E. coli ribonucleotide reductase catalyzes the conversion of nucleotides to deoxynucleotides, and consists of two subunits, alpha 2 and beta 2. beta 2 contains a stable diiron tyrosyl radical (Y-122 center dot) that is essential for catalysis. alpha 2 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 beta 2 containing the unnatural amino acid 3,4-dihydroxyphenylalanine (DOPA) at residue 356 (DOPA-beta beta'). In this heterodimer, the beta-monomer is full-length (residues 1-375), whereas the beta'-monomer is truncated and only contains residues 1-353. DOPA-beta beta', upon addition of alpha 2, CDP, and ATP effector, generates a DOPA center dot concomitant with loss of the Y-122 center dot. Analysis of DOPA center dot stability by EPR reveal that DOPA center dot-beta beta' can reoxidize Y-122 thereby regenerating the Y-122 center dot. These results, for the first time, directly demonstrate back electron transfer from residue 356 to Y-122.