Binding and Stabilization of a Semiquinone Radical by an Artificial Metalloenzyme Containing a Binuclear Copper (II) Cofactor.

A binuclear Cu(II) cofactor was covalently bound to a lauric acid anchor. The resulting conjugate was characterized then combined with beta-lactoglobulin (ßLG) to generate a new biohybrid following the so-called "Trojan horse" strategy. This biohybrid was examined for its effectiveness in the oxidation of a catechol derivative to the corresponding quinone. The resulting biohybrid did not exhibit the sought after catecholase activity, likely due to its ability to bind and stabilize the semiquinone radical intermediate DTB-SQ. This semi-quinone radical was stabilized only in the presence of the protein and was characterized using optical and magnetic spectroscopic techniques, demonstrating stability for over 16 hours. Molecular docking studies revealed that this stabilization could occur owing to interactions of the semi-quinone with hydrophobic amino acid residues of ßLG.

Proton Domino Reactions at an Imidazole Relay Control the Oxidation of a TyrZ-His190 Artificial Mimic of Photosystem II.

A close mimic of P680 and the TyrosineZ-Histidine190 pair in photosystem II (PS II) has been synthesized using a ruthenium chromophore and imidazole-phenol ligands. The intramolecular oxidation of the ligands by the photoproduced Ru(III) species is characterized by a small driving force, very similar to PS II where the complexity of kinetics was attributed to the reversibility of electron transfer steps. Laser flash photolysis revealed biphasic kinetics for ligand oxidation. The fast phase (τ<50 ns) corresponds to partial oxidation of the imidazole-phenol ligand, proton transfer within the hydrogen bond, and formation of a neutral phenoxyl radical. The slow phase (5-9 µs) corresponds to full oxidation of the ligand which is kinetically controlled by deprotonation of the distant 1-nitrogen of the imidazolium. These results show that imidazole with its two protonatable sites plays a special role as a proton relay in a 'proton domino' reaction.

Heterolytic O-O Bond Cleavage Upon Single Electron Transfer to a Nonheme Fe(III)-OOH Complex.

The one-electron reduction of the nonheme iron(III)-hydroperoxo complex, [FeIII (OOH)(L5 2 )]2+ (L5 2 =N-methyl-N,N',N'-tris(2-pyridylmethyl)ethane-1,2-diamine), carried out at -70 °C results in the release of dioxygen and in the formation of [FeII (OH)(L5 2 )]+ following a bimolecular process. This reaction can be performed either with cobaltocene as chemical reductant, or electrochemically. These experimental observations are consistent with the disproportionation of the hydroperoxo group in the putative FeII (OOH) intermediate generated upon reduction of the FeIII (OOH) starting complex. One plausible mechanistic scenario is that this disproportionation reaction follows an O-O heterolytic cleavage pathway via a FeIV -oxo species.

A Tale of Two Complexes: Electro-Assisted Oxidation of Thioanisole by an "O2 Activator/Oxidizing Species" Tandem System of Non-Heme Iron Complexes.

We report two new FeIII complexes [L1 FeIII (H2 O)](OTf)2 and [L2 FeIII (OTf)], obtained by replacing pyridines by phenolates in a known non-heme aminopyridine iron complex. While the original, starting aminopyridine [(L5 2 )FeII (MeCN)](PF6 ) complex is stable in air, the potentials of the new FeIII/II couples decrease to the point that [L2 FeII ] spontaneously reduces O2 to superoxide. We used it as an O2 activator in an electrochemical setup, as its presence allows to generate superoxide at a much more accessible potential (>500 mV gain). Our aim was to achieve substrate oxidation via the reductive activation of O2 . While L2 FeIII (OTf) proved to be a good O2 activator but a poor oxidation system, its association with another complex (TPEN)FeII (PF6 )2 generates a complementary tandem couple for electro-assisted oxidation of substrates, working at a very accessible potential: upon reduction, L2 FeIII (OTf) activates O2 to superoxide and transfers it to (TPEN)FeII (PF6 )2 leading in fine to the oxidation of thioanisole.

Proton-controlled Action of an Imidazole as Electron Relay in a Photoredox Triad.

