Abstract
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.
Highlights
Dithiolene moiety of a tricyclic pyranopterin, termed molybdopterin (MPT)1 [1]
The results indicated that anaerobically purified MOCS1A is a monomeric protein containing two oxygen-sensitive FeS clusters, each ligated by only three cysteine residues
The mutagenesis results presented demonstrate that all six of the conserved cysteines in MOCS1A are required for catalytic activity and that the N-terminal and C-terminal cysteine-binding motifs ligate distinct FeS clusters
Summary
Dithiolene moiety of a tricyclic pyranopterin, termed molybdopterin (MPT)1 [1]. In humans, defects in molybdenum cofactor biosynthesis lead to the pleiotropic loss of the molybdoenzymes sulfite oxidase, aldehyde oxidase, and xanthine dehydrogenase [2, 3]. Several mutations identified in molybdenum cofactor deficiency patients are located in these conserved cysteine motifs indicating their functional importance for protein activity [2, 3]. Based on sequence similarities to proteins such as biotin synthase, pyruvate formate-lyase-activating enzyme, and anaerobic ribonucleotide reductase-activating enzyme, MOCS1A has been classified as a member of the superfamily of S-adenosylmethionine (AdoMet)-dependent radical enzymes [16]. The results indicated that anaerobically purified MOCS1A is a monomeric protein containing two oxygen-sensitive FeS clusters, each ligated by only three cysteine residues. MOCS1A could be reconstituted in vitro to yield a form containing two [4Fe-4S]2ϩ clusters, and both clusters appeared to be degraded via [3Fe-4S]0 and/or [2Fe-2S]2ϩ cluster intermediates on exposure to oxygen
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