebknecht Strasse 24-25, Potsdam 14476, Germany.
Saggu
Miguel
M
Möller
Nadine
N
Anker
Stefan D
SD
Lendzian
Friedhelm
F
Hildebrandt
Peter
P
Leimkühler
Silke
S
D000596:Amino Acids / Q000737:chemistry; D001665:Binding Sites; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy; D004583:Electrons; D008954:Models, Biological; D008968:Molecular Conformation; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D016354:Rhodobacter capsulatus / Q000201:enzymology*; D014968:Xanthine Dehydrogenase / Q000378:metabolism*
D000596:Amino Acids; D014968:Xanthine Dehydrogenase; D003545:Cysteine