Mechanism of Assembly of the Tyrosyl Radical-Diiron(III) Cofactor of E. Coli Ribonucleotide Reductase. 1. Mössbauer Characterization of the Diferric Radical Precursor

The R2 subunit of Escherichia coli ribonucleotide reductase (RNR) contains a cofactor which consists of a stable tyrosyl radical (Y122) adjacent to a μ-oxo-bridged diiron(III) cluster. This cofactor assembles spontaneously when apo R2 is treated with Fe²⁺ and O₂. By using rapid kinetic techniques, two kinetically competent intermediates in this assembly process were recently identified (Bollinger, J. M., Jr. et al. Science 1991, 253, 292-298). One of the intermediates is a paramagnetic iron species. By using the rapid freeze-quench technique and a mutant R2 subunit in which Y122 is replaced with F, 1 equiv of this intermediate per R2 subunit can be trapped. Previous electron paramagnetic resonance (EPR) and Mössbauer studies suggested that this species is a spin coupled system involving two high-spin ferric ions and a free radical (a "diferric radical species") (Bollinger, J. M., Jr. et al. J. Am. Chem. Soc. 1991,113,6289-6291). In the present study, Mössbauer spectra have been recorded over a wide range of applied fields (60 mT-8 T), and detailed analysis of the Mössbauer data consolidates the initial finding. The observed isomer shifts (0.55 mm/s for iron site 1 and 0.36 mm/s for site 2) and quadrupole splittings (~1 mm/s) are typical for high-spin ferric species. The magnetic hyperfine coupling tensors, A, for the two iron sites are relatively isotropic (within 20%), a feature which is unique to high-spin Fe(III). By using a simple three-spin coupling model (and for the two iron sites, and for the radical), both the signs and magnitudes of the observed A values for the iron sites (A₁/g_nβ_n - -52.5 T and A₂/g_nβ_n = +24 T) can be explained with an intrinsic A value that is consistent with high-spin Fe(III). The asymmetry in the A values for the two iron sites is a result of the spin orientations: the spin of the radical is parallel to that of the negative-A-value Fe site and antiparallel to that of the positives-value Fe site. Since it has been suggested on the basis of functional analogy with heme-iron-dependent peroxidases that generation of ∗Y122 in RNR-R2 may involve a high-valent iron-oxo intermediate, the possibility that this paramagnetic iron species contains Fe(IV) has also been carefully examined. Both the EPR and the Mössbauer data are incompatible with such a possibility.