Study of Different Variants of Mo Enzyme crARC and the Interaction with Its Partners crCytb5-R and crCytb5-1
Author
Chamizo-Ampudia, Alejandro
Galván Cejudo, Aurora
Fernández Reyes, Emilio
Llamas Azúa, Ángel
Publisher
MDPIDate
2017Subject
mARCChlamydomonas
Amidoxine
HAP
Molybdenum
Partners
Interaction
Oligomers
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Show full item recordAbstract
The mARC (mitochondrial Amidoxime Reducing Component) proteins are recently
discovered molybdenum (Mo) Cofactor containing enzymes. They are involved in the reduction
of several N-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 alga Chlamydomonas reinhardtii (crARC). Interestingly, all the ARC proteins need
the reducing power supplied by other proteins. It is known that crARC requires a cytochrome b5
(crCytb5-1) and a cytochrome b5 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