RESUMEN
The oxidation of substrates via the cleavage of thermodynamically strong C-H bonds is an essential part of mammalian metabolism. These reactions are predominantly carried out by enzymes that produce high-valent metal-oxido species, which are directly responsible for cleaving the C-H bonds. While much is known about the identity of these transient intermediates, the mechanistic factors that enable metal-oxido species to accomplish such difficult reactions are still incomplete. For synthetic metal-oxido species, C-H bond cleavage is often mechanistically described as synchronous, proton-coupled electron transfer (PCET). However, data have emerged that suggest that the basicity of the M-oxido unit is the key determinant in achieving enzymatic function, thus requiring alternative mechanisms whereby proton transfer (PT) has a more dominant role than electron transfer (ET). To bridge this knowledge gap, the reactivity of a monomeric MnIV-oxido complex with a series of external substrates was studied, resulting in a spread of over 104 in their second-order rate constants that tracked with the acidity of the C-H bonds. Mechanisms that included either synchronous PCET or rate-limiting PT, followed by ET, did not explain our results, which led to a proposed PCET mechanism with asynchronous transition states that are dominated by PT. To support this premise, we report a semiempirical free energy analysis that can predict the relative contributions of PT and ET for a given set of substrates. These findings underscore why the basicity of M-oxido units needs to be considered in C-H functionalization.
RESUMEN
Although the activation of inert C-H bonds by metal-oxo complexes has been widely studied, important questions remain, particularly regarding the role of oxygen spin population (i.e., unpaired electrons on the oxo ligand) in facilitating C-H bond cleavage. In order to shed light on this issue, we have utilized 17O electron nuclear double resonance spectroscopy to measure the oxygen spin populations of three compound I intermediates in heme enzymes with different reactivities toward C-H bonds: chloroperoxidase, cytochrome P450, and a selenolate (selenocysteinyl)-ligated cytochrome P450. The experimental data suggest an inverse correlation between oxygen spin population and electron donation from the axial ligand. We have explored the implications of this result using a Hückel-type molecular orbital model and constrained density functional theory calculations. These investigations have allowed us to examine the relationship between oxygen spin population, oxygen charge, electron donation from the axial ligand, and reactivity.
Asunto(s)
Cloruro Peroxidasa , Complejos de Coordinación , Espectroscopía de Resonancia por Spin del Electrón , Electrones , Oxígeno/química , Ligandos , Hemo/química , Sistema Enzimático del Citocromo P-450/química , Complejos de Coordinación/químicaRESUMEN
Nitrogenase catalyzes the multielectron reduction of dinitrogen to ammonia. Electron transfer in the catalytic protein (MoFeP) proceeds through a unique [8Fe-7S] cluster (P-cluster) to the active site (FeMoco). In the reduced, all-ferrous (PN) state, the P-cluster is coordinated by six cysteine residues. Upon two-electron oxidation to the P2+ state, the P-cluster undergoes conformational changes in which a highly conserved oxygen-based residue (a Ser or a Tyr) and a backbone amide additionally ligate the cluster. Previous studies of Azotobacter vinelandii (Av) MoFeP revealed that when the oxygen-based residue, ßSer188, was mutated to a noncoordinating residue, Ala, the P-cluster became redox-labile and reversibly lost two of its eight Fe centers. Surprisingly, the Av strain with a MoFeP variant that lacked the serine ligand (Av ßSer188Ala MoFeP) displayed the same diazotrophic growth and in vitro enzyme turnover rates as wild-type Av MoFeP, calling into question the necessity of this conserved ligand for nitrogenase function. Based on these observations, we hypothesized that ßSer188 plays a role in protecting the P-cluster under nonideal conditions. Here, we investigated the protective role of ßSer188 both in vivo and in vitro by characterizing the ability of Av ßSer188Ala cells to grow under suboptimal conditions (high oxidative stress or Fe limitation) and by determining the tendency of ßSer188Ala MoFeP to be mismetallated in vitro. Our results demonstrate that ßSer188 (1) increases Av cell survival upon exposure to oxidative stress in the form of hydrogen peroxide, (2) is necessary for efficient Av diazotrophic growth under Fe-limiting conditions, and (3) may protect the P-cluster from metal exchange in vitro. Taken together, our findings suggest a structural adaptation of nitrogenase to protect the P-cluster via Ser ligation, which is a previously unidentified functional role of the Ser residue in redox proteins and adds to the expanding functional roles of non-Cys ligands to FeS clusters.
