Dioxygen Binding Research Articles

Trapping an Elusive Fe(IV)-Superoxo Intermediate Inside a Self-Assembled Nanocage in Water at Room Temperature.

Molecular cavities that mimic natural metalloenzymes have shown the potential to trap elusive reaction intermediates. Here, we demonstrate the formation of a rare yet stable Fe(IV)-superoxo intermediate at room temperature subsequent to dioxygen binding at the Fe(III) site of a (Et4N)2[FeIII(Cl)(bTAML)] complex confined inside the hydrophobic interior of a water-soluble Pd6L412+ nanocage. Using a combination of electron paramagnetic resonance, Mössbauer, Raman/IR vibrational, X-ray absorption, and emission spectroscopies, we demonstrate that the cage-encapsulated complex has a Fe(IV) oxidation state characterized by a stable S = 1/2 spin state and a short Fe-O bond distance of ∼1.70 Å. We find that the O2 reaction in confinement is reversible, while the formed Fe(IV)-superoxo complex readily reacts when presented with substrates having weak C-H bonds, highlighting the lability of the O-O bond. We envision that such optimally trapped high-valent superoxos can show new classes of reactivities catalyzing both oxygen atom transfer and C-H bond activation reactions.

The Unique Role of the Second Coordination Sphere to Unlock and Control Catalysis in Nonheme Fe(II)/2-Oxoglutarate Histone Demethylase KDM2A.

Nonheme Fe(II) and 2-oxoglutarate (2OG)-dependent histone lysine demethylases 2A (KDM2A) catalyze the demethylation of the mono- or dimethylated lysine 36 residue in the histone H3 peptide (H3K36me1/me2), which plays a crucial role in epigenetic regulation and can be involved in many cancers. Although the overall catalytic mechanism of KDMs has been studied, how KDM2 catalysis takes place in contrast to other KDMs remains unknown. Understanding such differences is vital for enzyme redesign and can help in enzyme-selective drug design. Herein, we employed molecular dynamics (MD) and combined quantum mechanics/molecular mechanics (QM/MM) to explore the complete catalytic mechanism of KDM2A, including dioxygen diffusion and binding, dioxygen activation, and substrate oxidation. Our study demonstrates that the catalysis of KDM2A is controlled by the conformational change of the second coordination sphere (SCS), specifically by a change in the orientation of Y222, which unlocks the 2OG rearrangement from off-line to in-line mode. The study demonstrates that the variant Y222A makes the 2OG rearrangement more favorable. Furthermore, the study reveals that it is the size of H3K36me3 that prevents the 2OG rearrangement, thus rendering the enzyme inactivity with trimethylated lysine. Calculations show that the SCS and long-range interacting residues that stabilize the HAT transition state in KDM2A differ from those in KDM4A, KDM7B, and KDM6A, thus providing the basics for the enzyme-selective redesign and modulation of KDM2A without influencing other KDMs.

QM/MM Study Into the Mechanism of Oxidative C=C Double Bond Cleavage by Lignostilbene-α,β-Dioxygenase.

The enzymatic biosynthesis of fragrance molecules from lignin fragments is an important reaction in biotechnology for the sustainable production of fine chemicals. In this work we investigated the biosynthesis of vanillin from lignostilbene by a nonheme iron dioxygenase using QM/MM and tested several suggested proposals via either an epoxide or dioxetane intermediate. Binding of dioxygen to the active site of the protein results in the formation of an iron(II)-superoxo species with lignostilbene cation radical. The dioxygenase mechanism starts with electrophilic attack of the terminal oxygen atom of the superoxo group on the central C=C bond of lignostilbene, and the second-coordination sphere effects in the substrate binding pocket guide the reaction towards dioxetane formation. The computed mechanism is rationalized with thermochemical cycles and valence bond schemes that explain the electron transfer processes during the reaction mechanism. Particularly, the polarity of the protein and the local electric field and dipole moments enable a facile electron transfer and an exergonic dioxetane formation pathway.

Oxygen Activation in Aromatic Ring Cleaving Salicylate Dioxygenase: Detection of Reaction Intermediates with a Nitro-substituted Substrate Analog.

