Site-Specific Insertion of 3-Aminotyrosine into Subunit α2 of <i>E. coli</i> Ribonucleotide Reductase:  Direct Evidence for Involvement of Y<sub>730</sub> and Y<sub>731</sub> in Radical Propagation

<i>E. coli</i> ribonucleotide reductase (RNR) catalyzes the production of deoxynucleotides using complex radical chemistry. Active RNR is composed of a 1:1 complex of two subunits:  α2 and β2. α2 binds nucleoside diphosphate substrates and deoxynucleotide/ATP allosteric effectors and is the site of nucleotide reduction. β2 contains the stable diiron tyrosyl radical (Y<sub>122</sub>·) cofactor that initiates deoxynucleotide formation. This process is proposed to involve reversible radical transfer over >35 Å between the Y<sub>122</sub>· in β2 and C<sub>439</sub> in the active site of α2. A docking model of α2β2, based on structures of the individual subunits, suggests that radical initiation involves a pathway of transient, aromatic amino acid radical intermediates, including Y<sub>730</sub> and Y<sub>731</sub> in α2. In this study the function of residues Y<sub>730</sub> and Y<sub>731</sub> is investigated by their site-specific replacement with 3-aminotyrosine (NH<sub>2</sub>Y). Using the in vivo suppressor tRNA/aminoacyl-tRNA synthetase method, Y<sub>730</sub>NH<sub>2</sub>Y-α2 and Y<sub>731</sub>NH<sub>2</sub>Y-α2 have been generated with high fidelity in yields of 4−6 mg/g of cell paste. These mutants have been examined by stopped flow UV−vis and EPR spectroscopies in the presence of β2, CDP, and ATP. The results reveal formation of an NH<sub>2</sub>Y radical (NH<sub>2</sub>Y<sub>730</sub>· or NH<sub>2</sub>Y<sub>731</sub>·) in a kinetically competent fashion. Activity assays demonstrate that both NH<sub>2</sub>Y-α2s make deoxynucleotides. These results show that the NH<sub>2</sub>Y· can oxidize C<sub>439</sub> suggesting a hydrogen atom transfer mechanism for the radical propagation pathway within α2. The observed NH<sub>2</sub>Y· may constitute the first detection of an amino acid radical intermediate in the proposed radical propagation pathway during turnover.