A 2.8 Å Fe–Fe Separation in the Fe₂^III/IV Intermediate, X, from <i>Escherichia coli</i> Ribonucleotide Reductase

Abstract

A class Ia ribonucleotide reductase (RNR) employs a μ-oxo-Fe₂^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 <i>Escherichia coli</i> RNR-β cofactor, reaction of the protein’s Fe₂^II/II complex with O₂ results in accumulation of an Fe₂^III/IV cluster, termed <b>X</b>, which oxidizes the adjacent tyrosine (Y₁₂₂) to the radical (Y₁₂₂^•) as the cluster is converted to the μ-oxo-Fe₂^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, <b>X</b> 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 (<i>d</i>_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 <i>d</i>_Fe–Fe ≥ 2.7 Å. To resolve this conundrum, we revisited the EXAFS characterization of <b>X</b>. 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 <i>in situ</i> from chlorite using the enzyme chlorite dismutase to prepare <b>X</b> at ∼2.0 mM, more than 2.5 times the concentration realized in the previous EXAFS study. The measured <i>d</i>_Fe–Fe = 2.78 Å is fully consistent with computational models containing a (μ-oxo)₂-Fe₂^III/IV core. Correction of the <i>d</i>_Fe–Fe brings the experimental data and computational models into full conformity and informs analysis of the mechanism by which <b>X</b> generates Y₁₂₂^•