Conspectus Mononuclear non-heme Fe (NHFe) enzymes play key roles in antibiotic biosynthesis, hypoxic response, DNA repair, anticancer therapy and many other biological processes. On a molecular level these enzymes catalyze a diverse range of oxidation reactions, including hydroxylation, halogenation, ring closure, desaturation and electrophilic aromatic substitution (EAS). Most of these enzymes use an FeII site to activate dioxygen. These ferrous active sites had been inaccessible to traditional spectroscopic methods. A methodology has been developed that provides detailed geometric and electronic structure insight for these NHFeII active sites. This has defined a general mechanistic strategy utilized by a wide range of these enzymes to control O2 activation by FeII coordination unsaturation only in the presence of cosubstrates to limit autooxidation and self-hydroxylation. Depending on the type of enzyme, O2 activation either involves a 2e− reduced FeIII–OOH intermediate or a 4e− reduced FeIV=O intermediate. The nature of these intermediates has been defined in terms of geometric structure using nuclear resonance vibrational spectroscopy (NRVS) and electronic structure using magnetic circular dichroism (MCD) to define the frontier molecular orbitals (FMOs) that control reactivity. For FeIII–OOH intermediates the anticancer drug Activated Bleomycin is shown to be the non-heme Fe analog of compound 0 in heme (e.g. P450) chemistry but undergoes different reactivity where the low-spin (LS) FeIII–OOH can directly abstract an H atom from DNA. It is also shown that the transition states of LS and high-spin (HS) FeIII–OOH are fundamentally different in that the former goes through a hydroxyl radical while the latter is activated for EAS without O-O cleavage, which is important in one class of NHFe enzymes that utilizes a HS FeIII–OOH intermediate in dioxygenation. For FeIV=O intermediates the LS form is shown to have a π-type FMO activated for attack perpendicular to the Fe–O bond while the HS form (present in the NHFe enzymes) has both π and σ FMOs that are activated for attack both perpendicular to and along the Fe–O bond, respectively. For the NHFe enzymes these π vs σ FMOs direct reactivity for EAS vs H-atom abstraction, and for the latter halogenation vs hydroxylation. This study emphasizes that experimental spectroscopy is critical in evaluating the results of electronic structure calculations and thus key to bridging structure and reactivity with mechanism.

The alpha-ketoglutarate (alpha-KG)-dependent oxygenases are a large and diverse class of mononuclear non-heme iron enzymes that require FeII, alpha-KG, and dioxygen for catalysis with the alpha-KG cosubstrate supplying the additional reducing equivalents for oxygen activation. While these systems exhibit a diverse array of reactivities (i.e., hydroxylation, desaturation, ring closure, etc.), they all share a common structural motif at the FeII active site, termed the 2-His-1-carboxylate facial triad. Recently, a new subclass of alpha-KG-dependent oxygenases has been identified that exhibits novel reactivity, the oxidative halogenation of unactivated carbon centers. These enzymes are also structurally unique in that they do not contain the standard facial triad, as a Cl- ligand is coordinated in place of the carboxylate. An FeII methodology involving CD, MCD, and VTVH MCD spectroscopies was applied to CytC3 to elucidate the active-site structural effects of this perturbation of the coordination sphere. A significant decrease in the affinity of FeII for apo-CytC3 was observed, supporting the necessity of the facial triad for iron coordination to form the resting site. In addition, interesting differences observed in the FeII/alpha-KG complex relative to the cognate complex in other alpha-KG-dependent oxygenases indicate the presence of a distorted 6C site with a weak water ligand. Combined with parallel studies of taurine dioxygenase and past studies of clavaminate synthase, these results define a role of the carboxylate ligand of the facial triad in stabilizing water coordination via a H-bonding interaction between the noncoordinating oxygen of the carboxylate and the coordinated water. These studies provide initial insight into the active-site features that favor chlorination by CytC3 over the hydroxylation reactions occurring in related enzymes.

Deployment of solar fuels derived from water requires robust oxygen-evolving catalysts made from earth abundant materials. Copper has recently received much attention in this regard. Mechanistic parallels between Cu and single-site Ru/Ir/Mn water oxidation catalysts, including intermediacy of terminal Cu oxo/oxyl species, are prevalent in the literature; however, intermediacy of late transition metal oxo species would be remarkable given the high d-electron count would fill antibonding orbitals, making these species high in energy. This may suggest alternate pathways are at work in copper-based water oxidation. This report characterizes a dinuclear copper water oxidation catalyst, {[(L)Cu(II)]-(μ-OH)}(OTf) (L = MeTMPA = bis((6-methyl-2-pyridyl)methyl)(2-pyridylmethyl)amine) in which water oxidation proceeds with high Faradaic efficiency (>90%) and moderate rates (33 s at ∼1 V overpotential, pH 12.5). A large kinetic isotope effect (k/k = 20) suggests proton coupled electron transfer in the initial oxidation as the rate-determining step. This species partially dissociates in aqueous solution at pH 12.5 to generate a mononuclear {[(L)Cu(II)(OH)]} adduct (K = 0.0041). Calculations that reproduce the experimental findings reveal that oxidation of either the mononuclear or dinuclear species results in a common dinuclear intermediate, {[LCu(III)]-(μ-O)}, which avoids formation of terminal Cu(IV)═O/Cu(III)-O intermediates. Calculations further reveal that both intermolecular water nucleophilic attack and redox isomerization of {[LCu(III)]-(μ-O)} are energetically accessible pathways for O-O bond formation. The consequences of these findings are discussed in relation to differences in water oxidation pathways between Cu catalysts and catalysts based on Ru, Ir, and Mn.

