Site-Specific Incorporation of 3-Nitrotyrosine as a Probe of pK[subscript a] Perturbation of Redox-Active Tyrosines in Ribonucleotide Reductase

E. coli ribonucleotide reductase catalyzes the reduction of nucleoside 5′-diphosphates into 2′-deoxynucleotides and is composed of two subunits: α2 and β2. During turnover, a stable tyrosyl radical (Y•) at Y[subscript 122-]β2 reversibly oxidizes C[subscript 439] in the active site of α2. This radical propagation step is proposed to occur over 35 Å, to use specific redox-active tyrosines (Y[subscript 122] and Y[subscript 356] in β2, Y[subscript 731] and Y[subscript 730] in α2), and to involve proton-coupled electron transfer (PCET). 3-Nitrotyrosine (NO[subscript 2]Y, pK[subscript a] 7.1) has been incorporated in place of Y[subscript 122], Y[subscript 731], and Y[subscript 730] to probe how the protein environment perturbs each pK[subscript a] in the presence of the second subunit, substrate (S), and allosteric effector (E). The activity of each mutant is 9.6. X-ray crystal structures have been obtained for all [NO[subscript 2]Y]-α2 mutants (2.1−3.1 Å resolution), which show minimal structural perturbation compared to wt-α2. Together with the pK[subscript a] of the previously reported NO[subscript 2]Y[subscript 356-]β2 (7.5 in the α2/S/E complex; Yee, C. et al. Biochemistry 2003, 42, 14541−14552), these studies provide a picture of the protein environment of the ground state at each Y in the PCET pathway, and are the starting point for understanding differences in PCET mechanisms at each residue in the pathway.National Institutes of Health (U.S.) (GM29595