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The cytochrome P450 (CYP) family of heme monooxygenase enzymes commonly catalyzes enantioselective hydroxylation and epoxidation reactions. Epoxidation reactions have been hypothesized to proceed via multiple mechanisms involving different reactive intermediates. Here, we use activity, spectroscopic, structural, and molecular dynamics data to investigate the activity and stereoselectivity of 4-vinylbenzoic acid epoxidation by the bacterial enzyme CYP199A4 from Rhodopseudomonas palustris HaA2. The epoxidation of 4-vinylbenzoic acid by CYP199A4 proceeded with high enantioselectivity, giving the (S)-epoxide in 99% ee at an activity of 220 nmol nmol-CYP–1 min–1. Optical and EPR spectroscopy, redox potential measurements, and the crystal structure of 4-vinylbenzoic acid-bound CYP199A4 indicated the partial retention of an aqua ligand at the heme center in the presence of the substrate, providing a justification of the lower activity (∼20%) compared to the oxidative demethylation of 4-methoxybenzoic acid. Mutagenesis at the conserved acid–alcohol pair (D251/T252), which perturbs the generation of the reactive oxygen intermediates, was employed to investigate their role in epoxidation reactions. The T252A mutant increased the rate of turnover of the catalytic cycle, but an elevation in hydrogen peroxide generation via uncoupling resulted in a similar rate of epoxide formation. The activity of epoxidation significantly reduced with the D251N mutant. The chemoselectivity and stereoselectivity of the epoxidation reaction were maintained in the turnovers by these mutants. Overall, there was little evidence that other intermediates, aside from the archetypal reactive ferryl porphyrin cation radical, Compound I, contributed significantly to the epoxidation reaction. The observation of the high selectivity for the (S)-enantiomer was rationalized by molecular dynamics simulations. When the arrangement of the alkene and the active intermediate approached an ideal transition state structure for epoxidation, one face of the alkene was more often exposed to the iron oxo unit.

The melting behaviour of metal-organic frameworks (MOFs) has aroused significant research interest in the areas of materials science, condensed matter physics and chemical engineering. This work first introduces a novel method to fabricate a bimetallic MOF glass, through melt-quenching of the cobalt-based zeolitic imidazolate framework (ZIF) [ZIF-62(Co)] with an adsorbed ferric coordination complex. The high-temperature chemically reactive ZIF-62(Co) liquid facilitates the formation of coordinative bonds between Fe and imidazolate ligands, incorporating Fe nodes into the framework after quenching. The resultant Co-Fe bimetallic MOF glass therefore shows a significantly enhanced oxygen evolution reaction performance. The novel bimetallic MOF glass, when combined with the facile and scalable mechanochemical synthesis technique for both discrete powders and surface coatings on flexible substrates, enables significant opportunities for catalytic device assembly.

This research was supported by the Engineering & Physical Sciences Research Council (EPSRC) and the Biotechnology & Biological Sciences Research Council (BBSRC), UK (EP/D048559). AMB and EOJD were supported by graduate studentships from the BBSRC (BB/F01709X/1) and NJH and JEL were supported by graduate studentships from the EPSRC, and JEL after her DPhil by EP/D048559. AMB gratefully acknowledges her current fellowship support from the Royal Society and EPSRC for a Dorothy Hodgkin Fellowship (DH160004). JRH acknowledges support from the ARC (FT120100421) and the Centre for Advanced Imaging, The University of Queensland. Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of chemically inert carbon-hydrogen bonds in diverse endogenous and exogenous organic compounds by atmospheric oxygen. This C–H bond oxy-functionalization activity has huge potential in biotechnological applications. Class I CYPs receive the two electrons required for oxygen activation from NAD(P)H via a ferredoxin reductase and ferredoxin. The interaction of Class I CYPs with their cognate ferredoxin is specific. In order to reconstitute the activity of diverse CYPs, structural characterization of CYP-ferredoxin complexes is necessary, but little structural information is available. Here we report a structural model of such a complex (CYP199A2-HaPux) in frozen solution derived from distance and orientation restraints gathered by the EPR technique of orientation-selective double electron-electron resonance (os-DEER). The long-lived oscillations in the os-DEER spectra were well modeled by a single orientation of the CYP199A2-HaPux complex. The structure is different from the two known Class I CYP-Fdx structures: CYP11A1-Adx and CYP101A1-Pdx. At the protein interface, HaPux residues in the [Fe2S2] cluster-binding loop and the α3 helix, and the C-terminus residue interact with CYP199A2 residues in the proximal loop and the C helix. These residue contacts are consistent with biochemical data on CYP199A2-ferredoxin binding and electron transfer. Electron-tunneling calculations indicate an efficient electron-transfer pathway from the [Fe2S2] cluster to the heme. This new structural model of a CYP-Fdx complex provides the basis for tailoring CYP enzymes for which the cognate ferredoxin is not known, to accept electrons from HaPux and display monooxygenase activity. Postprint

