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

Abstract

Fluorinated tyrosines (FnY’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. FnY (3,5; 2,3; 2,3,5; and 2,3,6) incorporation in place of Y122-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-FnY· cofactor is self-assembled from apo FnY-β2, Fe2+, and O2 to produce ∼1 Y·/β2 and ∼3 Fe3+/β2. The FnY· are stable and active in nucleotide reduction with activities that vary from 5% to 85% that of wt-β2. Each FnY·-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 19F nucleus provide unique signatures of each FnY· that are readily distinguishable from unlabeled Y·’s. The variability of the abiotic FnY pKa’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.

Glutamate 350 Plays an Essential Role in Conformational Gating of Long-Range Radical Transport in Escherichia coli Class Ia Ribonucleotide Reductase

“…20 The two important chemical properties that govern the chemistry at Y 356 -β and Y 731 -α are p K a and reduction potential. The p K a of the N -acetyl 3,5-difluoro-L-tyrosinamide is 7.2.…”

“…Finally, F 2 Y has been successfully incorporated site-specifically in place of each of the Ys in the RT pathway and the activities of these mutants have been determined to be 65 to 90% that of the wt enzyme. 20, 34 …”

“…As noted above, the last 34 amino acids of E. coli β2 (341–375) are structurally disordered. 17–20 Potential insight about the location of residues 360–375 of this tail was provided by crystallization of α2 with a peptide corresponding to residues 355–375 of β2, 7 but structural information is absent for residues 341 to 359, including Y 356 and E 350 (Figure 1). Studies by Sjöberg and coworkers were the first to demonstrate that the α/ β subunit affinity in E. coli RNR is largely governed by the last 20 amino acids of the β2 C terminus 23 and that the K d for the interaction is weak (~0.2 μM).…”

Formal Reduction Potentials of Difluorotyrosine and Trifluorotyrosine Protein Residues: Defining the Thermodynamics of Multistep Radical Transfer

“…If the asymmetrically labeled F n Y 32 side chains have multiple rotameric states, 18,49 it has little influence on their E °’s.…”

“…14 2,3-F 2 Y 122 •-β2 was expressed, purified and reconstituted as previously described. 18 Experimental conditions for all spectroscopic studies are described in detail in the SI.…”