Biophysical Characterization of Fluorotyrosine Probes Site-Specifically Incorporated into Enzymes: E. coli Ribonucleotide Reductase As an Example.
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, Fe(2+), and O2 to produce ∼1 Y·/β2 and ∼3 Fe(3+)/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 (19)F 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.
Department of Chemistry, University of California, Davis , One Shields Avenue, Davis, California 95616, United States.
Paul H
PH
Oyala
Kanchana R
KR
Ravichandran
Michael A
MA
Funk
Department of Chemistry, University of California, Davis , One Shields Avenue, Davis, California 95616, United States.
Paul A
PA
Stucky
Department of Chemistry, University of California, Davis , One Shields Avenue, Davis, California 95616, United States.
Troy A
TA
Stich
Howard Hughes Medical Institute, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
Catherine L
CL
Drennan
Department of Chemistry, University of California, Davis , One Shields Avenue, Davis, California 95616, United States.
R David
RD
Britt
JoAnne
J
Stubbe
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D003198:Computer Simulation; D003460:Crystallization; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005095:Exoribonucleases / Q000737:chemistry*; D005461:Fluorine / Q000737:chemistry*; D005609:Free Radicals / Q000737:chemistry; D006860:Hydrogen Bonding; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D063747:Methanocaldococcus / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D010766:Phosphorylation; D012343:RNA, Transfer / Q000737:chemistry; D012264:Ribonucleotide Reductases / Q000737:chemistry*; D013696:Temperature; D014443:Tyrosine / Q000737:chemistry*
D029968:Escherichia coli Proteins; D005609:Free Radicals; C489683:rnr protein, E coli; D005461:Fluorine; D014443:Tyrosine; D012343:RNA, Transfer; D012264:Ribonucleotide Reductases; D005095:Exoribonucleases; D000604:Amino Acyl-tRNA Synthetases; D010100:Oxygen