Cupin dioxygenases such as salicylate 1,2-dioxygense (SDO) perform aromatic C-C bond scission via a 3-His motif tethered iron cofactor. Here, transient kinetics measurements are used to monitor the catalytic cycle of SDO by using a nitro-substituted substrate analog, 3-nitrogentisate. Compared to the natural substrate, the nitro group reduces the enzymatic kcat by 500-fold, thereby facilitating the detection and kinetic characterization of reaction intermediates. Sums and products of reciprocal relaxation times derived from kinetic measurements were found to be linearly dependent on O2 concentration, suggesting reversible formation of two distinct intermediates. Dioxygen binding to the metal cofactor takes place with a forward rate of 5.9×103 M-1 s-1: two orders of magnitude slower than other comparable ring-cleaving dioxygenses. Optical chromophore of the first intermediate is distinct from the in situ generated SDO Fe(III)-O2⋅- complex but closer to the enzyme-substrate precursor.

Insights into dioxygen binding on metal centers: an ab initio multireference electronic structure analysis.

Why does binding of dioxygen (O2) to metal centers, the initial step of O2 storage, transportation, and activation, almost inevitably induce metal-to-O2 single-electron transfer and generate superoxo (O2-˙) species, instead of genuine O02 adducts? To address this question, this study describes highly correlated wavefunction-based ab initio calculations using CASSCF/NEVPT2 (CASSCF = complete active space self-consistent field, and NEVPT2 = N-electron valence state second-order perturbation theory) approaches to explore the electronic-structure evolution of O2 association on Fe(II)(BDPP) (H2BDPP = 2,6-bis((2-(S)-diphenylhydroxylmethyl-1-pyrrolidinyl)methyl)pyridine) and Co(II)(BDPP) to produce S = 3 Fe(III)(BDPP)(O2-˙) (1) and Co(III)(BDPP)(O2-˙) (2). CASSCF/NEVPT2 calculations suggest that the processes furnishing 1 and 2 feature an avoided crossing resulting from interactions of two diabatic curves, of which one is characterized as Co(II) and Fe(II) centers interacting with a triplet O2 ligand and the other as Co(III) and Fe(III) centers bound to a superoxo ligand. In both cases, the avoided crossing induces a one-electron transfer from the divalent metal center to the incoming O2 ligand and leads to formation of trivalent metal-O2-˙ complexes. To facilitate the interpretation of complicated multireference wavefunctions, we formulated two-fragment spin eigenfunctions utilizing Clebsch-Gordan coefficients (CGCs) to rationalize computed spin populations on the metal centers and the O2 ligand and compared these results with usual valence bonding (VB) analyses. It turns out that both methods give the same results and are complementary to each other. Finally, the limitation of DFT approaches in describing complex electronic structures involving metal-ligand magnetic couplings is delineated.

Reversible dioxygen uptake at [Cu4] clusters.

Dioxygen binding solely through non-covalent interactions is rare. In living systems, dioxygen transport takes place via iron or copper-containing biological cofactors. Specifically, a reversible covalent interaction is established when O2 binds to the mono or polynuclear metal center. However, O2 stabilization in the absence of covalent bond formation is challenging and rarely observed. Here, we demonstrate a unique example of reversible non-covalent binding of dioxygen within the cavity of a well-defined synthetic all-Cu(i) tetracopper cluster.

Oxygen reduction catalysis by [Cu(cyclen)Cl]Cl·H2O: The relationship of selectivity to proton in nonaqueous solution and aqueous solution

The reaction of 1,4,7,10‐tetraazacyclododecane (cyclen) with CuCl2·2H2O results in a novel five‐coordinated mononuclear copper complex, [Cu(cyclen)Cl]Cl·H2O, which has been characterized using physicochemical and spectroscopic methods. This copper complex can serve as a catalyst for oxygen reduction reactions (ORRs). The selectivity for 4e−/4H+ reduction to H2O νersus 2e−/2H+ reduction to H2O2 is determined by the relationship of the pKa value of the proton donor in nonaqueous solutions. The dissociation of Cl− from [Cu(cyclen)Cl]Cl·H2O creates an empty position on the copper centre, enabling to binding of dioxygen (O2) to form Cu(II)‐superoxo intermediate, which further reacts with a strong acid as a proton donor to form H2O and with a weak acid as a proton donor to form H2O2. Moreover, the faradaic efficiency for H2O2 production increases in an aqueous environment with the increase of pH.

