title: The Chlamydomonas reinhardtii molybdenum cofactor enzyme crARC has a Zn-dependent activity and protein partners similar to those of its human homologue.
abstract: The ARC (amidoxime reducing component) proteins are molybdenum cofactor (Moco) enzymes named hmARC1 and hmARC2 (human ARCs [hmARCs]) in humans and YcbX in Escherichia coli. They catalyze the reduction of a broad range of N-hydroxylated compounds (NHC) using reducing power supplied by other proteins. Some NHC are prodrugs or toxic compounds. YcbX contains a ferredoxin (Fd) domain and requires the NADPH flavin reductase CysJ to reduce NHC. In contrast, hmARCs lack the Fd domain and require a human cytochrome b5 (hCyt b5) and a human NADH Cyt b5 reductase (hCyt b5-R) to reduce NHC. The ARC proteins in the plant kingdom are uncharacterized. We demonstrate that Chlamydomonas reinhardtii mutants defective in Moco biosynthesis genes are sensitive to the NHC N(6)-hydroxylaminopurine (HAP). The Chlamydomonas reinhardtii ARC protein crARC has been purified and characterized. The six Chlamydomonas Fds were isolated, but none of them are required by crARC to reduce HAP. We have also purified and characterized five C. reinhardtii Cyt b5 (crCyt b5) and two flavin reductases, one that is NADPH dependent (crCysJ) and one that is NADH dependent (crCyt b5-R). The data show that crARC uses crCyt b5-1 and crCyt b5-R to reduce HAP. The crARC has a Zn-dependent activity, and the presence of Zn increases its V(max) more than 14-fold. In addition, all five cysteines of crARC were substituted by alanine, and we demonstrate that the fully conserved cysteine 252 is essential for both Moco binding and catalysis. Therefore, it is proposed that crARC belongs to the sulfite oxidase family of Moco enzymes.
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Córdoba, Campus de Rabanales, Edificio Severo Ochoa, 14071 Córdoba, Spain.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Chamizo-Ampudia
authors.forename: Aurora
authors.initials: A
authors.lastname: Galvan
authors.forename: Emilio
authors.initials: E
authors.lastname: Fernandez
authors.forename: Angel
authors.initials: A
authors.lastname: Llamas
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D016825:Chlamydomonas reinhardtii / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D003067:Coenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D042966:Cytochrome-B(5) Reductase / Q000378:metabolism; D015786:Cytochromes b5 / Q000378:metabolism; D005288:Ferredoxins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008667:Metalloproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D000090006:Molybdenum Cofactors; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011621:Pteridines / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D015032:Zinc / Q000378:metabolism*
chemical_list: D000225:Adenine; D003067:Coenzymes; D042966:Cytochrome-B(5) Reductase; D015786:Cytochromes b5; D005288:Ferredoxins; D008565:Membrane Proteins; D008667:Metalloproteins; D024101:Mitochondrial Proteins; D000090006:Molybdenum Cofactors; D010088:Oxidoreductases; D010940:Plant Proteins; D011621:Pteridines; D015032:Zinc; C032610:6-N-hydroxylaminopurine; C022261:molybdopterin; C570001:MTARC2 protein, human; C534493:amidoxime reducing component, human