Acetohydroxyacid synthase (AHAS) is the first enzyme in the biosynthesis pathway of the branched-chain amino acids, catalyzing the condensation of pyruvate with another molecule of pyruvate or with 2-ketobutyrate, to produce 2-acetolactate or 2-acetohydroxybutyrate, respectively. The catalytic subunit of the dimeric enzyme has thiamin diphosphate (ThDP), a divalent metal ion, flavin adenine dinucleotide (FAD), and two molecules of oxygen (O-2(I) and O-2(II)) as cofactors. Here, crystal structures of Saccharomyces cerevisiae AHAS in complex with pyruvate provide novel insights into the mechanistic features of this enzyme including: i) The precise position taken by pyruvate molecules as they enter the active site (i.e. prior to catalysis occurring) with conformations suitable for the transfer of electrons to/from O-2(I) and FAD; ii) The formation of ternary donor-acceptor-O-2(I) complexes and iii) The location of O-2(II) relative to the substrate showing that it plays a critical role in the organization of substrate for catalysis. These structural data, accompanied by electron paramagnetic resonance evidence that a radical is produced during AHAS catalysis, lead to the proposal that FAD and O-2 are involved in an indirect one-electron redox cycle. In this mechanism, the spatial configurations of O-2 and FAD in the active site can allow electrons to be exchanged with the substrates and catalytic intermediates to satisfy and control the overall AHAS catalyzed reaction.

Carbon phosphides, Cn Pm , may have highly promising electronic, optical, and mechanical properties, but they are experimentally almost unexplored materials. Phosphaheteroallenes stabilized by N-heterocyclic carbenes undergo a one-electron reduction to yield compounds of the type (L)2 C2 P2 with diverse structures. The use of imidazolylidenes as ligands L give complexes with a central four-membered ring C2 P2 , while more electrophilic cyclic diamidocarbenes (DAC) give a compound with an acyclic π-conjugated CP-PC unit. Cyclic C2 P2 compounds are best described as non-Kekule molecules that are stabilized by coordination to the NHC ligands NHC→(C2 P2 )←NHC. These species can be easily oxidized to give stable radical cations [(NHC)2 C2 P2 ]+. . The remarkably stable molecules with an acylic C2 P2 core are best described with electron-sharing bonds (DAC)=C=P-P=C=(DAC).

Derivatives of 1,2‐dithienylethene (DTE) have superb photochromic properties due to an efficient reversible photocyclization reaction of their hexatriene structure and, thus, have application potential in materials for optoelectronics and (multi‐responsive) molecular switches. Transition‐metal complexes bearing switchable DTE motifs commonly incorporate their coordination site rather distant from the hexatriene system. In this work the redox active ligand 1,2‐bis(2,5‐dimethylthiophen‐3‐yl)ethane‐1,2‐dione is described, which reacts with [V(TMEDA)2Cl2] to give a rare non‐oxido vanadium(IV) species 3(M,M/P,P). This blue complex has two bidentate en‐diolato ligands which chelate the VIV center and give rise to two five‐membered metallacycles with the adjacent hexatriene DTE backbone bearing axial chirality. Upon irradiation with UVA light or prolonged heating in solution, the blue compound 3(M,M/P,P) converts into the purple atropisomer 4(para,M/para,P). Both complexes were isolated and structurally characterized by single‐crystal X‐ray diffraction analysis (using lab source and synchrotron radiation). The antiparallel configuration (M or P helicity) present in both 3(M,M/P,P) and 4(para,M/para,P) is a prerequisite for (reversible) 6π cyclization reactions. A CW EPR spectroscopic study reveals the metalloradical character for 3(M,M/P,P) and 4(para,M/para,P) and indicates dynamic reversible cyclization of the DTE backbone in complex 3(M,M/P,P) at ambient temperature in solution., A rare species: In this work, the redox active ligand 1,2‐bis(2,5‐dimethylthiophen‐3‐yl)ethane‐1,2‐dione is described. Chelation of a vanadium metal center gives rise to an en‐diolate motif in two five‐membered VIV metallacycles with hexatriene DTE backbones bearing axial chirality. Two non‐oxido VIV atropisomeric complexes with metalloradical character were isolated. Their dynamic behavior (reversible cyclization) in solution was studied in a CW EPR spectroscopic study.

