Molybdenum Cofactor Biosynthesis in Humans:  Identification of a Persulfide Group in the Rhodanese-like Domain of MOCS3 by Mass Spectrometry

Abstract

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.

A Novel Role for Human Nfs1 in the Cytoplasm

“…Because the proteins for the second step of Moco biosynthesis, MOCS2A, MOCS2B, and MOCS3 are located in the cytosol (14), this would imply a novel function for cytosolic Nfs1 in humans. Human MOCS3 contains a C-terminal rhodanese-like domain which was shown to act as direct sulfur donor for the formation of the thiocarboxylate group on human MOCS2A (13,14). However, the low activity of MOCS3 with thiosulfate and detailed mutagenesis studies of the rhodanese active site loop suggested that thiosulfate is not the physiological sulfur source of MOCS3, and that proteins like L-cysteine desulfurases might perform this function in humans (12).…”

“…After incubation for 12 h at 4°C, L-cysteine was removed by gel filtration. In addition, the MOCS3-RLD-C412A variant was used as a control, to verify the persulfide formation on Cys412 of MOCS3-RLD, which was not detected in this mutant (13).…”

“…This RLD is present in all eukaryotic homologues, including the Saccharomyces cerevisiae Uba4 protein, and several bacterial homologues, but not in the E. coli MoeB protein (12). MOCS3 was shown to catalyze both the adenylation and the subsequent generation of a thiocarboxylate group at the C terminus of MOCS2A during Moco biosynthesis (13). All three proteins were shown to be localized in the cytosol in humans (14).…”

The Molybdenum Cofactor

“…At this stage, the sulfur transfer reaction in higher organisms appears to involve different protein components, as the eukaryotic genes cannot complement their bacterial counterparts. MPT synthase sulfurase is a two-domain protein consisting of an N-terminal adenylation domain (homologous to E. coli MoeB) and a C-terminal rhodanese-like domain (RLD), where the sulfur is bound to a conserved cysteine in the form of a persulfide (38,44). In analogy to the bacterial mechanism, this enzyme is supposed to activate the small subunit of MPT synthase by adenylation followed by sulfur transfer (coming from the RLD), thus forming the thiocarboxylate at the C terminus of the small subunit (2).…”

Characterization and Interaction Studies of Two Isoforms of the Dual Localized 3-Mercaptopyruvate Sulfurtransferase TUM1 from Humans

“…Maximal MPT production was obtained for assays consisting of MOCS3, MOCS2A, and MoaE with sodium sulfide as direct sulfur source or sodium thiosulfate as substrate for MOCS3 (27,43). The fluorescence intensity for the produced Form A in these assay mixtures was set to 100%, and all further assay mixtures were related to this value.…”