Asunto(s)
Nitrogenasa , Serina , LigandosRESUMEN
Diazophosphonates, readily prepared from α-ketophosphonates by oxidation of the corresponding hydrazones in batch or in flow, are useful partners in 1,3-dipolar cycloaddition reactions to alkynes to give N-H pyrazoles, including the first intramolecular examples of such a process. The phosphoryl group imbues a number of desirable properties into the diazo 1,3-dipole. The electron-withdrawing nature of the phosphoryl stabilizes the diazo compound making it easier to handle, whilst the ability of the phosphoryl group to migrate readily in a [1,5]-sigmatropic rearrangement enables its transfer from C to N to aromatize the initial cycloadduct, and hence its facile removal from the final pyrazole product. Overall, the diazophosphonate acts as a surrogate for the much less stable diazoalkane in cycloadditions, with the phosphoryl group playing a vital, but traceless, role. The cycloaddition proceeds more readily with alkynes bearing electron-withdrawing groups, and is regiospecific with asymmetrical alkynes. The potential of diazophosphonates for use in bioorthogonal cycloadditions is demonstrated by their facile addition to strained alkynes.
Asunto(s)
Alquinos , Pirazoles , Reacción de Cicloadición , HidrazonasRESUMEN
The protonation state of the iron(IV) oxo (or ferryl) form of ascorbate peroxidase compound II (APX-II) is a subject of debate. It has been reported that this intermediate is best described as an iron(IV) hydroxide species. Neutron diffraction data obtained from putative APX-II crystals indicate a protonated oxygenic ligand at 1.88 Å from the heme iron. This finding, if correct, would be unprecedented. A basic iron(IV) oxo species has yet to be spectroscopically observed in a histidine-ligated heme enzyme. The importance of ferryl basicity lies in its connection to our fundamental understanding of C-H bond activation. Basic ferryl species have been proposed to facilitate the oxidation of inert C-H bonds, reactions that are unknown for histidine-ligated hemes enzymes. To provide further insight into the protonation status of APX-II, we examined the intermediate using a combination of Mössbauer and X-ray absorption spectroscopies. Our data indicate that APX-II is an iron(IV) oxo species with an Fe-O bond distance of 1.68 Å, a K-edge pre-edge absorption of 18 units, and Mössbauer parameters of ΔEq = 1.65 mm/s and δ = 0.03 mm/s.
RESUMEN
High-valent nonheme FeIV-oxido species are key intermediates in biological oxidation, and their properties are proposed to be influenced by the unique microenvironments present in protein active sites. Microenvironments are regulated by noncovalent interactions, such as hydrogen bonds (H-bonds) and electrostatic interactions; however, there is little quantitative information about how these interactions affect crucial properties of high valent metal-oxido complexes. To address this knowledge gap, we introduced a series of FeIV-oxido complexes that have the same S = 2 spin ground state as those found in nature and then systematically probed the effects of noncovalent interactions on their electronic, structural, and vibrational properties. The key design feature that provides access to these complexes is the new tripodal ligand [poat]3-, which contains phosphinic amido groups. An important structural aspect of [FeIVpoat(O)]- is the inclusion of an auxiliary site capable of binding a Lewis acid (LAII); we used this unique feature to further modulate the electrostatic environment around the Fe-oxido unit. Experimentally, studies confirmed that H-bonds and LAII s can interact directly with the oxido ligand in FeIV-oxido complexes, which weakens the FeâO bond and has an impact on the electronic structure. We found that relatively large vibrational changes in the Fe-oxido unit correlate with small structural changes that could be difficult to measure, especially within a protein active site. Our work demonstrates the important role of noncovalent interactions on the properties of metal complexes, and that these interactions need to be considered when developing effective oxidants.
