%0 Journal Article %A Seyedsayamdost, Mohammad R. %A Xie, Jianming %A T. Y. Chan, Clement %A Schultz, Peter G. %A Stubbe, JoAnne %D 2007 %T Site-Specific Insertion of 3-Aminotyrosine into Subunit α2 of E. coli Ribonucleotide Reductase:  Direct Evidence for Involvement of Y730 and Y731 in Radical Propagation %U https://acs.figshare.com/articles/journal_contribution/Site_Specific_Insertion_of_3_Aminotyrosine_into_Subunit_2_of_i_E_coli_i_Ribonucleotide_Reductase_Direct_Evidence_for_Involvement_of_Y_sub_730_sub_and_Y_sub_731_sub_in_Radical_Propagation/2971054 %R 10.1021/ja076043y.s001 %2 https://acs.figshare.com/ndownloader/files/4670326 %K EPR %K propagation pathway %K 731NH %K residues Y 730 %K ATP %K nucleoside diphosphate substrates %K deoxynucleotide %K Y 730 %K Y 731 %K α2. %K oxidize C 439 %K α2 %K UV %K hydrogen atom transfer mechanism %K RNR %K NH 2Y %K coli ribonucleotide reductase %K 730NH %K CDP %X E. coli 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 (Y122·) cofactor that initiates deoxynucleotide formation. This process is proposed to involve reversible radical transfer over >35 Å between the Y122· in β2 and C439 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 Y730 and Y731 in α2. In this study the function of residues Y730 and Y731 is investigated by their site-specific replacement with 3-aminotyrosine (NH2Y). Using the in vivo suppressor tRNA/aminoacyl-tRNA synthetase method, Y730NH2Y-α2 and Y731NH2Y-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 NH2Y radical (NH2Y730· or NH2Y731·) in a kinetically competent fashion. Activity assays demonstrate that both NH2Y-α2s make deoxynucleotides. These results show that the NH2Y· can oxidize C439 suggesting a hydrogen atom transfer mechanism for the radical propagation pathway within α2. The observed NH2Y· may constitute the first detection of an amino acid radical intermediate in the proposed radical propagation pathway during turnover. %I ACS Publications