Activation of dioxygen at the carboxylate-bridged diiron(II) cluster in the R2 subunit of Escherichia coli class I ribonucleotide reductase produces the enzyme's catalytically essential stable tyrosyl radical by one-electron oxidation of tyrosine 122. An intermediate in the reaction, the formally Fe(IV)Fe(III) cluster X, can oxidize Y122 in the final and rate-limiting step. During formation of X, an "extra" electron must be transferred to an as-yet-uncharacterized adduct between O-2 and the diiron(II) cluster. It was previously shown that a transient, broad absorption band centered near 560 nm develops when the reaction is carried out without an obvious exogenous source of the extra electron, and this band was ascribed to a tryptophan cation radical (W+.) resulting from temporary donation of the electron by the near-surface tryptophan residue 48 during formation of X [Bollinger, J. M.; Jr.; Tong, W. H.; Ravi, N.; Huynh, B. H.; Edmondson, D. E.; Stubbe, J. J. Am. Chem. Sec. 1994, 116, 8024-8032]. In this work, we provide mon definitive evidence for the W+. assignment by showing that(1) the absorbing species reacts rapidly with reductants,(2) the species is associated with a g = 2.0 EPR signal and perturbs the EPR and Mossbauer spectra of X, and (3) most definitively, the absorption spectrum of the species from 310 to 650 nm closely matches the very distinctive spectrum of the tryptophan cation radical previously determined in pulse radiolysis studies [Solar, S.; Getoff, N.; Surdhar, P. S.; Armstrong, D. A.; Sing, A. J. Phys. Chern. 1991, 95, 3639-3643]. Quantitation of species at short reaction times by optical, EPR, and Mossbauer spectroscopies is consistent with the rapid formation of an intermediate containing both ii and the W+. tan X-W+. diradical species). Formation of the W+. land presumably of X) is kinetically first order in both O-2 and Fe(II)-R2 complex, even at the highest reactant concentrations examined, which give a formation rate constant approaching 200 s(-1). This observation implies that precursors to the diradical species must not accumulate to greater than similar to 10% of the initial Fe(II)-R2 reactant concentration and that the immediate precursor must generate the highly oxidizing W+. with a rate constant of at least 400 s(-1) at 5 degreesC.