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-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 indirectly by optical, Mossbauer, and EPR kinetic data, which show concomitant accumulation of the two constituents, and directly by the unique EPR and Mossbauer spectroscopic features of the X-Y-. species, which can be properly simulated by using the known magnetic properties of X and Y122(.) and introducing a spin-spin interaction between the two radicals. This analysis of the spectroscopic data provides an estimate of the distance between the two radical constituents that is consistent with the crystallographically defined distance between Y122 and the diiron cluster. These results suggest that substitution of W48 with phenylalanine impairs the pathway through which the extra electron is normally transferred. As a result, the two-electron-oxidized diiron species, designated as (Fe2O2)(4+), which in wild-type R2 would oxidize W48 to form X and the W48(+.), instead oxidizes Y122 to form the X-Y-.. Most of the Y122(.) that forms as part of the X-Y-. subsequently decays. Decay of the Y122(.) probably results from further reaction with the adjacent X, as indicated by the formation of altered diiron(LII) products and by the ability of the strong reductant, dithionite, to "rescue" the Y122(.) from decay by reducing X to form the normal mu -oxo diiron(III) cluster.