Abstract
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.
Highlights
Mo is the only second-row transition metal that participates in critical biological functions in most living beings from bacteria to humans [1]
Most of these Mo-containing enzymes occur in prokaryotes while only five of them have been identified in eukaryotes: nitrate reductase (NR), sulphite oxidase (SO), aldehyde oxidase (AO), xanthine oxidoreductase (XOR), and mARC the last one uncovered [4]
The N-hydroxylated compounds (NHC) substrate selected to be reduced in this assay was the model substrate benzamidoxime [4]
Summary
Mo is the only second-row transition metal that participates in critical biological functions in most living beings from bacteria to humans [1]. For gaining biological activity and fulfilling its function in enzymes, all studied eukaryotic Mo enzymes have Mo chelated by a tricyclic pyranopterin compound called molybdopterin (MPT), forming the Mo Cofactor [2] (Figure 1). The Mo Cofactor takes part in the active centre of more than fifty different enzymes involved in key reactions of nitrogen and sulphur metabolism, phytohormone biosynthesis and detoxification of xenobiotics. Most of these Mo-containing enzymes occur in prokaryotes while only five of them have been identified in eukaryotes: nitrate reductase (NR), sulphite oxidase (SO), aldehyde oxidase (AO), xanthine oxidoreductase (XOR), and mARC the last one uncovered [4]. SJ.CMsolu. pScei.r2fa01m7,i1ly8,,6i7n0cluding mARC, are proteins without any confirmed physiological functio2no. f 18
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