Spectroscopic and electronic structure studies of the class I Escherichia coli ribonucleotide reductase (RNR) intermediate X and three computationally derived model complexes are presented, compared, and evaluated to determine the electronic and geometric structure of the Fe-III-Fe-IV active site of intermediate X. Rapid freeze-quench (RFQ) EPR, absorption, and MCD were used to trap intermediate X in R2 wild-type (WT) and two variants, W48A and Y122F/Y356F. RFQ-EPR spin quantitation was used to determine the relative contributions of intermediate X and radicals present, while RFQ-MCD was used to specifically probe the Fe-III/Fe-IV active site, which displayed three Fe-IV d-d transitions between 16 700 and 22 600 cm(-1), two Fe-IV d-d spin-flip transitions between 23 500 and 24 300 cm(-1), and five oxo to Fe-IV and Fe-III charge transfer (CT) transitions between 25 000 and 32 000 cm(-1). The Fe-IV d-d transitions were perturbed in the two variants, confirming that all three d-d transitions derive from the d-pi manifold. Furthermore, the Fe-IV d-pi splittings in the WT are too large to correlate with a bis-mu-oxo structure. The assignment of the Fe-IV d-d transitions in WT intermediate X best correlates with a bridged mu-oxo/mu-hydroxo [Fe-III(mu-O)(mu-OH)Fe-IV] structure. The mu-oxo/mu-hydroxo core structure provides an important sigma/pi superexchange pathway, which is not present in the bis-mu-oxo structure, to promote facile electron transfer from Y122 to the remote Fe-IV through the bent oxo bridge, thereby generating the tyrosyl radical for catalysis.