Structural Examination of the Transient 3-Aminotyrosyl Radical on the PCET Pathway of E. coli Ribonucleotide Reductase by Multifrequency EPR Spectroscopy

Abstract

E. coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides, a process that requires long-range radical transfer over 35 Å from a tyrosyl radical (Y122•) within the β2 subunit to a cysteine residue (C439) within the α2 subunit. The radical transfer step is proposed to occur by proton-coupled electron transfer via a specific pathway consisting of Y122 → W48 → Y356 in β2, across the subunit interface to Y731 → Y730 → C439 in α2. Using the suppressor tRNA/aminoacyl-tRNA synthetase (RS) methodology, 3-aminotyrosine has been incorporated into position 730 in α2. Incubation of this mutant with β2, substrate, and allosteric effector resulted in loss of the Y122• and formation of a new radical, previously proposed to be a 3-aminotyrosyl radical (NH2Y•). In the current study [15N]- and [14N]-NH2Y730• have been generated in H2O and D2O and characterized by continuous wave 9 GHz EPR and pulsed EPR spectroscopies at 9, 94, and 180 GHz. The data give insight into the electronic and molecular structure of NH2Y730•. The g tensor (gx = 2.0052, gy = 2.0042, gz = 2.0022), the orientation of the β-protons, the hybridization of the amine nitrogen, and the orientation of the amino protons relative to the plane of the aromatic ring were determined. The hyperfine coupling constants and geometry of the NH2 moiety are consistent with an intramolecular hydrogen bond within NH2Y730•. This analysis is an essential first step in using the detailed structure of NH2Y730• to formulate a model for a PCET mechanism within α2 and for use of NH2Y in other systems where transient Y•s participate in catalysis.