Electron relays play a crucial role for efficient light-induced activation by a photo-redox moiety of catalysts for multi-electronic transformations. Their insertion between the two units reduces detrimental energy transfer quenching while establishing at the same time unidirectional electron flow. This rectifying function allows charge accumulation necessary for catalysis. Mapping these events in photophysical studies is an important step towards the development of efficient molecular photocatalysts. Three modular complexes comprised of a Ru-chromophore, an imidazole electron relay function, and a terpyridine unit as coordination site for a metal ion were synthesized and the light-induced electron transfer events studied by laser flash photolysis. In all cases, formation of an imidazole radical by internal electron transfer to the oxidized chromophore was observed. The effect of added base evidenced that the reaction sequence depends strongly on the possibility for deprotonation of the imidazole function in a proton-coupled electron transfer process. In the complex with MnII present as a proxy for a catalytic site, a strongly accelerated decay of the imidazole radical together with a decreased rate of back electron transfer from the external electron acceptor to the oxidized complex was observed. This transient formation of an imidazolyl radical is clear evidence for the function of the imidazole group as an electron relay. The implication of the imidazole proton and the external base for the kinetics and energetics of the electron trafficking is discussed.

Biosynthesis of the sactipeptide Ruminococcin C by the human microbiome: Mechanistic insights into thioether bond formation by radical SAM enzymes.

Despite its major importance in human health, the metabolic potential of the human gut microbiota is still poorly understood. We have recently shown that biosynthesis of Ruminococcin C (RumC), a novel ribosomally synthesized and posttranslationally modified peptide (RiPP) produced by the commensal bacterium Ruminococcus gnavus, requires two radical SAM enzymes (RumMC1 and RumMC2) catalyzing the formation of four Cα-thioether bridges. These bridges, which are essential for RumC's antibiotic properties against human pathogens such as Clostridium perfringens, define two hairpin domains giving this sactipeptide (sulfur-to-α-carbon thioether-containing peptide) an unusual architecture among natural products. We report here the biochemical and spectroscopic characterizations of RumMC2. EPR spectroscopy and mutagenesis data support that RumMC2 is a member of the large family of SPASM domain radical SAM enzymes characterized by the presence of three [4Fe-4S] clusters. We also demonstrate that this enzyme initiates its reaction by Cα H-atom abstraction and is able to catalyze the formation of nonnatural thioether bonds in engineered peptide substrates. Unexpectedly, our data support the formation of a ketoimine rather than an α,ß-dehydro-amino acid intermediate during Cα-thioether bridge LC-MS/MS fragmentation. Finally, we explored the roles of the leader peptide and of the RiPP precursor peptide recognition element, present in myriad RiPP-modifying enzymes. Collectively, our data support a more complex role for the peptide recognition element and the core peptide for the installation of posttranslational modifications in RiPPs than previously anticipated and suggest a possible reaction intermediate for thioether bond formation.

Evolution of Ate-Organoiron(II) Species towards Lower Oxidation States: Role of the Steric and Electronic Factors.

Ate-iron(II) species such as [Ar3 FeII ]- (Ar=aryl) are key intermediates in Fe-catalyzed couplings between aryl nucleophiles and organic electrophiles. They can be active species in the catalytic cycle, or lead to Fe0 and FeI oxidation states, which can themselves be catalytically active or lead to unwished organic byproducts. Analysis of the reactivity of the intermediates obtained by step-by-step displacement of the mesityl groups in high-spin [Mes3 FeII ]- by less hindered phenyl ligands was performed, and uncovered the crucial role of both steric and electronic parameters in the formation of the Fe0 and FeI oxidation states. The formation of quaternized [Ar4 FeII MgBr(THF)]- intermediates allows the bielectronic reductive elimination energy required for the formation of Fe0 to be reduced. Similarly, the small steric pressure of the aryl groups in [Ar3 FeII ]- enables the formation of aryl-bridged [{FeII (Ar)2 }2 (µ-Ar)2 ]2- species, which afford the FeI oxidation state by bimetallic reductive elimination. These results are supported by 1 H NMR, EPR, and 57 Fe Mössbauer spectroscopies, as well as by DFT calculations.

Hydroxylation of Aromatics by H2 O2 Catalyzed by Mononuclear Non-heme Iron Complexes: Role of Triazole Hemilability in Substrate-Induced Bifurcation of the H2 O2 Activation Mechanism.

