Ribonucleotide reductase from the higher plant Arabidopsis thaliana : expression of the R2 component and characterization of its iron-radical center

Abstract

 Deoxyribonucleotides synthesis has not been biochemically characterized in higher plants. From a cDNA of the small component (protein R2) of ribonucleotide reductase from Arabidopsis thaliana, an inducible overexpression plasmid has been constructed. A recombinant 78-kDa homodimeric protein containing very little iron was purified to homogeneity. Addition of ferrous iron and oxygen resulted in a protein containing 1.2 tyrosyl radicals and 4 iron atoms per dimer. Light absorption and low-temperature EPR spectra indicated close similarity of the iron-radical centers in plant and mouse R2 proteins. It is then suggested that, as in all class I eukaryotic ribonucleotide reductase, the active site of R2 component contains a μ-oxo bridged di-iron center in strong interaction with a tyrosyl radical. The stability of the radical seems, however, to be larger in the plant R2 protein, as shown by its resistance to hydroxyurea.