The mechanism and outcome of dioxygen activation by the carboxylate-bridged diiron(II) cluster in the W48F site-directed variant of protein R2 of ribonucleotide reductase from Escherichia coli has been investigated by kinetic, spectroscopic, and chemical methods. The data corroborate the hypothesis advanced in earlier work and in the preceding paper that W48 mediates, by a shuttling mechanism in which it undergoes transient one-electron oxidation, the transfer of the “extra” electron that is required for formation of the formally Fe(IV)Fe(III) cluster X on the reaction pathway to the tyrosyl radical/μ-oxodiiron(III) cofactor of the catalytically active protein. The transient 560-nm absorption, which develops in the reaction of the wild-type R2 protein and is ascribed to the W48 cation radical, is not observed in the reaction of R2-W48F. Instead, a diradical intermediate containing both X and the Y122 radical (X-Y•) accumulates rapidly to a high level. The formation of this X-Y• species is demonstrated ind...