The tyrosyl radical-diiron(III) cofactor of E. coli ribonucleotide reductase assembles Spontaneously in vitro when the iron-free (apo) form of the enzyme’s R2 Subunit is mixed with Fe2+ and O-2. In previous work (Bollinger, J. M., Jr. et al., Science, 1991, 253, 292-298), kinetic and spectroscopic evidence was presented that the cofactor assembly reaction partitions between two pathways and that the partition ratio depends on the availability of the "extra" reducing equivalent that is required to balance the four-electron reduction of O-2. In this study, stopped-flow absorption, rapid freeze-quench electron paramagnetic resonance, and rapid freeze-quench Mossbauer spectroscopies have been used to examine the kinetics of the reaction carried out with excess Fe2+ (Fe2+/R2 greater than or equal to 5.0). The kinetic data are consistent with a mechanism involving two Sequential first-order reactions; in Which the diferric radical species, X(Ravi, N. et al. J. Am. Chem. Soc., previous paper in this issue), accumulates rapidly(k(obs) = 5-10 s(-1)) and decays concomitantly with formation of *Y122 and the oxo-bridged diferric cluster (k(obs) 0.7-1.0 s(-1)). The simplest interpretation of these data is that oxidation of Y122 by X generates the product cofactor and therefore, that Y122 oxidation is not carried out by a high valent iron species. The Mossbauer kinetic data also suggest that a stable or slowly decaying Fe(III)-containing species, which is distinct from the diferric luster is produced concomitantly with X. It is proposed that this Fe(III) species may represent the product of donation of the "extra" election by Fe2+.