Synthesis, Structure and Reactivity of a Mononuclear N,N,O-Bound Fe(II) α-Keto-Acid Complex.

A bulky, tridentate phenolate ligand (ImPh2 NNOtBu ) was used to synthesise the first example of a mononuclear, facial, N,N,O-bound iron(II) benzoylformate complex, [Fe(ImPh2 NNOtBu )(BF)] (2). The X-ray crystal structure of 2 reveals that the iron centre is pentacoordinate (τ=0.5), with a vacant site located cis to the bidentate BF ligand. The Mössbauer parameters of 2 are consistent with high-spin iron(II), and are very close to those reported for α-ketoglutarate-bound non-heme iron enzyme active sites. According to NMR and UV-vis spectroscopies, the structural integrity of 2 is retained in both coordinating and non-coordinating solvents. Cyclic voltammetry studies show that the iron centre has a very low oxidation potential and is more prone to electrochemical oxidation than the redox-active phenolate ligand. Complex 2 reacts with NO to form a S=3 /2 {FeNO}7 adduct in which NO binds directly to the iron centre, according to EPR, UV-vis, IR spectroscopies and DFT analysis. Upon O2 exposure, 2 undergoes oxidative decarboxylation to form a diiron(III) benzoate complex, [Fe2 (ImPh2 NNOtBu )2 (µ2 -OBz)(µ2 -OH)2 ]+ (3). A small amount of hydroxylated ligand was also observed by ESI-MS, hinting at the formation of a high-valent iron(IV)-oxo intermediate. Initial reactivity studies show that 2 is capable of oxygen atom transfer reactivity with O2 , converting methyl(p-tolyl)sulfide to sulfoxide.

Superoxide Dismutase-like Activity of Zeolitic Imidazolate Framework Nanoparticles Comprising Biomimetic Imidazolato-bridged CuZn Units.

Nanoparticles exhibiting enzymatic functions have garnered considerable attention due to their structural robustness and the profusion of active sites that can be introduced to a single nanosized particle. Here we report that nanosized mixed-metal zeolitic imidazolate frameworks (ZIFs) show a superoxide dismutase (SOD)-like catalytic activity. We chose a ZIF composed of copper and zinc ions and 2-methylimidazole, CuZn-ZIF-8, in which the Cu and Zn ions are bridged by an imidazolato ligand. This coordination geometry closely mimics the active site of CuZn superoxide dismutase (CuZnSOD). The CuZn-ZIF-8 nanoparticles exhibit potent SOD-like activity, attributed to their porous nature and numerous copper active sites, and also possess exceptional recyclability.

A De Novo Designed Trimeric Metalloprotein as a Nip Model of the Acetyl-CoA Synthase.

We present a Nip site model of acetyl coenzyme-A synthase (ACS) within a de novo-designed trimer peptide that self-assembles to produce a homoleptic Ni(Cys)3 binding motif. Spectroscopic and kinetic studies of ligand binding demonstrate that Ni binding stabilizes the peptide assembly and produces a terminal NiI-CO complex. When the CO-bound state is reacted with a methyl donor, a new species is quickly produced with new spectral features. While the metal-bound CO is albeit unactivated, the presence of the methyl donor produces an activated metal-CO complex. Selective outer sphere steric modifications demonstrate that the physical properties of the ligand-bound states are altered differently depending on the location of the steric modification above or below the Ni site.

A New Thiolate-Bound Dimanganese Cluster as a Structural and Functional Model for Class Ib Ribonucleotide Reductases.

In class Ib ribonucleotide reductases (RNRs) a dimanganese(II) cluster activates superoxide (O2 ⋅- ) rather than dioxygen (O2 ), to access a high valent MnIII -O2 -MnIV species, responsible for the oxidation of tyrosine to tyrosyl radical. In a biomimetic approach, we report the synthesis of a thiolate-bound dimanganese complex [MnII 2 (BPMT)(OAc)2 ](ClO)4 (BPMT=(2,6-bis{[bis(2-pyridylmethyl)amino]methyl}-4-methylthiophenolate) (1) and its reaction with O2 ⋅- to form a [(BPMT)MnO2 Mn]2+ complex 2. Resonance Raman investigation revealed the presence of an O-O bond in 2, while EPR analysis displayed a 16-line St =1/2 signal at g=2 typically associated with a MnIII MnIV core, as detected in class Ib RNRs. Unlike all other previously reported Mn-O2 -Mn complexes, generated by O2 ⋅- activation at Mn2 centers, 2 proved to be a capable electrophilic oxidant in aldehyde deformylation and phenol oxidation reactions, rendering it one of the best structural and functional models for class Ib RNRs.

