Mechanism of Assembly of the Tyrosyl Radical-Diiron(III) Cofactor of E. coli Ribonucleotide Reductase. 2. Kinetics of The Excess Fe²⁺Reaction by Optical, EPR, and Mossbauer Spectroscopies

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 O₂. 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 O₂. In this study, stopped-flow absorption, rapid freeze-quench electron paramagnetic resonance, and rapid freeze-quench MSssbauer spectroscopies have been used to examine the kinetics of the reaction carried Out with excess Fe²⁺(Fe²⁺/R2 ≥ 5.0). The kinetic data are consistent with a mechanism involving two sequential first-order reactions, in which the diferric radical species,.Y(Ravi, N. et al. J. Am. Chem. Soc., previous paper in this issue), accumulates rapidly (f_obs= 5-10 s^-1) and decays concomitantly with formation of ∗Y122 and the oxo-bridged diferric cluster (f_obs= 0.7-1.0 s^-1)- The simplest interpretation of these data is that oxidation of Y122 by A'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 cluster, is produced concomitantly with X. It is proposed that this Fe(III) species may represent the product of donation of the "extra" electron by Fe²⁺.