MECHANISM OF ASSEMBLY OF THE TYROSYL RADICAL-DIIRON(III) COFACTOR OF ESCHERICHIA-COLI RIBONUCLEOTIDE REDUCTASE .3. KINETICS OF THE LIMITINGFE2+ REACTION BY OPTICAL, EPR, AND MOSSBAUER SPECTROSCOPIES

The R2 subunit of E. coli ribonucleotide reductase contains a tyrosyl radical-diiron(III) cofactor which assembles spontaneously when apo R2 is mixed with Fe2+ and O-2. The kinetic/spectroscopic characteristics of this assembly reaction were previously shown to depend on the Fe2/apo R2 ratio (Bollinger, J. M., Jr. et al. Science 1991, 253, 292-298 ). In the case of the reaction carried out with limiting Fe2+ (Fe2+/ap o R2 2.0-2.4), two intermediates were proposed to accumulate. On the b asis of its broad absorption feature at 560 nm, the first intermediate was suggested to contain a mu-peroxodiferric cluster. The one-electro n oxidation of tyrosine 122 by this cluster was postulated to generate the second intermediate, which was proposed to contain the tyrosyl ra dical (Y122) and the diferric radical species (Ravi, N. et al. J. Am. Chem. Soc., first of three papers in this issue; Boilinger, J. M., Jr . et al. J. Am, Chem. Sec. 1991, 113, 6289-6291). In this study, stopp ed-flow absorption, rapid freeze-quench electron paramagnetic resonanc e, and rapid freeze-quench Mossbauer spectroscopies have been used to characterize in detail the kinetics of the assembly reaction carried o ut with limiting Fe2+, The time course of development and decay of the 560 nm absorption transient, and the quantities of diferric radical s pecies, Y122, and product diferric cluster as functions of time, are consistent with the proposal that the 560 nm absorbing species generat es Y122. The data indicate, in contrast to our previous hypothesis, t hat the 560 nm absorbing species is not iron based. It is proposed, in stead, that the species is a tryptophan radical cation produced by the one-electron oxidation of W48. The results of this study are consiste nt with our previous proposal that Fe(IV) intermediates are not involv ed in generation of Y122.