Modulation of the Band Gap and Redox Properties by Mixed Addenda in Sandwich Polyoxometalates.

Due to the wide range of applications of band-gap engineering in optoelectronics and photocatalysis, the rational design of polyoxometalate (POM) frameworks is highly desired. Here, we have successfully synthesized a series of mixed addenda (Mo and W) sandwich POMs by systematically varying pH, concentrations of salts, and counterions in Weakley-type sandwich POMs by incorporating Mo into the framework of tetrasubstituted sandwich POMs. Crystallographic analysis reveals the centrosymmetric structure; with variation in the Mo to W ratio, Mo preferentially binds to μ2 oxygen connected to transition metals in the sandwich position. UV-visible spectroscopy, electrochemical, and theoretical modeling rationalize the band-gap modulations. Theoretical studies and cyclic voltammograms indicate that during the reduction, the incoming electrons preferentially go to substituted transition metals followed by Mo. Flat band potential calculated from the Mott-Schottky enables tuning of the electronic properties of composites based on these sandwich POMs. Moreover, the dioxygen binding and activation studies of these polyoxometalates have been highlighted.

Detection of a Geminate Photoproduct of Bovine Cytochrome c Oxidase by Time-Resolved Serial Femtosecond Crystallography.

Cytochrome c oxidase (CcO) is a large membrane-bound hemeprotein that catalyzes the reduction of dioxygen to water. Unlike classical dioxygen binding hemeproteins with a heme b group in their active sites, CcO has a unique binuclear center (BNC) composed of a copper atom (CuB) and a heme a3 iron, where O2 binds and is reduced to water. CO is a versatile O2 surrogate in ligand binding and escape reactions. Previous time-resolved spectroscopic studies of the CO complexes of bovine CcO (bCcO) revealed that photolyzing CO from the heme a3 iron leads to a metastable intermediate (CuB-CO), where CO is bound to CuB, before it escapes out of the BNC. Here, with a pump-probe based time-resolved serial femtosecond X-ray crystallography, we detected a geminate photoproduct of the bCcO-CO complex, where CO is dissociated from the heme a3 iron and moved to a temporary binding site midway between the CuB and the heme a3 iron, while the locations of the two metal centers and the conformation of Helix-X, housing the proximal histidine ligand of the heme a3 iron, remain in the CO complex state. This new structure, combined with other reported structures of bCcO, allows for a clearer definition of the ligand dissociation trajectory as well as the associated protein dynamics.

Physico- and Electrochemical Properties of First-Row Transition-Metal-Substituted Sandwich Polyoxometalates.

The physico- and electrochemical behaviors of a series of [WZn3(H2O)2(ZnW9O34)2]12- (Zn-WZn3) and its first-row transition-metal-substituted analogues [WZn(TM)2(H2O)2(ZnW9O34)2]12- (Zn-WZn(TM)2; TM = MnII, CoII, FeIII, NiII and CuII) are reported. Various spectroscopic studies, including Fourier transform infrared (FTIR) spectroscopy, UV-visible spectroscopy, electrospray ionization (ESI)-mass spectrometry, and Raman spectroscopy, show similar spectral patterns in all sandwich polyoxometalates (POMs) because of their isostructural geometry and constancy of the overall negative charge (-12). However, the electronic properties highly depend on the transition metals at the "sandwich core" and correlate well with the density functional theory (DFT) study. Further, depending on the substituted TM atoms, there is a decrease in the highest occupied molecular orbital-lowest unoccupied molecular orbital (HOMO-LUMO) band-gap energy in these transition-metal-substituted POM (TMSP) complexes wrt Zn-WZn3, as confirmed by diffuse reflectance spectroscopy and DFT study. Cyclic voltammetry reveals that the electrochemistry of these sandwich POMs (Zn-WZn3 and TMSPs) is highly dependent on the pH of the solution. Moreover, the dioxygen binding/activation studies of these polyoxometalates show that Zn-WZn3 and Zn-WZnFe2 have better efficiency toward dioxygen binding, as confirmed by FTIR spectroscopy, Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), and thermogravimetric analysis (TGA), which is also reflected in their catalytic activity toward imine synthesis.

