Site-specific replacement of Y₃₅₆ with 3,4- dihydroxyphenylalanine in the β2 subunit of E. coli ribonucleotide reductase

E. coli ribonucleotide reductase (RNR), composed of the homodimeric subunits α2 and β2, catalyzes the conversion of nucleotides to deoxynucleotides via complex radical chemistry. The radical initiation process involves a putative proton-coupled electron transfer (PCET) pathway over 35 Å between α2 and β2. Y₃₅₆ in β2 has been proposed to lie on this pathway. To test this model, intein technology has been used to make β2 semi-synthetically in which Y₃₅₆ is replaced with a DOPA-amino acid. Analysis of this mutant with α2 and various combinations of substrate and effector by SF UV-vis spectroscopy and EPR methods demonstrates formation of a DOPA radical concomitant with disappearance of the tyrosyl radical, which initiates the reaction. The results reveal that Y₃₅₆ lies on the PCET pathway and demonstrate the first kinetically competent conformational changes prior to ET. They further show that substrate binding brings about rapid conformational changes which place the complex into its active form(s) and suggest that the RNR complex is asymmetric.