Biochemical and Spectroscopic Characterization of the Human Mitochondrial Amidoxime Reducing Components hmARC-1 and hmARC-2 Suggests the Existence of a New Molybdenum Enzyme Family in Eukaryotes

Abstract

The mitochondrial amidoxime reducing component mARC is a newly discovered molybdenum enzyme that is presumed to form the catalytical part of a three-component enzyme system, consisting of mARC, heme/cytochrome b 5 , and NADH/FADdependent cytochrome b 5 reductase. mARC proteins share a significant degree of homology to the molybdenum cofactorbinding domain of eukaryotic molybdenum cofactor sulfurase proteins, the latter catalyzing the post-translational activation of aldehyde oxidase and xanthine oxidoreductase. The human genome harbors two mARC genes, referred to as hmARC-1/ MOSC-1 and hmARC-2/MOSC-2, which are organized in a tandem arrangement on chromosome 1. Recombinant expression of hmARC-1 and hmARC-2 proteins in Escherichia coli reveals that both proteins are monomeric in their active forms, which is in contrast to all other eukaryotic molybdenum enzymes that act as homo-or heterodimers. Both hmARC-1 and hmARC-2 catalyze the N-reduction of a variety of N-hydroxylated substrates such as N-hydroxy-cytosine, albeit with different specificities. Reconstitution of active molybdenum cofactor onto recombinant hmARC-1 and hmARC-2 proteins in the absence of sulfur indicates that mARC proteins do not belong to the xanthine oxidase family of molybdenum enzymes. Moreover, they also appear to be different from the sulfite oxidase family, because no cysteine residue could be identified as a putative ligand of the molybdenum atom. This suggests that the hmARC proteins and sulfurase represent members of a new family of molybdenum enzymes.In eukaryotes the trace element molybdenum is essential for a number of enzymes where the molybdenum atom is part of the so-called molybdenum cofactor (Moco) 2 in the active site of these enzymes (1). Moco is a pterin-based cofactor with a C6-substituted pyrano ring, a terminal phosphate, and a unique dithiolate group that binds the molybdenum atom. Moco-containing enzymes (Mo-enzymes) catalyze important reactions in the global carbon, sulfur, and nitrogen cycles that are characterized by transfer of an oxygen atom to or from a substrate. In mammals, one Mo-enzyme is sulfite oxidase (SO), which catalyzes the last step in the degradation of sulfur-containing amino acids and sulfatides (2). The active SO protein is a homodimer with each monomer of ϳ52 kDa consisting of a N-terminal cytochrome b 5 (cyt b 5 )/heme-binding domain and a C-terminal Moco-binding domain, the latter also harboring the dimerization interface. Both the Moco-and the heme-binding domain of mammalian SO are similar to the respective domains of nitrate reductase (NR), which catalyzes the first and rate-limiting step in nitrate assimilation in autotrophic organisms like plants, algae, and fungi (3). In addition to its N-terminal Mocobinding domain and the cytb 5 /heme-binding domain, each NR monomer possesses a C-terminal FAD-binding domain. Xanthine oxidoreductase (XOR) is another mammalian Mo-enzyme, and it is active as a homodimer with each ϳ145-kDa monomer consisting of several distinct domains: an N-terminal domain...

Amidoxime Reductase System Containing Cytochrome b5 Type B (CYB5B) and MOSC2 Is of Importance for Lipid Synthesis in Adipocyte Mitochondria

“…Although soluble CYB5R3 can support the amidoxime reductase activity in vitro together with truncated soluble MOSC2 and CYB5B (13,14), we were unable to confirm its involvement in the adipocyte cell system. This is despite the fact that we identified CYB5R3 as one of the proteins that was crosslinked by the radiolabeled substrate in the OMM and might possibly reflect not direct binding of the substrate to CYB5R3 but background caused by nonspecific cross-linking background.…”

“…MOSC1 was like its homolog MOSC2 up-regulated in differentiated adipocytes, but down-regulation of MOSC1 in these cells did not affect the amidoxime reductase activity. MOSC1 has previously been shown to be able to reduce amidoximes to amidines in vitro (13,14). The reason for this discrepancy is unclear, but in the in vitro system the amidoxime reductase activity was reconstituted with recombinantly expressed truncated and soluble components CYB5B and CYB5R3 and MOSC1 or MOSC2.…”

“…Secondary horseradish peroxidase-coupled antirabbit and anti-mouse antibodies were from Dako (Dako Sweden AB, Stockholm, Sweden). AR-H069637, AR-H069927, and AZ13228184- 14 C were from AstraZeneca R&D Mölndal, Sweden.…”

“…Several studies have attempted to identify the third component, and the involvement of stearoyl-CoA desaturase (11) and cytochrome P450 isoenzyme CYP2D (10) in the microsomal fraction as well as a novel molybdenum-containing enzyme in the mitochondrial fraction (12) was suggested. In subsequent studies, two highly homologous molybdenum-containing proteins, namely molybdenum cofactor sulfurase C-terminal containing 1 and 2 (MOSC1 and MOSC2, respectively, referred to as mARC1 and mARC2), were shown to display amidoxime reductase activity in vitro when incubated in a reconstituted system with CYB5 and CYB5R3 (cytochrome b 5 reductase 3) (13,14). In addition, Kurian et al (15) showed that in a reconstituted system purified soluble CYB5 and purified soluble CYB5R were able to reduce amidoximes to amidines without the involvement of a third component.…”

Molybdenum Cofactor Deficiency: A New HPLC Method for Fast Quantification of S-Sulfocysteine in Urine and Serum

“…Xanthine dehydrogenase, another important Moco enzyme, catalyzes the conversion of hypoxanthine to xanthine and further to uric acid (Hille 2005). Aldehyde oxidase catalyzes the hydroxylation of various compounds (Garattini et al 2009), while mARC was identified as a general detoxifying and pro-drug metabolizing enzyme with yet unknown physiological substrates (Wahl et al 2010).…”