A Copper Cage-Complex as Mimic of the pMMO CuC Site.

The active site of particulate methane monooxygenase (pMMO) and its mechanism of action are not known. Recently, the CuC site emerged as a potential active site, but to date it lacks any study on biomimetic resemblance of the coordination environment provided by the enzyme. Here, the synthesis of a cage ligand providing such an environment is reported. Copper is incorporated, and coordination occurs by the two imidazole and one carboxylate group offered by the ligand. Depending on the oxidation state, it can adopt different coordination modes, as evidenced by the solid-state structures and computational investigation. The copper(I) state readily reacts with dioxygen and thereby undergoes CH activation. Moreover, the catalytic aerobic oxidation of hydroquinones as ubiquinol mimics is shown. Clean one-electron oxidation occurs under mild conditions and EPR analysis of the copper(II) state in the presence of water reveals striking similarities to the data obtained from pMMO.

Moderated Basicity of Endohedral Amine Groups in an Octa-Cationic Self-Assembled Cage.

A self-assembled FeII4 L6 cage was synthesized with 12 internal amines in the cavity. The cage forms as the dodeca-ammonium salt, despite the cage carrying an overall 8+ charge at the metal centers, extracting protons from displaced water in the reaction. Despite this, the basicity of the internal amines is lower than their counterparts in free solution. The 12 amines have a sliding scale of basicity, with a ≈6 pKa unit difference between the first and last protons to be removed. This moderation of side-chain basicity in an active site is a hallmark of enzymatic catalysis.

The Role of Heme Peroxo Oxidants in the Rational Mechanistic Modeling of Nitric Oxide Synthase: Characterization of Key Intermediates and Elucidation of the Mechanism.

Mammalian nitric oxide synthase (NOS) mediates the two-step O2 -dependent oxidative degradation of arginine, and has been linked to a medley of disease situations in humans. Nonetheless, its exact mechanism of action still remains unclear. This work presents the first NOS model system where biologically proposed heme superoxo and peroxo intermediates are assessed as active oxidants against oxime substrates. Markedly, heme peroxo intermediates engaged in a bioinspired oxime oxidation reaction pathway, converting oximes to ketones and nitroxyl anions (NO- ). Detailed thermodynamic, kinetic, and mechanistic interrogations all evince a rate-limiting step primarily driven by the nucleophilicity of the heme peroxo moiety. Coherent with other findings, 18 O and 15 N isotope substitution experiments herein suffice compelling evidence toward a detailed mechanism, which draw close parallels to one of the enzymatic proposals. Intriguingly, recent enzymatic studies also lend credence to these findings, and several relevant reaction intermediates have been observed during NOS turnover.

A Promising Approach for Controlling the Second Coordination Sphere of Biomimetic Metal Complexes: Encapsulation in a Dynamic Hydrogen-Bonded Capsule.

The design of biomimetic models of metalloenzymes needs to take into account many factors and is therefore a challenging task. We propose in this work an original strategy to control the second coordination sphere of a metal centre and its distal environment. A biomimetic complex, reproducing the first coordination sphere, is encapsulated in a self-assembled hydrogen-bonded capsule. The cationic complex is co-encapsulated with its counter-anion or with solvent molecules. The capsule is dynamic, allowing a fast in/out exchange of the co-encapsulated species. It also provides both a hydrogen-bonding site in the second coordination sphere and a source of proton as it can be deprotonated in the presence of the complex, providing a globally neutral host-guest assembly. This simple and broad scope strategy is unprecedented in biomimetic studies. The approach appears to be a very promising method for the stabilisation of reactive species and for the study of their reactivity.

Proton Switch in the Secondary Coordination Sphere to Control Catalytic Events at the Metal Center: Biomimetic Oxo Transfer Chemistry of Nickel Amidate Complex.

High-valent metal-oxo species are key intermediates for the oxygen atom transfer step in the catalytic cycles of many metalloenzymes. While the redox-active metal centers of such enzymes are typically supported by anionic amino acid side chains or porphyrin rings, peptide backbones might function as strong electron-donating ligands to stabilize high oxidation states. To test the feasibility of this idea in synthetic settings, we have prepared a nickel(II) complex of new amido multidentate ligand. The mononuclear nickel complex of this N5 ligand catalyzes epoxidation reactions of a wide range of olefins by using mCPBA as a terminal oxidant. Notably, a remarkably high catalytic efficiency and selectivity were observed for terminal olefin substrates. We found that protonation of the secondary coordination sphere serves as the entry point to the catalytic cycle, in which high-valent nickel species is subsequently formed to carry out oxo-transfer reactions. A conceptually parallel process might allow metalloenzymes to control the catalytic cycle in the primary coordination sphere by using proton switch in the secondary coordination sphere.