Asunto(s)
Compuestos de Hierro/química , Óxidos/química , Teoría Funcional de la Densidad , Ácidos de Lewis/química , Conformación MolecularRESUMEN
An important class of non-heme dioxygenases contains a conserved Fe binding site that consists of a 2-His-1-carboxylate facial triad. Results from structural biology show that, in the resting state, these proteins are six-coordinate with aqua ligands occupying the remaining three coordination sites. We have utilized biotin-streptavidin (Sav) technology to design new artificial Fe proteins (ArMs) that have many of the same structural features found within active sites of these non-heme dioxygenases. An Sav variant was isolated that contains the S112E mutation, which installed a carboxylate side chain in the appropriate position to bind to a synthetic FeII complex confined within Sav. Structural studies using X-ray diffraction (XRD) methods revealed a facial triad binding site that is composed of two N donors from the biotinylated ligand and the monodentate coordination of the carboxylate from S112E. Two aqua ligands complete the primary coordination sphere of the FeII center with both involved in hydrogen bond networks within Sav. The corresponding FeIII protein was also prepared and structurally characterized to show a six-coordinate complex with two exogenous acetato ligands. The FeIII protein was further shown to bind an exogenous azido ligand through replacement of one acetato ligand. Spectroscopic studies of the ArMs in solution support the results found by XRD.
Asunto(s)
Dioxigenasas/química , Proteínas de Hierro no Heme/química , Sitios de Unión , Dioxigenasas/metabolismo , Compuestos Férricos/química , Compuestos Férricos/metabolismo , Ligandos , Conformación Molecular , Proteínas de Hierro no Heme/metabolismoRESUMEN
We present a mixed experimental/theoretical determination of the bond strengths and redox potentials that define the ground-state thermodynamics for C-H bond activation in cytochrome P450 catalysis. Using redox titrations with [Ir(IV)Cl6]2-, we have determined the compound II/ferric (or Fe(IV)OH/Fe(III)OH2) couple and its associated D(O-H)Ferric bond strength in CYP158. Knowledge of this potential as well as the compound II/ferric (or Fe(IV)O/Fe(III)OH) reduction potential in horseradish peroxidase and the two-electron compound I/ferric (or Fe(IV)O(Porâ¢)/Fe(III)OH2(Por)) reduction potential in aromatic peroxidase has allowed us to gauge the accuracy of theoretically determined bond strengths. Using the restricted open shell (ROS) method as proposed by Wright and co-workers, we have obtained O-H bond strengths and associated redox potentials for charge-neutral H-atom reductions of these iron(IV)-hydroxo and -oxo porphyrin species that are within 1 kcal/mol of experimentally determined values, suggesting that the ROS method may provide accurate values for the P450-II O-H bond strength and P450-I reduction potential. The efforts detailed here indicate that the ground-state thermodynamics of C-H bond activation in P450 are best described as follows: E0'Comp-I = 1.22 V (at pH 7, vs NHE) with D(O-H)Comp-II = 95 kcal/mol and E0'Comp-II = 0.99 V (at pH 7, vs NHE) with D(O-H)Ferric = 90 kcal/mol.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Termodinámica , Sistema Enzimático del Citocromo P-450/química , Teoría Funcional de la Densidad , Compuestos de Hierro/química , Compuestos de Hierro/metabolismo , Oxidación-ReducciónRESUMEN
Terminal iron nitrides (Fe≡N) have been proposed as intermediates of (bio)catalytic nitrogen fixation, yet experimental evidence to support this hypothesis has been lacking. In particular, no prior synthetic examples of terminal Fe≡N species have been derived from N2. Here we show that a nitrogen-fixing Fe-N2 catalyst can be protonated to form a neutral Fe(NNH2) hydrazido(2-) intermediate, which, upon further protonation, heterolytically cleaves the N-N bond to release [FeIV≡N]+ and NH3. These observations provide direct evidence for the viability of a Chatt-type (distal) mechanism for Fe-mediated N2-to-NH3 conversion. The physical oxidation state range of the Fe complexes in this transformation is buffered by covalency with the ligand, a feature of possible relevance to catalyst design in synthetic and natural systems that facilitate multiproton/multielectron redox processes.