Factor Inhibiting Hypoxia–Inducible Factor (FIH) is an α-ketoglutarate (αKG) dependent enzyme which catalyzes hydroxylation of residue Asn803 in the C-terminal transactivation domain (CAD) of hypoxia–inducible factor 1α (HIF-1α) and plays an important role in cellular oxygen sensing and hypoxic response. Circular dichroism (CD), magnetic circular dichroism (MCD) and variable–temperature, variable–field (VTVH) MCD spectroscopies are used to determine the geometric and electronic structures of FIH in its (FeII), (FeII/αKG) and (FeII/αKG/CAD) forms. (FeII)FIH and (FeII/αKG)FIH are found to be six-coordinate (6C), whereas (FeII/αKG/CAD)FIH is found to be a 5C/6C mixture. Thus, FIH follows the general mechanistic strategy of nonheme FeII enzymes. Modeling shows that when Arg238 of FIH is removed the facial triad carboxylate binds to FeII in a bidentate mode with concomitant lengthening of the FeII–αKG-carbonyl bond, which would inhibit the O2 reaction. Correlations over α-keto acid-dependent enzymes and with the extradiol dioxygenases shows that members of these families (where both the electron source and O2 bind to FeII) have a second-sphere residue H-bonding to the terminal oxygen of the carboxylate, which stays monodentate. Alternatively, structures of the pterin-dependent and Rieske dioxygenases, which do not have substrate binding to FeII, lack H-bonds to the carboxylate, and thus allow its bidentate coordination which would direct O2 reactivity. Finally, Vis-UV MCD spectra show an unusually high-energy FeII→αKG π* metal-to-ligand charge transfer transition in (FeII/αKG)FIH which is red-shifted upon CAD binding. This red shift indicates formation of H bonds to the αKG that lower the energy of its carbonyl LUMO, activating it for nucleophilic attack by the Fe-O2 intermediate formed along the reaction coordinate.

Nitrile hydratases (NHases) are mononuclear nonheme enzymes that catalyze the hydration of nitriles to amides. NHase is unusual in that it utilizes a low-spin (LS) FeIII center and a unique ligand set comprised of two deprotonated backbone amides, cysteine-based sulfenic acid (RSO(H)) and sulfinic acid (RSO2−), and an unmodified cysteine trans to an exogenous ligand site. Electron paramagnetic resonance (EPR), magnetic circular dichroism (MCD) and low-temperature absorption (LT-Abs) spectroscopies are used to determine the geometric and electronic structures of butyrate-bound (NHaseBA) and active (NHaseAq) NHase. These data calibrate DFT models, which are then extended to explore the mechanism of nitrile hydration by NHase. In particular, the nitrile is activated by coordination to the LS FeIII and the sulfenate group is found to be deprotonated and a significantly better nucleophile than water that can attack the coordinated nitrile to form a cyclic species. Attack at the sulfenate S atom of the cyclic species is favorable and leads to a lower kinetic barrier than attack by water on coordinated, uncyclized nitrile, while attack at the C of the cyclic species is unfavorable. The roles of the unique ligand set and low-spin nature of the NHase active site in function are also explored. It is found that the oxidized thiolate ligands are crucial to maintaining the LS state, which is important in the binding and activation of nitrile susbtrates. The dominant role of the backbone amidate ligands appears to be as a chelate in keeping the sulfenate properly oriented for nucleophilic attack on the coordinated substrate.

The mono-μ-hydroxo complex {[Cu(tmpa)]-(μ-OH)} (1) can undergo reversible deprotonation at -30 °C to yield {[Cu(tmpa)]-(μ-O)} (2). This species is basic with a pK of 24.3. 2 is competent for concerted proton-electron transfer from TEMPOH, but is an intrinsically poor hydrogen atom abstractor (BDFE(OH) of 77.2 kcal/mol) based on kinetic and thermodynamic analyses. Nonetheless, DFT calculations experimentally calibrated against 2 reveal that [CuO] is likely thermodynamically viable in copper-dependent methane monoxygenase enzymes.

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Mononuclear nonheme Fe(II) (MNH) and α-ketoglutarate (α-KG) dependent halogenases activate O2 to perform oxidative halogenations of activated and nonactivated carbon centers. While the mechanism of halide incorporation into a substrate has been investigated, the mechanism by which halogenases prevent oxidations in the absence of chloride is still obscure. Here, we characterize the impact of chloride on the metal center coordination and reactivity of the fatty acyl-halogenase HctB. Stopped-flow kinetic studies show that the oxidative transformation of the Fe(II)-α-KG-enzyme complex is >200-fold accelerated by saturating concentrations of chloride in both the absence and presence of a covalently bound substrate. By contrast, the presence of substrate, which generally brings about O2 activation at enzymatic MNH centers, only has an ∼10-fold effect in the absence of chloride. Circular dichroism (CD) and magnetic CD (MCD) studies demonstrate that chloride binding triggers changes in the metal center ligation: chloride binding induces the proper binding of the substrate as shown by variable-temperature, variable-field (VTVH) MCD studies of non-α-KG-containing forms and the conversion from six-coordinate (6C) to 5C/6C mixtures when α-KG is bound. In the presence of substrate, a site with square pyramidal five-coordinate (5C) geometry is observed, which is required for O2 activation at enzymatic MNH centers. In the absence of substrate an unusual trigonal bipyramidal site is formed, which accounts for the observed slow, uncoupled reactivity. Molecular dynamics simulations suggest that the binding of chloride to the metal center of HctB leads to a conformational change in the enzyme that makes the active site more accessible to the substrate and thus facilitates the formation of the catalytically competent enzyme–substrate complex. Results are discussed in relation to other MNH dependent halogenases.