CO dehydrogenase (CODH) from the Gram-negative bacterium Oligotropha carboxidovorans is a complex metalloenzyme from the xanthine oxidase family of molybdenum-containing enzymes, bearing a unique binuclear Mo-S-Cu active site in addition to two [2Fe-2S] clusters (FeSI and FeSII) and one equivalent of FAD. CODH catalyzes the oxidation of CO to CO2 with the concomitant introduction of reducing equivalents into the quinone pool, thus enabling the organism to utilize CO as sole source of both carbon and energy. Using a variety of EPR monitored redox titrations and spectroelectrochemistry, we report the redox potentials of CO dehydrogenase at pH 7.2 namely MoVI/V, MoV/IV, FeSI2+/+, FeSII2+/+, FAD/FADH and FADH/FADH−. These potentials are systematically higher than the corresponding potentials seen for other members of the xanthine oxidase family of Mo enzymes, and are in line with CODH utilising the higher potential quinone pool as an electron acceptor instead of pyridine nucleotides. CODH is also active when immobilised on a modified Au working electrode as demonstrated by cyclic voltammetry in the presence of CO.

The facile preparation of alkali salts of phosphanyl cyanophosphides [NHP-PCN]- (NHP=N-heterocyclic phosphenium) is reported. Their formation is achieved by isoelectronic replacement of O for [N]- in the phosphaketenes NHP-PCO using alkaline hexamethyldisilazide M[N(SiMe3)2] (M=Na, K) as reagent. The new anionic entities are versatile PCN building blocks which allow the formation of a diversity of new cyanophosphine derivates including the first example of a PCNB hetero-cumulene and a PCN-ligated transition metal complex.

The Ley–Griffith reaction is utilized extensively in the selective oxidation of alcohols to aldehydes or ketones. The central catalyst is commercially available tetra-n-propylammonium perruthenate (TPAP, n-Pr4N[RuO4]) which is used in combination with the co-oxidant N-methylmorpholine N-oxide (NMO). Although this reaction has been employed for more than 30 years, the mechanism remains unknown. Herein we report a comprehensive study of the oxidation of diphenylmethanol using the Ley–Griffith reagents to show that the rate determining step involves a single alcohol molecule, which is oxidised by a single perruthenate anion; NMO does not appear in rate law. A key finding of this study is that when pure n-Pr4N[RuO4] is employed in anhydrous solvent, alcohol oxidation initially proceeds very slowly. After this induction period, water produced by alcohol oxidation leads to partial formation of insoluble RuO2, which dramatically accelerates catalysis via a heterogeneous process. This is particularly relevant in a synthetic context where catalyst degradation is usually problematic. In this case a small amount of n-Pr4N[RuO4] must decompose to RuO2 to facilitate catalysis.

Antibiotic resistance has emerged as a major threat to global health care. This is largely due to the fact that many pathogens have developed strategies to acquire resistance to antibiotics. Metallo-β-lactamases (MBL) have evolved to inactivate most of the commonly used β-lactam antibiotics. AIM-1 is one of only a few MBLs from the B3 subgroup that is encoded on a mobile genetic element in a major human pathogen. Here, its mechanism of action was characterised with a combination of spectroscopic and kinetic techniques and compared to that of other MBLs. Unlike other MBLs it appears that AIM-1 has two avenues available for the turnover of the substrate nitrocefin, distinguished by the identity of the rate-limiting step. This observation may be relevant with respect to inhibitor design for this group of enzymes as it demonstrates that at least some MBLs are very flexible in terms of interactions with substrates and possibly inhibitors.

