Study of Different Variants of Mo Enzyme crARC and the Interaction with Its Partners crCytb5-R and crCytb5-1.
The mARC (mitochondrial Amidoxime Reducing Component) proteins are recently discovered molybdenum (Mo) Cofactor containing enzymes. They are involved in the reduction of severalN-hydroxylated compounds (NHC) and nitrite. Some NHC are prodrugs containing an amidoxime structure or mutagens such as 6-hydroxylaminopurine (HAP). We have studied this protein in the green algaChlamydomonas reinhardtii(crARC). Interestingly, all the ARC proteins need the reducing power supplied by other proteins. It is known that crARC requires a cytochromeb₅ (crCytb5-1) and a cytochromeb₅ reductase (crCytb5-R) that form an electron transport chain from NADH to the substrates. Here, we have investigated NHC reduction by crARC, the interaction with its partners and the function of important conserved amino acids. Interactions among crARC, crCytb5-1 and crCytb5-R have been studied by size-exclusion chromatography. A protein complex between crARC, crCytb5-1 and crCytb5-R was identified. Twelve conserved crARC amino acids have been substituted by alanine by in vitro mutagenesis. We have determined that the amino acids D182, F210 and R276 are essential for NHC reduction activity, R276 is important and F210 is critical for the Mo Cofactor chelation. Finally, the crARC C-termini were shown to be involved in protein aggregation or oligomerization.
Departamento de Bioquímica y Biología Molecular, Universidad de Córdoba, Campus de Rabanales, Edificio Severo Ochoa, 14071 Córdoba, Spain. b42chaaa@uco.es.
Alejandro
A
Chamizo-Ampudia
Departamento de Bioquímica y Biología Molecular, Universidad de Córdoba, Campus de Rabanales, Edificio Severo Ochoa, 14071 Córdoba, Spain. bb1gacea@uco.es.
Aurora
A
Galvan
Departamento de Bioquímica y Biología Molecular, Universidad de Córdoba, Campus de Rabanales, Edificio Severo Ochoa, 14071 Córdoba, Spain. bb1feree@uco.es.
Emilio
E
Fernandez
Departamento de Bioquímica y Biología Molecular, Universidad de Córdoba, Campus de Rabanales, Edificio Severo Ochoa, 14071 Córdoba, Spain. allamas@uco.es.
Angel
A
Llamas
D019943:Amino Acid Substitution; D001665:Binding Sites; D016825:Chlamydomonas reinhardtii / Q000201:enzymology / Q000378:metabolism; D003067:Coenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015786:Cytochromes b5 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008667:Metalloproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000090006:Molybdenum Cofactors; D011485:Protein Binding; D055503:Protein Multimerization; D011621:Pteridines / Q000737:chemistry / Q000378:metabolism*
D003067:Coenzymes; D015786:Cytochromes b5; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D011621:Pteridines; C022261:molybdopterin
Chlamydomonas; HAP; amidoxime; interaction; mARC; molybdenum; oligomers; partners