A 2.8 Å Fe–Fe Separation in the Fe2III/IV Intermediate, X, from Escherichia coli Ribonucleotide Reductase

A class Ia ribonucleotide reductase (RNR) employs a μ-oxo-Fe₂ᴵᴵᴵ/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 Fe₂ᴵᴵ/ᴵᴵ complex with O₂ results in accumulation of an Fe₂ᴵᴵᴵ/ᴵⱽ cluster, termed X, which oxidizes the adjacent tyrosine (Y₁₂₂) to the radical (Y₁₂₂•) as the cluster is converted to the μ-oxo-Fe₂ᴵᴵᴵ/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 (dFₑ–Fₑ) of 2.5 Å, which was interpreted to imply the presence of three single-atom bridges (O²–, HO–, and/or μ-1,1-carboxylates). This short distance has been irreconcilable with computational and synthetic models, which all have dFₑ–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 dFₑ–Fₑ = 2.78 Å is fully consistent with computational models containing a (μ-oxo)₂-Fe₂ᴵᴵᴵ/ᴵⱽ core. Correction of the dFₑ–Fₑ brings the experimental data and computational models into full conformity and informs analysis of the mechanism by which X generates Y₁₂₂•.