Dioxygen Binding Is Controlled by the Protein Environment in Non-heme FeII and 2-Oxoglutarate Oxygenases: A Study on Histone Demethylase PHF8 and an Ethylene-Forming Enzyme.

Invited for the cover of this issue are Christo Z. Christov and co-workers at Michigan Technological University, University of Oxford, and Michigan State University. The image depicts the oxygen diffusion channel in class 7 histone demethylase (PHF8) and ethylene-forming enzyme (EFE) and changes in the enzymes' conformations upon binding. Read the full text of the article at 10.1002/chem.202300138.

Front Cover: Dioxygen Binding Is Controlled by the Protein Environment in Non‐heme FeII and 2‐Oxoglutarate Oxygenases: A Study on Histone Demethylase PHF8 and an Ethylene‐Forming Enzyme (Chem. Eur. J. 24/2023)

The cover shows the oxygen diffusion channel in class 7 histone demethylase (PHF8) and ethylene-forming enzyme (EFE). PHF8 catalyzes the hydroxylation of its H3 K9me2 histone substrate, and EFE catalyzes two competing reactions of ethylene generation and substrate l-Arg hydroxylation. Although both enzymes initially have an off-line 2-oxoglutarate (2OG) conformation, upon oxygen binding, PHF8 forms an in-line superoxo complex, whereas EFE uses the off-line superoxo complex. More information can be found in the Research Article by C. Z. Christov and co-workers. (DOI: 10.1002/chem.202300138).

QM Calculations Revealed that Outer-Sphere Electron Transfer Boosted O-O Bond Cleavage in the Multiheme-Dependent Cytochrome bd Oxygen Reductase.

The cytochrome bd oxygen reductase catalyzes the four-electron reduction of dioxygen to two water molecules. The structure of this enzyme reveals three heme molecules in the active site, which differs from that of heme-copper cytochrome c oxidase. The quantum chemical cluster approach was used to uncover the reaction mechanism of this intriguing metalloenzyme. The calculations suggested that a proton-coupled electron transfer reduction occurs first to generate a ferrous heme b595. This is followed by the dioxygen binding at the heme d center coupled with an outer-sphere electron transfer from the ferrous heme b595 to the dioxygen moiety, affording a ferric ion superoxide intermediate. A second proton-coupled electron transfer produces a heme d ferric hydroperoxide, which undergoes efficient O-O bond cleavage facilitated by an outer-sphere electron transfer from the ferrous heme b595 to the O-O σ* orbital and an inner-sphere proton transfer from the heme d hydroxyl group to the leaving hydroxide. The synergistic benefits of the two types of hemes rationalize the highly efficient oxygen reduction repertoire for the multi-heme-dependent cytochrome bd oxygen reductase family.

Dioxygen Binding Is Controlled by the Protein Environment in Non-heme FeII and 2-Oxoglutarate Oxygenases: A Study on Histone Demethylase PHF8 and an Ethylene-Forming Enzyme.

This study investigates dioxygen binding and 2-oxoglutarate (2OG) coordination by two model non-heme FeII /2OG enzymes: a class 7 histone demethylase (PHF8) that catalyzes the hydroxylation of its H3K9me2 histone substrate leading to demethylation reactivity and the ethylene-forming enzyme (EFE), which catalyzes two competing reactions of ethylene generation and substrate l-Arg hydroxylation. Although both enzymes initially bind 2OG by using an off-line 2OG coordination mode, in PHF8, the substrate oxidation requires a transition to an in-line mode, whereas EFE is catalytically productive for ethylene production from 2OG in the off-line mode. We used classical molecular dynamics (MD), quantum mechanics/molecular mechanics (QM/MM) MD and QM/MM metadynamics (QM/MM-MetD) simulations to reveal that it is the dioxygen binding process and, ultimately, the protein environment that control the formation of the in-line FeIII -OO⋅- intermediate in PHF8 and the off-line FeIII -OO⋅- intermediate in EFE.