Asunto(s)
Fijación del Nitrógeno , Nitrógeno/química , Amoníaco/química , Catálisis , Electrones , Hierro/química , Ligandos , Oxidación-Reducción , ProtonesRESUMEN
The rebound mechanism for alkane hydroxylation was invoked over 40 years ago to help explain reactivity patterns in cytochrome P450, and subsequently has been used to provide insight into a range of biological and synthetic systems. Efforts to model the rebound reaction in a synthetic system have been unsuccessful, in part because of the challenge in preparing a suitable metal-hydroxide complex at the correct oxidation level. Herein we report the synthesis of such a complex. The reaction of this species with a series of substituted radicals allows for the direct interrogation of the rebound process, providing insight into this uniformly invoked, but previously unobserved process.
Asunto(s)
Hidróxidos/química , Compuestos de Hierro/química , Oxígeno/química , Conformación MolecularRESUMEN
Protonated ferryl (or iron(IV)hydroxide) intermediates have been characterized in several thiolate-ligated heme proteins that are known to catalyze C-H bond activation. The basicity of the ferryl intermediates in these species has been proposed to play a critical role in facilitating this chemistry, allowing hydrogen abstraction at reduction potentials below those that would otherwise lead to oxidative degradation of the enzyme. In this contribution, we discuss the events that led to the assignment and characterization of the unusual iron(IV)hydroxide species, highlighting experiments that provided a quantitative measure of the ferryl basicity, the iron(IV)hydroxide pKa. We then turn to the importance of the iron(IV)hydroxide state, presenting a new way of looking at the role of thiolate ligation in these systems.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Compuestos de Sulfhidrilo/química , Sistema Enzimático del Citocromo P-450/química , Compuestos Férricos/química , Compuestos Férricos/metabolismo , Compuestos de Sulfhidrilo/metabolismoRESUMEN
We report on the protonation state of Helicobacter pylori catalase compound II. UV/visible, Mössbauer, and X-ray absorption spectroscopies have been used to examine the intermediate from pH 5 to 14. We have determined that HPC-II exists in an iron(IV) hydroxide state up to pH 11. Above this pH, the iron(IV) hydroxide complex transitions to a new species (pKa = 13.1) with Mössbauer parameters that are indicative of an iron(IV)-oxo intermediate. Recently, we discussed a role for an elevated compound II pKa in diminishing the compound I reduction potential. This has the effect of shifting the thermodynamic landscape toward the two-electron chemistry that is critical for catalase function. In catalase, a diminished potential would increase the selectivity for peroxide disproportionation over off-pathway one-electron chemistry, reducing the buildup of the inactive compound II state and reducing the need for energetically expensive electron donor molecules.
Asunto(s)
Catalasa/química , Helicobacter pylori/enzimología , Hidróxidos/química , Compuestos de Hierro/química , Sitios de Unión , Catalasa/metabolismo , Concentración de Iones de Hidrógeno , Hidróxidos/metabolismo , Compuestos de Hierro/metabolismo , Estructura Molecular , Espectrofotometría Ultravioleta , Espectroscopía de Mossbauer , Espectroscopía de Absorción de Rayos XRESUMEN
High-valent Fe-OH species are often invoked as key intermediates but have only been observed in Compound II of cytochrome P450s. To further address the properties of non-heme FeIV-OH complexes, we demonstrate the reversible protonation of a synthetic FeIV-oxo species containing a tris-urea tripodal ligand. The same protonated FeIV-oxo species can be prepared via oxidation, suggesting that a putative FeV-oxo species was initially generated. Computational, Mössbauer, XAS, and NRVS studies indicate that protonation of the FeIV-oxo complex most likely occurs on the tripodal ligand, which undergoes a structural change that results in the formation of a new intramolecular H-bond with the oxido ligand that aids in stabilizing the protonated adduct. We suggest that similar protonated high-valent Fe-oxo species may occur in the active sites of proteins. This finding further argues for caution when assigning unverified high-valent Fe-OH species to mechanisms.
