Ribonudeotide reductases (RNRs) catalyze conversion of ribonucleotides to deoxyribonudeotides in all organisms via a free-radical mechanism that is essentially conserved. In class I RNRs, the reaction is initiated and terminated by radical translocation (RT) between the alpha and beta subunits. In the class I-c RNR from Chlamydia trachomatis (Ct RNR), the initiating event converts the active S = 1 Mn(IV)/Fe(III) cofactor to the S = 1/2 Mn(III)/Fe(III) "RT-product" form in the beta subunit and generates a cysteinyl radical in the alpha active site. The radical can be trapped via the well-described decomposition reaction of the mechanism-based inactivator, 2'-azido-2'-deoxyuridine-5'-diphosphate, resulting in the generation of a long-lived, nitrogen-centered radical (N-center dot) in alpha. In this work, we have determined the distance between the Mn(III)/Fe(III) cofactor in beta and N-center dot in alpha to be 43 +/- 1 angstrom by using double-electron electron resonance experiments. This study provides the first structural data on the Ct RNR holoenzyme complex and the first direct experimental measurement of the inter-subunit RT distance in any class I RNR.