Structurally Modelling the 2-His-1-Carboxylate Facial Triad with a Bulky N,N,O Phenolate Ligand.

We present the synthesis and coordination chemistry of a bulky, tripodal N,N,O ligand, ImPh2 NNOtBu (L), designed to model the 2-His-1-carboxylate facial triad (2H1C) by means of two imidazole groups and an anionic 2,4-di-tert-butyl-subtituted phenolate. Reacting K-L with MCl2 (M = Fe, Zn) affords the isostructural, tetrahedral non-heme complexes [Fe(L)(Cl)] (1) and [Zn(L)(Cl)] (2) in high yield. The tridentate N,N,O ligand coordination observed in their X-ray crystal structures remains intact and well-defined in MeCN and CH2 Cl2 solution. Reacting 2 with NaSPh affords a tetrahedral zinc thiolate complex, [Zn(L)(SPh)] (4), that is relevant to isopenicillin N synthase (IPNS) biomimicry. Cyclic voltammetry studies demonstrate the ligand's redox non-innocence, where phenolate oxidation is the first electrochemical response observed in K-L, 2 and 4. However, the first electrochemical oxidation in 1 is iron-centred, the assignment of which is supported by DFT calculations. Overall, ImPh2 NNOtBu provides access to well-defined mononuclear, monoligated, N,N,O-bound metal complexes, enabling more accurate structural modelling of the 2H1C to be achieved.

Bioinspired Electrolysis for Green Molecular Transformations of Organic Halides Catalyzed by B12 Complex.

Naturally-occurring B12 -dependent enzymes catalyze various molecular transformations that are of particular interest from the viewpoint of biological chemistry as well as synthetic organic chemistry. Inspired by the unique property of the B12 -dependent enzymes, various catalytic reactions have been developed using its model complex. Among the B12 model complexes, heptamethyl cobyrinate, synthesized from natural vitamin B12 , is highly soluble in various organic solvents and a redox active cobalt complex with an excellent catalysis in electroorganic synthesis. The electrochemical dechlorination of pollutant organic chlorides, such as DDT, was effectively catalyzed by the B12 complex. Modification of the electrode surface by the sol-gel method to immobilize the B12 complex was also developed. The B12 modified electrodes were effective for the dehalogenation of organic halides with high turnover numbers based on the immobilized B12 complex. Electrolysis of an organic halide catalyzed by the B12 complex provided dechlorinated products under anaerobic conditions, while the electrolysis under aerobic conditions afforded oxygen incorporated products, such as an ester and amide along with dechlorination. Benzotrichloride was transformed into ethylbenzoate or N,N-diethylbenzamide in the presence of ethanol or diethylamine, respectively. This amide formation was further expanded to a unique paired electrolysis. Electrochemical reductions of an alkene and alkyne were also catalyzed by the B12 complex. A cobalt-hydrogen complex should be formed as a bioinspired intermediate. Using the B12 complex, light-assisted electrosynthesis was also developed to save the applied energy.

Rationalizing the Unprecedented Stereochemistry of an Enzymatic Nitrile Synthesis through a Combined Computational and Experimental Approach.

In this contribution, the unique and unprecedented stereochemical phenomenon of an aldoxime dehydratase-catalyzed enantioselective dehydration of racemic E- and Z-aldoximes with selective formation of both enantiomeric forms of a chiral nitrile is rationalized by means of molecular modelling, comprising in silico mutations and docking studies. This theoretical investigation gave detailed insight into why with the same enzyme the use of racemic E- and Z-aldoximes leads to opposite forms of the chiral nitrile. The calculated mutants with a larger or smaller cavity in the active site were then prepared and used in biotransformations, showing the theoretically predicted decrease and increase of the enantioselectivities in these nitrile syntheses. This validated model also enabled the rational design of mutants with a smaller cavity, which gave superior enantioselectivities compared to the known wild-type enzyme, with excellent E-values of up to E>200 when the mutant OxdRE-Leu145Phe was utilized.

Multiple-Site Concerted Proton-Electron Transfer in a Manganese-Based Complete Functional Model for [FeFe]-Hydrogenase.