Mechanistic Insight into Peptidyl-Cysteine Oxidation by the Copper-Dependent Formylglycine-Generating Enzyme.

The copper-dependent formylglycine-generating enzyme (FGE) catalyzes the oxygen-dependent oxidation of specific peptidyl-cysteine residues to formylglycine. Our QM/MM calculations provide a very likely mechanism for this transformation. The reaction starts with dioxygen binding to the tris-thiolate CuI center to form a triplet CuII -superoxide complex. The rate-determining hydrogen atom abstraction involves a triplet-singlet crossing to form a CuII -OOH species that couples with the substrate radical, leading to a CuI -alkylperoxo intermediate. This is accompanied by proton transfer from the hydroperoxide to the S atom of the substrate via a nearby water molecule. The subsequent O-O bond cleavage is coupled with the C-S bond breaking that generates the formylglycine and a CuII -oxyl complex. Moreover, our results suggest that the aldehyde oxygen of the final product originates from O2 , which will be useful for future experimental work.

Mechanistic Insight into Peptidyl‐Cysteine Oxidation by the Copper‐Dependent Formylglycine‐Generating Enzyme

AbstractThe copper‐dependent formylglycine‐generating enzyme (FGE) catalyzes the oxygen‐dependent oxidation of specific peptidyl‐cysteine residues to formylglycine. Our QM/MM calculations provide a very likely mechanism for this transformation. The reaction starts with dioxygen binding to the tris‐thiolate CuI center to form a triplet CuII‐superoxide complex. The rate‐determining hydrogen atom abstraction involves a triplet‐singlet crossing to form a CuII−OOH species that couples with the substrate radical, leading to a CuI‐alkylperoxo intermediate. This is accompanied by proton transfer from the hydroperoxide to the S atom of the substrate via a nearby water molecule. The subsequent O−O bond cleavage is coupled with the C−S bond breaking that generates the formylglycine and a CuII‐oxyl complex. Moreover, our results suggest that the aldehyde oxygen of the final product originates from O2, which will be useful for future experimental work.

Reversible Dioxygen Binding to Co(II) Complexes with Noninnocent Ligands.

A series of mononuclear Co(II) complexes with noninnocent (redox-active) ligands are prepared that exhibit metal-ligand cooperativity during the reversible binding of O2. The complexes have the general formula, [CoII(LS,N)(TpR2)] (R = Me, Ph), where LS,N is a bidentate o-aminothiophenolate and TpR2 is a hydrotris(pyrazol-1-yl)borate scorpionate with R-substituents at the 3- and 5-positions. Exposure to O2 at room temperature results in one-electron oxidation and deprotonation of LS,N. The oxidized derivatives possess substantial "singlet diradical" character arising from antiferromagnetic coupling between an iminothiosemiquinonate (ITSQ•-) ligand radical and a low-spin Co(II) ion. The [CoII(TpMe2)(X2ITSQ)] complexes, where X = H or tBu, coordinate O2 reversibly at reduced temperatures to provide Co/O2 adducts. The O2 binding reactions closely resemble those previously reported by our group (Kumar et al., J. Am. Chem. Soc. 2019,141, 10984-10987) for the related complexes [CoII(TpMe2)(tBu2SQ)] and [CoII(TpMe2)(tBu2ISQ)], where tBu2(I)SQ represents 4,6-di-tert-butyl-(2-imino)semiquinonate radicals. In each case, the oxygenation reaction proceeds via the addition of O2 to both the cobalt ion and the ligand radical, generating metallocyclic cobalt(III)-alkylperoxo structures. Thermodynamic measurements elucidate the relationship between O2 affinity and redox potentials of the (imino)(thio)semiquinonate radicals, as well as energetic differences between these reactions and conventional metal-based oxygenations. The results highlight the utility and versatility of noninnocent ligands in the design of O2-absorbing compounds.