RESUMEN
The iron(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) oxygenases catalyze an array of challenging transformations, but how individual members of the enzyme family direct different outcomes is poorly understood. The Fe/2OG halogenase, SyrB2, chlorinates C4 of its native substrate, l-threonine appended to the carrier protein, SyrB1, but hydroxylates C5 of l-norvaline and, to a lesser extent, C4 of l-aminobutyric acid when SyrB1 presents these non-native amino acids. To test the hypothesis that positioning of the targeted carbon dictates the outcome, we defined the positions of these three substrates by measuring hyperfine couplings between substrate deuterium atoms and the stable, EPR-active iron-nitrosyl adduct, a surrogate for reaction intermediates. The Fe-(2)H distances and N-Fe-(2)H angles, which vary from 4.2 Å and 85° for threonine to 3.4 Å and 65° for norvaline, rationalize the trends in reactivity. This experimental correlation of position to outcome should aid in judging from structural data on other Fe/2OG enzymes whether they suppress hydroxylation or form hydroxylated intermediates on the pathways to other outcomes.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Pseudomonas syringae/enzimología , Conformación Molecular , Especificidad por SustratoRESUMEN
Cfr-dependent methylation of C8 of A2503 in 23S ribosomal RNA confers bacterial resistance to an array of clinically important antibiotics that target the large subunit of the ribosome, including the synthetic oxazolidinone antibiotic linezolid. The key element of the proposed mechanism for Cfr, a radical S-adenosylmethionine enzyme, is the addition of a methylene radical, generated by hydrogen-atom abstraction from the methyl group of an S-methylated cysteine, onto C8 of A2503 to form a protein-nucleic acid crosslinked species containing an unpaired electron. Herein we use continuous-wave and pulsed EPR techniques to provide direct spectroscopic evidence for this intermediate, showing a spin-delocalized radical with maximum spin density at N7 of the adenine ring. In addition, we use rapid freeze-quench EPR to show that the radical forms and decays with rate constants that are consistent with the rate of formation of the methylated product.
Asunto(s)
Proteínas Bacterianas/química , Farmacorresistencia Microbiana , ARN Ribosómico 23S/química , Staphylococcus aureus/química , Catálisis , Campos Electromagnéticos , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli/metabolismo , Radicales Libres , Hidrógeno/química , Modelos Químicos , Ácidos Nucleicos/química , EspectrofotometríaRESUMEN
Recently, we reported the spectroscopic and kinetic characterizations of cytochrome P450 compound I in CYP119A1, effectively closing the catalytic cycle of cytochrome P450-mediated hydroxylations. In this minireview, we focus on the developments that made this breakthrough possible. We examine the importance of enzyme purification in the quest for reactive intermediates and report the preparation of compound I in a second P450 (P450ST). In an effort to bring clarity to the field, we also examine the validity of controversial reports claiming the production of P450 compound I through the use of peroxynitrite and laser flash photolysis.
Asunto(s)
Proteínas Arqueales/química , Sistema Enzimático del Citocromo P-450/química , Proteínas Arqueales/aislamiento & purificación , Biocatálisis , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Humanos , Hidroxilación , Hierro/química , Hierro/aislamiento & purificación , Oxidación-Reducción , TermodinámicaRESUMEN
To provide insight into the iron(IV)hydroxide pK(a) of histidine ligated heme proteins, we have probed the active site of myoglobin compound II over the pH range of 3.9-9.5, using EXAFS, Mössbauer, and resonance Raman spectroscopies. We find no indication of ferryl protonation over this pH range, allowing us to set an upper limit of 2.7 on the iron(IV)hydroxide pK(a) in myoglobin. Together with the recent determination of an iron(IV)hydroxide pK(a) â¼ 12 in the thiolate-ligated heme enzyme cytochrome P450, this result provides insight into Nature's ability to tune catalytic function through its choice of axial ligand.