The active site of [FeFe]-hydrogenase (H2 ase) is preorganized with an amine (azadithiolate) as a proton relay and a [4Fe4S] subunit as an electron reservoir, which together lower the overpotential for proton reduction and hydrogen oxidation by multiple-site concerted proton-electron transfer (MS-CPET). Herein, we report a mononuclear manganese complex, fac-[Mn(CO)3 (6-(2-hydroxyphenol)-2-pyridine-2-quinoline) Br] (1), as a rare model to fully mimic the functions of the H2 ase. In 1, a redox-active bidentate ligand with a pendent phenol replicates the roles of the electron reservoir and the proton relay in the enzyme. Experimental and theoretical studies revealed two consecutive MS-CPET processes in the catalytic cycle, in each of which an electron stored in the reductive ligand and a proton at the proximal phenol moiety are transferred to the Mn center in a concerted way. By virtue of this mechanism, complex 1 exhibited a low overpotential comparable to that of natural enzyme in electrochemical hydrogen production using phenol as a proton source.

A Pseudotetrahedral Terminal Oxoiron(IV) Complex: Mechanistic Promiscuity in C-H Bond Oxidation Reactions.

S=2 oxoiron(IV) species act as reactive intermediates in the catalytic cycle of nonheme iron oxygenases. The few available synthetic S=2 FeIV =O complexes known to date are often limited to trigonal bipyramidal and very rarely to octahedral geometries. Herein we describe the generation and characterization of an S=2 pseudotetrahedral FeIV =O complex 2 supported by the sterically demanding 1,4,7-tri-tert-butyl-1,4,7-triazacyclononane ligand. Complex 2 is a very potent oxidant in hydrogen atom abstraction (HAA) reactions with large non-classical deuterium kinetic isotope effects, suggesting hydrogen tunneling contributions. For sterically encumbered substrates, direct HAA is impeded and an alternative oxidative asynchronous proton-coupled electron transfer mechanism prevails, which is unique within the nonheme oxoiron community. The high reactivity and the similar spectroscopic parameters make 2 one of the best electronic and functional models for a biological oxoiron(IV) intermediate of taurine dioxygenase (TauD-J).

Biomimetic Iron Complex Achieves TET Enzyme Reactivity*.

The epigenetic marker 5-methyl-2'-deoxycytidine (5mdC) is the most prevalent modification to DNA. It is removed inter alia via an active demethylation pathway: oxidation by Ten-Eleven Translocation 5-methyl cytosine dioxygenase (TET) and subsequent removal via base excision repair or direct demodification. Recently, we have shown that the synthetic iron(IV)-oxo complex [FeIV (O)(Py5 Me2 H)]2+ (1) can serve as a biomimetic model for TET by oxidizing the nucleobase 5-methyl cytosine (5mC) to its natural metabolites. In this work, we demonstrate that nucleosides and even short oligonucleotide strands can also serve as substrates, using a range of HPLC and MS techniques. We found that the 5-position of 5mC is oxidized preferably by 1, with side reactions occurring only at the strand ends of the used oligonucleotides. A detailed study of the reactivity of 1 towards nucleosides confirms our results; that oxidation of the anomeric center (1') is the most common side reaction.

Biocatalytic Micro- and Nanomotors.

Enzyme-powered micro- and nanomotors are tiny devices inspired by nature that utilize enzyme-triggered chemical conversion to release energy stored in the chemical bonds of a substrate (fuel) to actuate it into active motion. Compared with conventional chemical micro-/nanomotors, these devices are particularly attractive because they self-propel by utilizing biocompatible fuels, such as glucose, urea, glycerides, and peptides. They have been designed with functional material constituents to efficiently perform tasks related to active targeting, drug delivery and release, biosensing, water remediation, and environmental monitoring. Because only a small number of enzymes have been exploited as bioengines to date, a new generation of multifunctional, enzyme-powered nanorobots will emerge in the near future to selectively search for and utilize water contaminants or disease-related metabolites as fuels. This Minireview highlights recent progress in enzyme-powered micro- and nanomachines.

Enhancing Tris(pyrazolyl)borate-Based Models of Cysteine/Cysteamine Dioxygenases through Steric Effects: Increased Reactivities, Full Product Characterization and Hints to Initial Superoxide Formation.

