Biophysical Characterization of Fluorotyrosine Probes Site-Specifically Incorporated into Enzymes:
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jacs.6b03605.pdf
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Author(s)
Oyala, Paul H.
Ravichandran, Kanchana R.
Stucky, Paul A.
Stich, Troy A.
Britt, R. David
Funk, Michael Andrew
Drennan, Catherine L
Stubbe, JoAnne
Date Issued
June 2016
Journal
Journal of the American Chemical Society
Publisher
American Chemical Society (ACS)
Citation
Oyala, Paul H. et al. “Biophysical Characterization of Fluorotyrosine Probes Site-Specifically Incorporated into Enzymes: E. Coli Ribonucleotide Reductase As an Example.” Journal of the American Chemical Society 138, 25 (June 2016): 7951–7964 © 2016 American Chemical Society
Version
Final published version
Abstract
Fluorinated tyrosines (F[subscript 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[subscript n]Y (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-F[subscript n]Y· cofactor is self-assembled from apo F[subscript n]Y-β2, Fe ²⁺, and O₂ to produce ∼1 Y·/β2 and ∼3 Fe ³⁺ /β2. The F[subscript n]Y· are stable and active in nucleotide reduction with activities that vary from 5% to 85% that of wt-β2. Each F[subscript 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[subscript n]Y· that are readily distinguishable from unlabeled Y·'s. The variability of the abiotic F[subscript 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.
MIT Department
Massachusetts Institute of Technology. Department of Biology
Massachusetts Institute of Technology. Department of Chemistry
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DOI of Published Version
https://doi.org/10.1021/JACS.6B03605
