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

E coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides, a process that requires long-range radical transfer over 35 angstrom from a tyrosyl radical (Y-122 center dot) within the beta 2 subunit to a cysteine residue (C-439) within the alpha 2 subunit. The radical transfer step is proposed to occur by proton-coupled electron transfer via a specific pathway consisting of Y-122 -> W-48 -> Y-356 in beta 2, across the subunit interface to Y-731 -> Y-730 -> C-439 in alpha 2. Using the suppressor tRNA/aminoacyl-tRNA synthetase (RS) methodology, 3-aminotyrosine has been incorporated into position 730 in alpha 2. Incubation of this mutant with beta 2, substrate, and allosteric effector resulted in loss of the Y-122 center dot and formation of a new radical, previously proposed to be a 3-aminotyrosyl radical, (NH2Y center dot). In the current study [N-15]- and [N-14]-NH2Y730 center dot 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 center dot. The g tensor (g(x) = 2.0052, g(y) = 2.0042, g(z) = 2.0022), the orientation of the beta-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 center dot. This analysis is an essential first step in using the detailed structure of NH2Y730 center dot to formulate a model for a PCET mechanism within alpha 2 and for use of NH2Y in other systems where transient Y center dot s participate in catalysis.