Escherichia coli ribonucleotide reductase (RNR) catalyzes the reduction of nucleotides to 2'-deoxynucleotides. The active enzyme a 1:1 complex of two homodimeric subunits, alpha 2 and beta 2. The alpha 2 is the site of nucleotide reduction, and beta 2 harbors a diferric tyrosyl radical (Y122 *) cofactor. Turnover requires formation of a cysteiny) radical (C-439 *) in the active site of alpha 2 at the expense of the Y-122 * in beta 2. A docking model for the alpha 2 beta 2 interaction and a pathway for radical transfer from beta 2 to alpha 2 have been proposed. This pathway contains three Y-S: Y-356 in beta(2) and Y-731/Y-730 in alpha 2. We have previously incorporated 3-hydroxytyrosine and 3-aminotyrosine into these residues and showed that they act as radical traps. In this study, we use these alpha 2/beta 2 variants and PELDOR spectroscopy to measure the distance between the Y-122 * in one alpha beta pair and the newly formed radical in the second alpha beta pair. The results yield distances that are similar to those predicted by the docking model for radical transfer. Further, they support a long-range radical initiation process for C-439 * generation and provide a structural constraint for residue Y-356, which is thermally labile in all beta 2 structures solved to date.