Substrate turnover in class Ia ribonucleotide reductase (RNR) requires reversible radical transport across two subunits over 35 Å, which occurs by a multistep proton-coupled electron-transfer mechanism. Using a photooxidant-labeled β2 subunit of Escherichia coli class Ia RNR, we demonstrate photoinitiated oxidation of a tyrosine in an α2:β2 complex, which results in substrate turnover. Using site-directed mutations of the redox-active tyrosines at the subunit interface, Y356F(β) and Y731F(α), this oxidation is identified to be localized on Y356. The rate of Y356 oxidation depends on the presence of Y731 across the interface. This observation supports the proposal that unidirectional PCET across the Y356(β)–Y731(α)–Y730(α) triad is crucial to radical transport in RNR.

The class Ia ribonucleotide reductase (RNR) from Escherichia coli employs a free-radical mechanism, which involves bidirectional translocation of a radical equivalent or “hole” over a distance of ∼35 Å from the stable diferric/tyrosyl-radical (Y122•) cofactor in the β subunit to cysteine 439 (C439) in the active site of the α subunit. This long-range, intersubunit electron transfer occurs by a multistep “hopping” mechanism via formation of transient amino acid radicals along a specific pathway and is thought to be conformationally gated and coupled to local proton transfers. Whereas constituent amino acids of the hopping pathway have been identified, details of the proton-transfer steps and conformational gating within the β sununit have remained obscure; specific proton couples have been proposed, but no direct evidence has been provided. In the key first step, the reduction of Y122• by the first residue in the hopping pathway, a water ligand to Fe1 of the diferric cluster was suggested to donate a proton to yield the neutral Y122. Here we show that forward radical translocation is associated with perturbation of the Mössbauer spectrum of the diferric cluster, especially the quadrupole doublet associated with Fe1. Density functional theory (DFT) calculations verify the consistency of the experimentally observed perturbation with that expected for deprotonation of the Fe1-coordinated water ligand. The results thus provide the first evidence that the diiron cluster of this prototypical class Ia RNR functions not only in its well-known role as generator of the enzyme’s essential Y122•, but also directly in catalysis.

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•.

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•.

A class I ribonucleotide reductase (RNR) uses either a tyrosyl radical (Y•) or a MnIV/FeIII cluster in its β subunit to oxidize a cysteine residue ∼35 Å away in its α subunit, generating a thiyl radical that abstracts hydrogen (H•) from the substrate. With either oxidant, the inter-subunit “hole-transfer” or “radical-translocation” (RT) process is thought to occur by a “hopping” mechanism involving multiple tyrosyl (and perhaps one tryptophanyl) radical intermediates along a specific pathway. The hopping intermediates have never been directly detected in a Mn/Fe-dependent (class Ic) RNR nor in any wild-type (wt) RNR. The MnIV/FeIII cofactor of Chlamydia trachomatis RNR assembles via a MnIV/FeIV intermediate. Here we show that this cofactor-assembly intermediate can propagate a hole into the RT pathway when α is present, accumulating radicals with EPR spectra characteristic of Y•’s. The dependence of Y• accumulation on the presence of substrate suggests that RT within this “super-oxidized” enzyme form is gated by the protein, and the failure of a β variant having the subunit-interfacial pathway Y substituted by phenylalanine to support radical accumulation implies that the Y•(s) in the wt enzyme reside(s) within the RT pathway. Remarkably, two variant β proteins having pathway substitutions rendering them inactive in their MnIV/FeIII states can generate the pathway Y•’s in their MnIV/FeIV states and also effect nucleotide reduction. Thus, the use of the more oxidized cofactor permits the accumulation of hopping intermediates and the “hurdling” of engineered defects in the RT pathway.

The class Ia ribonucleotide reductase (RNR) from Escherichia coli employs a free-radical mechanism, which involves bidirectional translocation of a radical equivalent or “hole” over a distance of ∼35 Å from the stable diferric/tyrosyl-radical (Y122•) cofactor in the β subunit to cysteine 439 (C439) in the active site of the α subunit. This long-range, intersubunit electron transfer occurs by a multistep “hopping” mechanism via formation of transient amino acid radicals along a specific pathway and is thought to be conformationally gated and coupled to local proton transfers. Whereas constituent amino acids of the hopping pathway have been identified, details of the proton-transfer steps and conformational gating within the β sununit have remained obscure; specific proton couples have been proposed, but no direct evidence has been provided. In the key first step, the reduction of Y122• by the first residue in the hopping pathway, a water ligand to Fe1 of the diferric cluster was suggested to donate a proton to yield the neutral Y122. Here we show that forward radical translocation is associated with perturbation of the Mössbauer spectrum of the diferric cluster, especially the quadrupole doublet associated with Fe1. Density functional theory (DFT) calculations verify the consistency of the experimentally observed perturbation with that expected for deprotonation of the Fe1-coordinated water ligand. The results thus provide the first evidence that the diiron cluster of this prototypical class Ia RNR functions not only in its well-known role as generator of the enzyme’s essential Y122•, but also directly in catalysis.