The design of biomimetic model complexes for the cysteine dioxygenase (CDO) and cysteamine dioxygenase (ADO) is reported, where the 3-His coordination of the iron ion is simulated by three pyrazole donors of a trispyrazolyl borate ligand (Tp) and protected cysteine and cysteamine represent substrate ligands. It is found that the replacement of phenyl groups-attached at the 3-positions of the pyrazole units in a previous model-by mesityl residues has massive consequences, as the latter arrange to a more spacious reaction pocket. Thus, the reaction with O2 proceeds much faster and afterwards the first structural characterization of an iron(II) η2 -O,O-sulfinate product became possible. If one of the three Tp-mesityl groups is placed in the 5-position, an even larger reaction pocket results, which leads to yet faster rates and accumulation of a reaction intermediate at low temperatures, as shown by UV/Vis and Mössbauer spectroscopy. After comparison with the results of investigations on the cobalt analogues this intermediate is tentatively assigned to an iron(III) superoxide species.

How Do Metal Ions Modulate the Rate-Determining Electron-Transfer Step in Cytochrome P450 Reactions?

Cytochrome P450 (CYP450) enzymes play important roles in maintaining human health and their reaction rates are dependent on the first electron transfer from the reduction partner. Interestingly, experimental work has shown that this step is highly influenced by the addition of metal ions. To understand the effect of external perturbations on the CYP450 first reduction step, we have performed a computational study with model complexes in the presence of metal and organic ions, solvent molecules, and an electric field. The results show that these medium-range interactions affect the driving force as well as electron-transfer rates dramatically. Based on the location, distance, and direction of the ions/electric field, the catalytic reaction rates are enhanced or impaired. Calculations on a large crystal structure with bonded alkali metal ions indicated inhibition patterns of the ions. Therefore, we predict that the active forms of the natural CYP450 isozymes will not have more than one alkali metal ion bound in the second-coordination sphere. As such, this study provides an insight into the activity of CYP450 enzymes and the effects of ions and electric field perturbations on their activity.

How Does Replacement of the Axial Histidine Ligand in Cytochrome c Peroxidase by Nδ-Methyl Histidine Affect Its Properties and Functions? A Computational Study.

Heme peroxidases have important functions in nature related to the detoxification of H2O2. They generally undergo a catalytic cycle where, in the first stage, the iron(III)-heme-H2O2 complex is converted into an iron(IV)-oxo-heme cation radical species called Compound I. Cytochrome c peroxidase Compound I has a unique electronic configuration among heme enzymes where a metal-based biradical is coupled to a protein radical on a nearby Trp residue. Recent work using the engineered Nδ-methyl histidine-ligated cytochrome c peroxidase highlighted changes in spectroscopic and catalytic properties upon axial ligand substitution. To understand the axial ligand effect on structure and reactivity of peroxidases and their axially Nδ-methyl histidine engineered forms, we did a computational study. We created active site cluster models of various sizes as mimics of horseradish peroxidase and cytochrome c peroxidase Compound I. Subsequently, we performed density functional theory studies on the structure and reactivity of these complexes with a model substrate (styrene). Thus, the work shows that the Nδ-methyl histidine group has little effect on the electronic configuration and structure of Compound I and little changes in bond lengths and the same orbital occupation is obtained. However, the Nδ-methyl histidine modification impacts electron transfer processes due to a change in the reduction potential and thereby influences reactivity patterns for oxygen atom transfer. As such, the substitution of the axial histidine by Nδ-methyl histidine in peroxidases slows down oxygen atom transfer to substrates and makes Compound I a weaker oxidant. These studies are in line with experimental work on Nδ-methyl histidine-ligated cytochrome c peroxidases and highlight how the hydrogen bonding network in the second coordination sphere has a major impact on the function and properties of the enzyme.

Crystals in Minutes: Instant On-Site Microcrystallisation of Various Flavours of the CYP102A1 (P450BM3) Haem Domain.

Despite CYP102A1 (P450BM3) representing one of the most extensively researched metalloenzymes, crystallisation of its haem domain upon modification can be a challenge. Crystal structures are indispensable for the efficient structure-based design of P450BM3 as a biocatalyst. The abietane diterpenoid derivative N-abietoyl-l-tryptophan (AbiATrp) is an outstanding crystallisation accelerator for the wild-type P450BM3 haem domain, with visible crystals forming within 2 hours and diffracting to a near-atomic resolution of 1.22 Å. Using these crystals as seeds in a cross-microseeding approach, an assortment of P450BM3 haem domain crystal structures, containing previously uncrystallisable decoy molecules and diverse artificial metalloporphyrins binding various ligand molecules, as well as heavily tagged haem-domain variants, could be determined. Some of the structures reported herein could be used as models of different stages of the P450BM3 catalytic cycle.