The R2 subunit of Escherichia coli ribonucleotide reductase (RNR) contains a stable tyrosyl radical (•Y122) diferric cluster cofactor. Earlier studies on the cofactor assembly reaction detected a paramagnetic intermediate, X, that was found to be kinetically competent to oxidize Y122. Studies using rapid freeze-quench (RFQ) Mössbauer and EPR spectroscopies led to the proposal that X is comprised of two high spin ferric ions and a S = 1/2 ligand radical, mutually spin coupled to give a S = 1/2 ground state (Ravi, N.; Bollinger, J. M., Jr.; Huynh, B. H.; Edmondson, D. E.; Stubbe, J. J. Am. Chem. Soc. 1994, 116, 8007−8014). An extension of RFQ methodology to Q-band ENDOR spectroscopy using 57Fe has shown that one of the irons has a very nearly isotropic hyperfine tensor (A(FeA) = −[74.2(2), 72.2(2), 73.2(2)] MHz) as expected for FeIII, but that the other iron site displays considerable anisotropy (A(FeB) = +[27.5(2), 36.8(2), 36.8(2)] MHz), indicative of substantial FeIV character. Reanalysis of the Mössbauer data using these results leads to isomer shifts of δ(FeA) = 0.56(3) mm/s and δ(FeB) = 0.26(4) mm/s. Based on the hyperfine anisotropy of FeB plus the reduced isomer shift, X is now best described as a spin-coupled FeIII/FeIV center without a radical, but with significant spin delocalization onto the oxygen ligand(s).

The assembly of the essential diferric cluster/tyrosyl radical cofactor of the R2 subunit of Escherichia coli ribonucleotide reductase from apoR2 with Fe2+ and O2 or diferrous R2 with O2 has been studied by a variety of rapid kinetic methods. An intermediate X, formally an Fe3+/Fe4+ diiron cluster, oxidizes tyrosine 122 to the tyrosyl radical concomitant with its own reduction to the diferric cluster generating the R2 cofactor. To characterize the properties of X, rapid freeze quench methods have been used in conjunction with Mössbauer, ENDOR, and EPR spectroscopies. These studies are extended here to include rapid freeze quench EXAFS. A short, 2.5 Å Fe−Fe vector and a 1.8 Å Fe−O interaction have been identified in nine independent samples of X. These samples have been generated using both wild-type and a mutant protein in which the essential tyrosine has been replaced by phenylalanine (Y122F). The short Fe−Fe interaction is neither present in diferrous or diferric R2 nor in samples of X that have aged to decay the intermediate. Several structural models which are consistent with the data are presented.