Characterization of MOCS1A, an oxygen-sensitive iron-sulfur protein involved in human molybdenum cofactor biosynthesis.
The human proteins MOCS1A and MOCS1B catalyze the conversion of a guanosine derivative to precursor Z during molybdenum cofactor biosynthesis. MOCS1A shares homology with S-adenosylmethionine (AdoMet)-dependent radical enzymes, which catalyze the formation of protein and/or substrate radicals by reductive cleavage of AdoMet through a [4Fe-4S] cluster. Sequence analysis of MOCS1A showed two highly conserved cysteine motifs, one near the N terminus and one near the C terminus. MOCS1A was heterologously expressed in Escherichia coli and purified under aerobic and anaerobic conditions. Individual mutations of the conserved cysteines to serine revealed that all are essential for synthesis of precursor Z in vivo. The type and properties of the iron-sulfur (FeS) clusters were investigated using a combination of UV-visible absorption, variable temperature magnetic circular dichroism, resonance Raman, Mössbauer, and EPR spectroscopies coupled with iron and acid-labile sulfide analyses. The results indicated that anaerobically purified MOCS1A is a monomeric protein containing two oxygen-sensitive FeS clusters, each coordinated by only three cysteine residues. A redox-active [4Fe-4S](2+,+) cluster is ligated by an N-terminal CX(3)CX(2)C motif as is the case with all other AdoMet-dependent radical enzymes investigated thus far. A C-terminal CX(2)CX(13)C motif that is unique to MOCS1A and its orthologs primarily ligates a [3Fe-4S](0) cluster. However, MOCS1A could be reconstituted in vitro under anaerobic conditions to yield a form containing two [4Fe-4S](2+) clusters. The N-terminal [4Fe-4S](2+) cluster was rapidly degraded by oxygen via a semistable [2Fe-2S](2+) cluster intermediate, and the C-terminal [4Fe-4S](2+) cluster was rapidly degraded by oxygen to yield a semistable [3Fe-4S](0) cluster intermediate.
Hänzelmann
Petra
P
Department of Biochemistry and Center for Structural Biology, State University of New York, Stony Brook, NY 11794-5215, USA. petra@csb.sunysb.edu
Hernández
Heather L
HL
Menzel
Christian
C
García-Serres
Ricardo
R
Huynh
Boi Hanh
BH
Johnson
Michael K
MK
Mendel
Ralf R
RR
Schindelin
Hermann
H
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019755:Carbon-Carbon Lyases; D002384:Catalysis; D002942:Circular Dichroism; D003067:Coenzymes / Q000378:metabolism*; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007501:Iron / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000378:metabolism; D008280:Magnetics; D008667:Metalloproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D000090006:Molybdenum Cofactors; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry* / Q000502:physiology*; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011621:Pteridines / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D013053:Spectrophotometry; D015204:Spectroscopy, Mossbauer; D013059:Spectrum Analysis, Raman; D014466:Ultraviolet Rays
D019755:Carbon-Carbon Lyases; D003067:Coenzymes; D003545:Cysteine; D007501:Iron; D007506:Iron-Sulfur Proteins; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D009687:Nuclear Proteins; D010100:Oxygen; D011621:Pteridines; D012694:Serine; C022261:molybdopterin; C114840:MOCS1 protein, human