Asunto(s)
Histidina/química , Hidróxidos/química , Hierro/química , Mioglobina/química , Catálisis , Dominio Catalítico , Concentración de Iones de Hidrógeno , Ligandos , Estructura Molecular , Espectroscopía de Mossbauer , Espectrometría Raman , Espectroscopía de Absorción de Rayos XRESUMEN
RlmN and Cfr are methyltransferases/methylsynthases that belong to the radical S-adenosylmethionine superfamily of enzymes. RlmN catalyzes C2 methylation of adenosine 2503 (A2503) of 23S rRNA, while Cfr catalyzes C8 methylation of the exact same nucleotide, and will subsequently catalyze C2 methylation if the site is unmethylated. A key feature of the unusual mechanisms of catalysis proposed for these enzymes is the attack of a methylene radical, derived from a methylcysteine residue, onto the carbon center undergoing methylation to generate a paramagnetic protein-nucleic acid cross-linked species. This species has been thoroughly characterized during Cfr-dependent C8 methylation, but does not accumulate to detectible levels in RlmN-dependent C2 methylation. Herein, we show that inactive C118S/A variants of RlmN accumulate a substrate-derived paramagnetic species. Characterization of this species by electron paramagnetic resonance spectroscopy in concert with strategic isotopic labeling shows that the radical is delocalized throughout the adenine ring of A2503, although predominant spin density is on N1 and N3. Moreover, (13)C hyperfine interactions between the radical and the methylene carbon of the formerly [methyl-(13)C]Cys355 residue show that the radical species exists in a covalent cross-link between the protein and the nucleic acid substrate. X-ray structures of RlmN C118A show that, in the presence of SAM, the substitution does not alter the active site structure compared to that of the wild-type enzyme. Together, these findings have new mechanistic implications for the role(s) of C118 and its counterpart in Cfr (C105) in catalysis, and suggest involvement of the residue in resolution of the cross-linked species via a radical mediated process.
Asunto(s)
Reactivos de Enlaces Cruzados/química , Proteínas de Escherichia coli/química , Metiltransferasas/química , ARN Ribosómico 23S/química , Adenosina/química , Biocatálisis , Cristalización , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Metilación , Modelos Moleculares , Conformación Proteica , Especificidad por SustratoRESUMEN
Addition of anionic donors to the manganese(V)-oxo corrolazine complex Mn(V)(O)(TBP8Cz) has a dramatic influence on oxygen-atom transfer (OAT) reactivity with thioether substrates. The six-coordinate anionic [Mn(V)(O)(TBP8Cz)(X)](-) complexes (X = F(-), N3(-), OCN(-)) exhibit a â¼5 cm(-1) downshift of the Mn-O vibrational mode relative to the parent Mn(V)(O)(TBP8Cz) complex as seen by resonance Raman spectroscopy. Product analysis shows that the oxidation of thioether substrates gives sulfoxide product, consistent with single OAT. A wide range of OAT reactivity is seen for the different axial ligands, with the following trend determined from a comparison of their second-order rate constants for sulfoxidation: five-coordinate ≈ thiocyanate ≈ nitrate < cyanate < azide < fluoride ⪠cyanide. This trend correlates with DFT calculations on the binding of the axial donors to the parent Mn(V)(O)(TBP8Cz) complex. A Hammett study was performed with p-X-C6H4SCH3 derivatives and [Mn(V)(O)(TBP8Cz)(X)](-) (X = CN(-) or F(-)) as the oxidant, and unusual "V-shaped" Hammett plots were obtained. These results are rationalized based upon a change in mechanism that hinges on the ability of the [Mn(V)(O)(TBP8Cz)(X)](-) complexes to function as either an electrophilic or weak nucleophilic oxidant depending upon the nature of the para-X substituents. For comparison, the one-electron-oxidized cationic Mn(V)(O)(TBP8Cz(â¢+)) complex yielded a linear Hammett relationship for all substrates (ρ = -1.40), consistent with a straightforward electrophilic mechanism. This study provides new, fundamental insights regarding the influence of axial donors on high-valent Mn(V)(O) porphyrinoid complexes.
Asunto(s)
Manganeso/química , Compuestos Organometálicos/química , Oxígeno/química , Porfirinas/química , Ligandos , Estructura Molecular , Teoría CuánticaRESUMEN
A class Ia ribonucleotide reductase (RNR) employs a µ-oxo-Fe2(III/III)/tyrosyl radical cofactor in its ß 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-ß cofactor, reaction of the protein's Fe2(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(â¢)) 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, 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(â¢).