A class Ia ribonucleotide reductase (RNR) employs a mu-oxo-Fe-2(III/III)/tyrosyl radical cofactor in its beta subunit to oxidize a cysteine residue similar to 35 angstrom away in its a subunit; the resultant cysteine radical initiates substrate reduction. During self-assembly of the Escherichia coil RNR-beta cofactor, reaction of the protein's Fe-2(II/II) complex with O-2 results in accumulation of an Fe-2(III/IV) cluster, termed X, which oxidizes the adjacent tyrosine (Y-122) to the radical (Y-122(center dot)) converted as the cluster is conveed to the mu-oxo-Fe-2(III/III) product. As the first high-valent non-heme-iron enzyme complex to be identified and the key activating intermediate of class la RNRs, X has been the focus of intensive efforts to determine its structure. Initial characterization by extended X-ray absorption fine structure (EXAFS) spectroscopy yielded a Fe-Fe separation (d(Fe-Fe)) of 2.5 angstrom, which was interpreted to imply the presence of three single-atom bridges (O2-, HO-, and/or mu-1,1-carboxylates). This short distance has been irreconcilable with computational and synthetic models, which all have d(F-eFe) >= 2.7 angstrom. To resolve this conundrum, we revisited the EXAFS characterization of X. Assuming that samples containing increased concentrations of the intermediate would yield EXAFS data of improved quality, we applied our recently developed method of generating O-2 in situ from chlorite using the enzyme chlorite dismutase to prepare X at similar to 2.0 mM, more than 2.5 times the concentration realized in the previous EXAFS study. The measured d(Fe-Fe) = 2.78 angstrom is fully consistent with computational models containing a (mu-oxo)(2)-Fe-2(III/IV) core. Correction of the d(Fe-Fe) brings the experimental data and computational models into full conformity and informs analysis of the mechanism by which X generates Y-122(center dot).