Reconsideration of X, the diiron intermediate formed during cofactor assembly in E. coli ribonucleotide reductase

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) Mossbauer and EPR spectroscopies led to the proposal that X is comprised of two high spin ferric ions and a S = 1/4 ligand radical, mutually spin coupled to give a S = 1/4 ground state (Ravi, N.; Bollinger, J.M., Jr.; Huyhn, 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(Fe(A)) = -[74.2(2), 72.2(2), 73.2(2)] MHz) as expected for Fe(III), but that the other iron site displays considerable anisotropy (A(Fe(B)) = +[27.5(2), 36.8(2), 36.8(2)] MHz), indicative of substantial Fe(IV) chracter. Reanalysis of the Mossbauer data using these results leads to isomer shifts of δ(Fe(A)) = 0.56(3) mm/s and δ(Fe(B)) = 0.26(4) mm/s. Based on the hyperfine anisotropy of Fe(B) plus the reduced isomer shift, X is now best described as a spin-coupled Fe(III)/Fe(IV) center without a radical, but with significant spin delocalization onto the oxygen ligand(s).