Reconsideration of X, the Diiron Intermediate Formed during Cofactor Assembly in E. coli Ribonucleotide Reductase

Abstract

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 57 Fe 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 character. Reanalysis of the Mössbauer 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).