Rieske dioxygenases are metalloenzymes capable of achieving cis-dihydroxylation of aromatics under mild conditions using O2 and a source of electrons. The intermediate responsible for this reactivity is proposed to be a cis-FeV (O)(OH) moiety. Molecular models allow the generation of a FeIII (OOH) species with H2 O2 , to yield a FeV (O)(OH) species with tetradentate ligands, or {FeIV (O); OH. } pairs with pentadentate ones. We have designed a new pentadentate ligand, mtL4 2 , bearing a labile triazole, to generate an "in-between" situation. Two iron complexes, [(mtL4 2 )FeCl](PF6 ) and [(mtL4 2 )Fe(OTf)2 ]), were obtained and their reactivity towards aromatic substrates was studied in the presence of H2 O2 . Spectroscopic and kinetic studies reflect that triazole is bound at the FeII state, but decoordinates in the FeIII (OOH). The resulting [(mtL4 2 )FeIII (OOH)(MeCN)]2+ then lies on a bifurcated decay pathway (end-on homolytic vs. side-on heterolytic) depending on the addition of aromatic substrate: in the absence of substrate, it is proposed to follow a side-on pathway leading to a putative (N4 )FeV (O)(OH), while in the presence of aromatics it switches to an end-on homolytic pathway yielding a {(N5 )FeIV (O); OH. } reactive species, through recoordination of triazole. This switch significantly impacts the reaction regioselectivity.

FeIII and FeII Phosphasalen Complexes: Synthesis, Characterization, and Catalytic Application for 2-Naphthol Oxidative Coupling.

We report on the synthesis and characterization of three iron(III) phosphasalen complexes, [FeIII (Psalen)(X)] differing in the nature of the counter-anion/exogenous ligand (X- =Cl- , NO3 - , OTf- ), as well as the neutral iron(II) analogue, [FeII (Psalen)]. Phosphasalen (Psalen) differs from salen by the presence of iminophosphorane (P=N) functions in place of the imines. All the complexes were characterized by single-crystal X-ray diffraction, UV/Vis, EPR, and cyclic voltammetry. The [FeII (Psalen)] complex was shown to remain tetracoordinated even in coordinating solvent but surprisingly exhibits a magnetic moment in line with a FeII high-spin ground state. For the FeIII complexes, the higher lability of triflate anion compared to nitrate was demonstrated. As they exhibit lower reduction potentials compared to their salen analogues, these complexes were tested for the coupling of 2-naphthol using O2 from air as oxidant. In order to shed light on this reaction, the interaction between 2-naphthol and the FeIII (Psalen) complexes was studied by cyclic voltammetry as well as UV/Vis spectroscopy.

Photoswitchable 11 nm CsCoFe Prussian Blue Analogue Nanocrystals with High Relaxation Temperature.

Photoswitchable 11 nm nanocrystals with the coordination network Cs{Co[Fe(CN)6]} were obtained using a template-free method. The nanocrystals were recovered from the colloidal solutions as solid materials surrounded by cetyltrimethylammonium (CTA) cations or embedded in the organic polymer polyvinylpyrrolidone (PVP). Complementary magnetic, spectroscopic, and structural techniques, including EPR spectroscopy, reveal a majority (∼70%) of the low-spin and photoactive diamagnetic CoIIIFeII pairs located in the core of the nanocrystals and a mixture of CoIIFeII and CoIIFeIII species present mainly within the shell of the objects. While bulk compounds with similar vacancy concentration do not exhibit noticeable photoinduced charge transfer, the observed photoactivity of the nanocrystals is ascribed to their nanometric size. The relaxation temperature of the photoinduced state shifts upward by ∼55 K when PVP is replaced by CTA. This is ascribed to the larger rigidity of the dense CsCoFe_CTA material, whose metastable state is lower than that for CsCoFe_PVP, leading to a larger relaxation energy barrier and, therefore, to a higher relaxation temperature.

Characterization in aqueous medium of an FMN semiquinone radical stabilized by the enzyme-like microenvironment of a modified polyethyleneimine.

The elusive flavin semiquinone intermediate found in flavoproteins such as cryptochromes has been obtained in aqueous solution by single electron reduction of the natural FMN cofactor using sodium ascorbate. This species was formed in the local hydrophobic microenvironment of a modified polyethyleneimine and characterized by UV-Visible, fluorescence and EPR spectroscopies.

Non-Heme FeII Diastereomeric Complexes Bearing a Hexadentate Ligand: Unexpected Consequences for the Spin State and Catalytic Oxidation Properties.

