Biophysical Characterization of Fluorotyrosine Probes Site-Specifically Incorporated into Enzymes: E. coli Ribonucleotide Reductase As an Example

[Image: see text] Fluorinated tyrosines (F(n)Y’s, n = 2 and 3) have been site-specifically incorporated into E. coli class Ia ribonucleotide reductase (RNR) using the recently evolved M. jannaschii Y-tRNA synthetase/tRNA pair. Class Ia RNRs require four redox active Y’s, a stable Y radical (Y·) in the β subunit (position 122 in E. coli), and three transiently oxidized Y’s (356 in β and 731 and 730 in α) to initiate the radical-dependent nucleotide reduction process. F(n)Y (3,5; 2,3; 2,3,5; and 2,3,6) incorporation in place of Y(122)-β and the X-ray structures of each resulting β with a diferric cluster are reported and compared with wt-β2 crystallized under the same conditions. The essential diferric-F(n)Y· cofactor is self-assembled from apo F(n)Y-β2, Fe(2+), and O(2) to produce ∼1 Y·/β2 and ∼3 Fe(3+)/β2. The F(n)Y· are stable and active in nucleotide reduction with activities that vary from 5% to 85% that of wt-β2. Each F(n)Y·-β2 has been characterized by 9 and 130 GHz electron paramagnetic resonance and high-field electron nuclear double resonance spectroscopies. The hyperfine interactions associated with the (19)F nucleus provide unique signatures of each F(n)Y· that are readily distinguishable from unlabeled Y·’s. The variability of the abiotic F(n)Y pK(a)’s (6.4 to 7.8) and reduction potentials (−30 to +130 mV relative to Y at pH 7.5) provide probes of enzymatic reactions proposed to involve Y·’s in catalysis and to investigate the importance and identity of hopping Y·’s within redox active proteins proposed to protect them from uncoupled radical chemistry.