A class I ribonucleotide reductase (RNR) uses either a tyrosyl radical (Y-center dot) or a Mn-IV/Fe-III cluster in its beta subunit to oxidize a cysteine residue similar to 35 angstrom away in its alpha subunit, generating a thiyl radical that abstracts hydrogen (H-center dot) from the substrate. With either oxidant, the inter-subunit "hole-transfer" or "radical-translocation" (RT) process is thought to occur by a "hopping" mechanism involving multiple tyrosyl (and perhaps one tryptophanyl) radical intermediates along a specific pathway. The hopping intermediates have never been directly detected in a Mn/Fe-dependent (class Ic) RNR nor in any wildtype (wt) RNR The Mn-IV/Fe-III cofactor of Chlamydia trachomatis RNR assembles via a Mn-IV/Fe-IV intermediate. Here we show that this cofactor-assembly intermediate can propagate a hole into the RT pathway when a is present, accumulating radicals with EPR spectra characteristic of Y-center dot's. The dependence of Y-center dot accumulation on the presence of substrate suggests that RT within this "super-oxidized" enzyme form is gated by the protein, and the failure of a beta variant having the subunit-interfacial pathway Y substituted by phenylalanine to support radical accumulation implies that the Y-center dot(s) in the wt enzyme reside(s) within the RT pathway. Remarkably, two variant beta proteins having pathway substitutions rendering them inactive in their Mn-IV/Fe-III states can generate the pathway Y-center dot's in their Mn-IV/Fe-IV states and also effect nucleotide reduction. Thus, the use of the more oxidized cofactor permits the accumulation of hopping intermediates and the "hurdling" of engineered defects in the RT pathway.