The reactivity and selectivity of non-heme FeII complexes as oxidation catalysts can be substantially modified by alteration of the ligand backbone or introduction of various substituents. In comparison with the hexadentate ligand N,N,N',N'-tetrakis(pyridin-2-ylmethyl)ethane-1,2-diamine (TPEN), N,N'-bis[1-(pyridin-2-yl)ethyl]-N,N'-bis(pyridin-2-ylmethyl)ethane-1,2-diamine (2Me L6 2 ) has a methyl group on two of the four picolyl positions. FeII complexation by 2Me L6 2 yields two diastereomeric complexes with very similar structures, which only differ in the axial/equatorial positions occupied by the methylated pyridyl groups. In solution, these two isomers exhibit different magnetic behaviors. Whereas one isomer exhibits temperature-dependent spin-state conversion between the S=0 and S=2 states, the other is more reluctant towards this spin-state equilibrium and is essentially diamagnetic at room temperature. Their catalytic properties for the oxidation of anisole by H2 O2 are very different and correlate with their magnetic properties, which reflect their lability/inertness. These different properties most likely depend on the different steric constraints of the methylated pyridyl groups in the two complexes.

Selective Formation of an FeIV O or an FeIII OOH Intermediate From Iron(II) and H2 O2 : Controlled Heterolytic versus Homolytic Oxygen-Oxygen Bond Cleavage by the Second Coordination Sphere.

We demonstrate that the devised incorporation of an alkylamine group into the second coordination sphere of an FeII complex allows to switch its reactivity with H2 O2 from the usual formation of FeIII species towards the selective generation of an FeIV -oxo intermediate. The FeIV -oxo species was characterized by UV/Vis absorption and Mössbauer spectroscopy. Variable-temperature kinetic analyses point towards a mechanism in which the heterolytic cleavage of the O-O bond is triggered by a proton transfer from the proximal to the distal oxygen atom in the FeII -H2 O2 complex with the assistance of the pendant amine. DFT studies reveal that this heterolytic cleavage is actually initiated by an homolytic O-O cleavage immediately followed by a proton-coupled electron transfer (PCET) that leads to the formation of the FeIV -oxo and release of water through a concerted mechanism.

Electron transfer activity of a de novo designed copper center in a three-helix bundle fold.

In this work, we characterized the intermolecular electron transfer (ET) properties of a de novo designed metallopeptide using laser-flash photolysis. α3D-CH3 is three helix bundle peptide that was designed to contain a copper ET site that is found in the ß-barrel fold of native cupredoxins. The ET activity of Cuα3D-CH3 was determined using five different photosensitizers. By exhibiting a complete depletion of the photo-oxidant and the successive formation of a Cu(II) species at 400 nm, the transient and generated spectra demonstrated an ET transfer reaction between the photo-oxidant and Cu(I)α3D-CH3. This observation illustrated our success in integrating an ET center within a de novo designed scaffold. From the kinetic traces at 400 nm, first-order and bimolecular rate constants of 10(5) s(-1) and 10(8) M(-1) s(-1) were derived. Moreover, a Marcus equation analysis on the rate versus driving force study produced a reorganization energy of 1.1 eV, demonstrating that the helical fold of α3D requires further structural optimization to efficiently perform ET. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

Electronic Structures of Mono-Oxidized Copper and Nickel Phosphasalen Complexes.

Non-innocent ligands render the determination of the electronic structure in metal complexes difficult. As such, a combination of experimental techniques and quantum chemistry are required to correctly elucidate them. This paper deals with the one-electron oxidation of copper(II) and nickel(II) complexes featuring a phosphasalen ligand (Psalen), which differs from salen analogues by the presence of iminophosphorane groups (P=N) instead of imines. Various experimental techniques (X-ray diffraction, cyclic voltammetry, NMR, EPR, and UV/Vis spectroscopies, and magnetic measurements) as well as quantum chemical calculations were used to define the electronic structure of the oxidized complexes. These can be modified by a small change in the ligand structure, that is, the replacement of a tert-butyl group by a methoxy on the phenoxide ring. The different techniques have allowed quantifying the amount of spin density located on the metal center and on the Psalen ligands. All complexes were found to possess a multi-configurational ground state, in which the ratio of the +II versus +III oxidation state of the metal center, and therefore the phenolate versus phenoxyl radical ligand character, varies upon the substituents. The tert-butyl group favors a strong localization on the metal center whereas with the methoxy group the metallic configurations decrease and the ligand configurations increase. The importance of the geometrical considerations compared with the electronic substituent effect is highlighted by the differences observed between the solid-state (EPR, magnetic measurements) and solution characterizations (EPR and NMR data).

αRep A3: A Versatile Artificial Scaffold for Metalloenzyme Design.

