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.
All Science Journal Classification (ASJC) codes
- Colloid and Surface Chemistry