MECHANISM OF ASSEMBLY OF THE TYROSYL RADICAL-DIIRON(III) COFACTOR OF ESCHERICHIA-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 red uctase 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 wor k (Bollinger, J. M., Jr. et al., Science, 1991, 253, 292-298), kinetic and spectroscopic evidence was presented that the cofactor assembly r eaction partitions between two pathways and that the partition ratio d epends on the availability of the ''extra'' reducing equivalent that i s required to balance the four-electron reduction of O-2. In this stud y, 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 F e2+ (Fe2+/R2 greater than or equal to 5.0). The kinetic data are consi stent 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) = 5-1 0 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 interpre tation of these data is that oxidation of Y122 by X generates the prod uct cofactor and therefore, that Y122 oxidation is not carried out by a high valent iron species. The Mossbauer kinetic data also suggest th at a stable or slowly decaying Fe(III)-containing species, which is di stinct from the diferric luster is produced concomitantly with X. It i s proposed that this Fe(III) species may represent the product of dona tion of the ''extra'' election by Fe2+.