Forward and reverse electron transfer with the Y₃₅₆DOPA-β2 heterodimer of E. coli ribonucleotide reductase

E. coli ribonucleotide reductase catalyzes the conversion of nucleotides to deoxynucleotides, and consists of two subunits, α2 and β2. β2 contains a stable diiron tyrosyl radical (Y₁₂₂•) that is essential for catalysis. α2 harbors the active site, where nucleotide reduction occurs, as well as effector and activity sites which control substrate specificity and turnover rates. In this study, we have used intein methodology to generate a heterodimer of β2 containing the unnatural amino acid 3,4-dihydroxyphenylalanine (DOPA) at residue 356 (DOPA-ββ-). In this heterodimer, the β-monomer is full-length (residues 1-375), whereas the β-monomer is truncated and only contains residues 1-353. DOPA-ββ-, upon addition of α2, CDP, and ATP effector, generates a DOPA• concomitant with loss of the Y₁₂₂•. Analysis of DOPA• stability by EPR reveal that DOPA•-ββ- can reoxidize Y₁₂₂ thereby regenerating the Y₁₂₂•. These results, for the first time, directly demonstrate back electron transfer from residue 356 to Y₁₂₂.