αRep refers to a new family of artificial proteins based on a thermostable α-helical repeated motif. One of its members, αRep A3, forms a stable homo-dimer with a wide cleft that is able to accommodate metal complexes and thus appears to be suitable for generating new artificial biocatalysts. Based on the crystal structure of αRep A3, two positions (F119 and Y26) were chosen, and independently changed into cysteine residues. A phenanthroline ligand was covalently attached to the unique cysteine residue of each protein variant, and the corresponding biohybrids were purified and characterized. Once mutated and coupled to phenanthroline, the protein remained folded and dimeric. Copper(II) was specifically bound by the two biohybrids with two different binding modes. Furthermore, the holo-biohybrid A3F119NPH was found to be capable of enantioselectively catalyzing Diels-Alder (D-A) cycloadditions with up to 62 % ee. This study validates the choice of the αRep A3 dimer as a protein scaffold and provides a promising new route for the design and production of new enantioselective biohybrids based on entirely artificial proteins obtained from a highly diverse library.

Characterization and Subsequent Reactivity of an Fe-Peroxo Porphyrin Generated by Electrochemical Reductive Activation of O2.

Reductive activation of O2 is achieved by using the [FeIII(F20TPP)Cl] (F20TPP = 5,10,15,20-tetrakis(pentafluorophenyl) porphyrinate) porphyrin through electrochemical reduction of the [FeIII(F20TPP)(O2•-)] superoxo complex. Formation of the [FeIII(F20TPP)(OO)]- peroxo species is monitored by using low-temperature electronic absorption spectroscopy, electron paramagnetic resonance, and cyclic voltammetry. Its subsequent protonation to yield the [FeIII(F20TPP)(OOH)] hydroperoxo intermediate is probed using low-temperature electronic absorption spectroscopy and electron paramagnetic resonance.

An Artificial Enzyme Made by Covalent Grafting of an Fe(II) Complex into ß-Lactoglobulin: Molecular Chemistry, Oxidation Catalysis, and Reaction-Intermediate Monitoring in a Protein.

An artificial metalloenzyme based on the covalent grafting of a nonheme Fe(II) polyazadentate complex into bovine ß-lactoglobulin has been prepared and characterized by using various spectroscopic techniques. Attachment of the Fe(II) catalyst to the protein scaffold is shown to occur specifically at Cys121. In addition, spectrophotometric titration with cyanide ions based on the spin-state conversion of the initial high spin (S=2) Fe(II) complex into a low spin (S=0) one allows qualitative and quantitative characterization of the metal center's first coordination sphere. This biohybrid catalyst activates hydrogen peroxide to oxidize thioanisole into phenylmethylsulfoxide as the sole product with an enantiomeric excess of up to 20 %. Investigation of the reaction between the biohybrid system and H2 O2 reveals the generation of a high spin (S=5/2) Fe(III) (η(2) -O2 ) intermediate, which is proposed to be responsible for the catalytic sulfoxidation of the substrate.

Synergistic "ping-pong" energy transfer for efficient light activation in a chromophore-catalyst dyad.

The synthesis of a porphyrin-Ru(II) polypyridine complex where the porphyrin acts as a photoactive unit and the Ru(II) polypyridine as a catalytic precursor is described. Comparatively, the free base porphyrin was found to outperform the ruthenium based chromophore in the yield of light induced electron transfer. Mechanistic insights indicate the occurrence of a ping-pong energy transfer from the (1)LC excited state of the porphyrin chromophore to the (3)MCLT state of the catalyst and back to the (3)LC excited state of the porphyrin unit. The latter, triplet-triplet energy transfer back to the chromophore, efficiently competes with fast radiationless deactivation of the excited state at the catalyst site. The energy thus recovered by the chromophore allows improved yield of formation of the oxidized form of the chromophore and concomitantly of the oxidation of the catalytic unit by intramolecular charge transfer. The presented results are among the rare examples where a porphyrin chromophore is successfully used to drive an oxidative activation process where reductive processes prevail in the literature.

Carbon dioxide reduction via light activation of a ruthenium-Ni(cyclam) complex.

In this paper we report the synthesis of a chromophore-catalyst assembly designed for the photoreduction of carbon dioxide. The chromophore unit is made up of a ruthenium trisbipyridyl-like unit covalently attached to a nickel cyclam (cyclam = 1,4,8,11-tetraazacyclotetradecane) via a triazole ring. The intramolecular electron transfer activation of the catalyst unit by visible light was studied by nanosecond flash photolysis and EPR spectroscopy. In aqueous solutions (pH = 6.5), activation of the Ru(II)-Ni(II) modular assembly with 450 nm visible light in the presence of a sacrificial electron donor accomplishes the reduction of CO2 into CO and H2 in a ratio of 2.7 to 1.