A 2.8 Å Fe-Fe separation in the Fe₂^III/IV intermediate, X, from Escherichia coli ribonucleotide reductase

A class Ia ribonucleotide reductase (RNR) employs a μ-oxo-Fe ₂^III/III/tyrosyl radical cofactor in its β subunit to oxidize a cysteine residue ∼35 Å away in its α subunit; the resultant cysteine radical initiates substrate reduction. During self-assembly of the Escherichia coli RNR-β cofactor, reaction of the protein's Fe ₂^II/II complex with O₂ results in accumulation of an Fe₂^III/IV cluster, termed X, which oxidizes the adjacent tyrosine (Y₁₂₂) to the radical (Y₁₂₂ ^•) as the cluster is converted to the μ-oxo-Fe ₂^III/III product. As the first high-valent non-heme-iron enzyme complex to be identified and the key activating intermediate of class Ia RNRs, X has been the focus of intensive efforts to determine its structure. Initial characterization by extended X-ray absorption fine structure (EXAFS) spectroscopy yielded a Fe-Fe separation (d_Fe-Fe) of 2.5 Å, which was interpreted to imply the presence of three single-atom bridges (O ^2-, HO^-, and/or μ-1,1-carboxylates). This short distance has been irreconcilable with computational and synthetic models, which all have d_Fe-Fe ≥ 2.7 Å. To resolve this conundrum, we revisited the EXAFS characterization of X. Assuming that samples containing increased concentrations of the intermediate would yield EXAFS data of improved quality, we applied our recently developed method of generating O₂ in situ from chlorite using the enzyme chlorite dismutase to prepare X at ∼2.0 mM, more than 2.5 times the concentration realized in the previous EXAFS study. The measured d_Fe-Fe = 2.78 Å is fully consistent with computational models containing a (μ-oxo)₂-Fe₂ ^III/IV core. Correction of the d_Fe-Fe brings the experimental data and computational models into full conformity and informs analysis of the mechanism by which X generates Y₁₂₂^•.