Molybdenum cofactor biosynthesis in humans: identification of a persulfide group in the rhodanese-like domain of MOCS3 by mass spectrometry

Biochemistry. 2005 May 31;44(21):7912-20. doi: 10.1021/bi0503448.


The human MOCS3 protein contains an N-terminal domain similar to the Escherichia coli MoeB protein and a C-terminal segment displaying similarities to the sulfurtransferase rhodanese. MOCS3 is proposed to catalyze both the adenylation and the subsequent generation of a thiocarboxylate group at the C-terminus of the smaller subunit of molybdopterin (MPT) synthase during Moco biosynthesis in humans. Recent studies have shown that the MOCS3 rhodanese-like domain (MOCS3-RLD) catalyzes the transfer of sulfur from thiosulfate to cyanide and is also able to provide the sulfur for the thiocarboxylation of MOCS2A in a defined in vitro system for the generation of MPT from precursor Z. MOCS3-RLD contains four cysteine residues of which only C412 in the six amino acid active loop is conserved in homologous proteins from other organisms. ESI-MS/MS studies gave direct evidence for the formation of a persulfide group that is exclusively formed on C412. Simultaneous mutagenesis of the remaining three cysteine residues showed that none of them is involved in the sulfur transfer reaction in vitro. A disulfide bridge was identified to be formed between C316 and C324, and possible roles of the three noncatalytic cysteine residues are discussed. By ESI-MS/MS a partially gluconoylated N-terminus of the His6-tagged MOCS3-RLD was identified (mass increment of 178 Da) which resulted in a heterogeneity of the protein but did not influence sulfurtransferase activity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Coenzymes / biosynthesis*
  • Cysteine / chemistry
  • Cysteine / genetics
  • Disulfides / chemistry
  • Histidine / genetics
  • Humans
  • Metalloproteins / biosynthesis*
  • Mice
  • Molecular Sequence Data
  • Molybdenum Cofactors
  • Mutagenesis, Site-Directed
  • Nucleotidyltransferases / biosynthesis*
  • Nucleotidyltransferases / chemistry*
  • Nucleotidyltransferases / genetics
  • Peptide Mapping
  • Protein Structure, Tertiary
  • Pteridines
  • Sequence Homology, Amino Acid
  • Spectrometry, Mass, Electrospray Ionization / methods
  • Sulfhydryl Compounds / chemistry
  • Sulfides / chemistry*
  • Sulfurtransferases / biosynthesis*
  • Sulfurtransferases / chemistry*
  • Sulfurtransferases / genetics
  • Thiosulfate Sulfurtransferase / chemistry*
  • Thiosulfate Sulfurtransferase / genetics


  • Coenzymes
  • Disulfides
  • Metalloproteins
  • Molybdenum Cofactors
  • Pteridines
  • Sulfhydryl Compounds
  • Sulfides
  • persulfides
  • Histidine
  • molybdenum cofactor
  • MOCS3 protein, human
  • Nucleotidyltransferases
  • Sulfurtransferases
  • Thiosulfate Sulfurtransferase
  • Cysteine