Oxygenation of copper(I) complexes containing fluorine tagged tripodal tetradentate chelates: significant ligand electronic effects

Copper-dioxygen (O2) interactions are of great importance in biological and chemical transformations involving reversible dioxygen binding, activation, or reduction. In this report, we describe O2-reactions with the mononuclear copper(I) complexes containing two new analogues of the known nitrogen-containing tetradentate tripodal chelate, tris[(2-pyridyl)methyl]amine (TMPA). In both derivatives, one electron-rich and one electron-deficient, fluorine atoms are attached to the ligand framework, allowing for the use of 19F-NMR spectroscopy to probe the oxygenation process. Variations of ligand electronic properties are manifested in the electrochemical behavior of copper complexes and their reactivities toward O2. Our NMR spectroscopic studies, along with variable-temperature electronic absorption measurements, revealed that the copper(I) complexes reversibly react with O2 to form the corresponding 1:1 copper-O2 (i.e., end-on superoxo) intermediates which can further react reversibly with second equivalents of copper(I) complexes to form the related dinuclear 2:1 copper-O2 (i.e., trans-peroxo) adducts. However, considerable differences exist in detail at various temperatures, depending on the chelate. All three end-on superoxo and trans-peroxo species described here possess similar spectroscopic features, although small but significant shifts in the energy of their signature bands were observed, suggesting that the variation in the chelates directly affects the electronic properties of the copper-O2 cores.

Oxygen Binding by Co(II) Complexes with Oxime-Containing Schiff Bases in Solution

The present work describes the complexation properties of two oxime-containing Schiff bases (used as ligands), viz. 2-hydroxyimino-N′-[1-(2-pyridyl)ethylidene]propanohydrazone (Hpop) and 2-hydroxyimino-N′-[(pyridine-2-yl)methylidene]propanohydrazone (Hpoa), with Co(II) ions in DMSO/water solution. Volumetric (oxygenation) studies were carried out to determine the uptake of molecular oxygen O2 in the formation of the complexes Co(II)-Hpop and Co(II)-Hpoa. The acquired data can be useful in the development of oxygen bioinorganic complexes of metal ions with Schiff base ligands in solution. Their properties allow them to be used as synthetic oxygen transporters. Moreover, the binding of dioxygen could play an important role in the research of catalytic activity by such systems.

Electronic Structure and Reactivity of Dioxygen-Derived Aliphatic Thiolate-Ligated Fe-Peroxo and Fe(IV) Oxo Compounds.

Herein, we examine the electronic and geometric structural properties of O2-derived aliphatic thiolate-ligated Fe-peroxo, Fe-hydroxo, and Fe(IV) oxo compounds. The latter cleaves strong C-H bonds (96 kcal mol-1) on par with the valine C-H bond cleaved by isopencillin N synthase (IPNS). Stopped-flow kinetics studies indicate that the barrier to O2 binding to [FeII(SMe2N4(tren))]+ (3) is extremely low (Ea = 36(2) kJ mol-1), as theoretically predicted for IPNS. Dioxygen binding to 3 is shown to be reversible, and a superoxo intermediate, [FeIII(SMe2N4(tren))(O2)]+ (6), forms in the first 25 ms of the reaction at -40 °C prior to the rate-determining (Ea = 46(2) kJ mol-1) formation of peroxo-bridged [(SMe2N4(tren))Fe(III)]2(μ-O2)2+ (7). A log(kobs) vs log([Fe]) plot for the formation of 7 is consistent with the second-order dependence on iron, and H2O2 assays are consistent with a 2:1 ratio of Fe/H2O2. Peroxo 7 is shown to convert to ferric-hydroxo [FeIII(SMe2N(tren))(OH)]+ (9, g⊥ = 2.24, g∥ = 1.96), the identity of which was determined via its independent synthesis. Rates of the conversion 7 → 9 are shown to be dependent on the X-H bond strength of the H-atom donor, with a kH/kD = 4 when CD3OD is used in place of CH3OH as a solvent. A crystallographically characterized cis thiolate-ligated high-valent iron oxo, [FeIV(O)(SMe2N4(tren))]+ (11), is shown to form en route to hydroxo 9. Electronic structure calculations were shown to be consistent with 11 being an S = 1 Fe(IV)═O with an unusually high νFe-O stretching frequency at 918 cm-1 in line with the extremely short Fe-O bond (1.603(7) Å).