E.coli class I ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides and is composed of two subunits: α2 and β2. β2 contains a stable di-iron tyrosyl radical (Y122•) cofactor required to generate a thiyl radical (C439•) in α2 over a distance of 35 Å, which in turn initiates the chemistry of the reduction process. The radical transfer process is proposed to occur by proton-coupled electron transfer (PCET) via a specific pathway: Y122 ⇆ W48[?] ⇆ Y356 in β2, across the subunit interface to Y731⇆ Y730 ⇆ C439 in α2. Within α2 a co-linear PCET model has been proposed. To obtain evidence for this model, 3-amino tyrosine (NH2Y) replaced Y730 in α2 and this mutant was incubated with β2, CDP and ATP to generate a (NH2Y730•) in D2O. [2H]-Electron-nuclear double resonance (ENDOR) spectra at 94 GHz of this intermediate were obtained and together with DFT models of α2 and quantum chemical calculations allowed assignment of the prominent ENDOR features to two hydrogen bonds likely associated with C439 and Y731. A third proton was assigned to a water molecule in close proximity (2.2 Å O-H---O distance) to residue 730. The calculations also suggest that the unusual g-values measured for NH2Y730• are consistent with the combined effect of the hydrogen bonds to Cys439 and Tyr731, both nearly perpendicular to the ring plane of NH2Y730. The results provide the first experimental evidence for the hydrogen bond network between the pathway residues in α2 of the active RNR complex, for which no structural data is available.

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.

For aromatic organic radicals, pulsed electron-electron double resonance (PELDOR) experiments at high magnetic fields provide information not only about the distance between the paramagnetic species but also about their relative orientation. However, the three-dimensional biradical structure is encoded in a complex pattern of orientation-selected PELDOR traces and the execution of the experiment is generally aggravated by constraints posed by the available hardware and the intrinsically low modulation depth observed. We present a 94 GHz PELDOR experiment performed with a commercial spectrometer and probe heads that permit separation of pump and detection frequencies up to 150 MHz. The setup is employed to examine the orientation selections on a general case of rigid biradicals with non-collinear g axes. The interacting radicals, a tyrosyl radical (Y 122 Á) located in the b2 subunit and an 3-aminotyrosyl radical (NH 2 Y 731 Á) located in the a2 subunit, are generated by Escherichia coli ribonucleotide reductase with a 3-aminotyrosine (NH 2 Y) site specifically incorporated into a2 in the presence of cytidine 5 0-diphosphate and adenosine 5 0-triphosphate. The experimental designs as well as some characteristic features of the observed modulation pattern are discussed.

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