MtsZ is a molybdenum-containing methionine sulfoxide reductase that supports virulence in the human respiratory pathogen Haemophilus influenzae (Hi). HiMtsZ belongs to a group of structurally and spectroscopically uncharacterized S-/N-oxide reductases, all of which are found in bacterial pathogens. Here, we have solved the crystal structure of HiMtsZ, which reveals that the HiMtsZ substrate-binding site encompasses a previously unrecognized part that accommodates the methionine sulfoxide side chain via interaction with His182 and Arg166. Charge and amino acid composition of this side chain-binding region vary and, as indicated by electrochemical, kinetic, and docking studies, could explain the diverse substrate specificity seen in closely related enzymes of this type. The HiMtsZ Mo active site has an underlying structural flexibility, where dissociation of the central Ser187 ligand affected catalysis at low pH. Unexpectedly, the two main HiMtsZ electron paramagnetic resonance (EPR) species resembled not only a related dimethyl sulfoxide reductase but also a structurally unrelated nitrate reductase that possesses an Asp-Mo ligand. This suggests that contrary to current views, the geometry of the Mo center and its primary ligands, rather than the specific amino acid environment, is the main determinant of the EPR properties of mononuclear Mo enzymes. The flexibility in the electronic structure of the Mo centers is also apparent in two of three HiMtsZ EPR-active Mo(V) species being catalytically incompetent off-pathway forms that could not be fully oxidized.

More than 80 years after Paneth's report of dimethyl bismuth, the first monomeric Bi(II) radical that is stable in the solid state has been isolated and characterized. Reduction of the diamidobismuth(III) chloride Bi(NON(Ar))Cl (NON(Ar)=[O(SiMe2NAr)2](2-); Ar=2,6-iPr2C6H3) with magnesium affords the Bi(II) radical ˙Bi(NON(Ar)). X-ray crystallographic measurements are consistent with a two-coordinate bismuth in the +2 oxidation state with no short intermolecular contacts, and solid-state SQUID magnetic measurements indicate a paramagnetic compound with a single unpaired electron. EPR and density functional calculations show a metal-centered radical with >90% spin density in a p-type orbital on bismuth.

Alkynes usually oligomerize to give rings with a conjugated π-electron system. In contrast, phosphaalkynes, R-C≡P, frequently give compounds with polycyclic structures, which are thermodynamically more stable than the corresponding π-conjugated isomers. The syntheses of the first CP tricyclic compounds are reported with either radical or cationic ground states stabilized by cyclic (alkyl)(amino)carbenes (CAACs). These compounds may be considered as examples of tricarbontriphosphide coordinated by carbenes and are likely formed via trimerization of the corresponding mono-radicals CAAC-CP. The mechanism for the formation of these tricarbontriphosphide radicals has been rationalized by a combination of experiments and DFT calculations.

Antibiotic resistance is a major global health problem, one that threatens to derail the benefits garnered from arguably the greatest success of modern medicine, the discovery of antibiotics. Among the most potent agents contributing to antibiotic resistance are metallo-β-lactamases (MBLs). The discovery of MBL-like enzymes in microorganisms that are not in contact with the human population is of particular concern as these proteins already have the in-built capacity to inactivate antibiotics, even though they may not need MBL activity for their survival. Here, we demonstrate that a microbiome from a remote and frozen environment in Alaska harbours at least one highly efficient MBL, LRA-8. LRA-8 is homologous to the B3 subgroup of MBLs and has a substrate profile and catalytic properties similar to well-known members of this enzyme family, which are expressed by major human pathogens. LRA-8 is predominantly a penicillinase, but is also active towards carbapenems, but not cephalosporins. Spectroscopic studies indicate that LRA-8 has an active site structure similar to that of other MBLs (in particular B3 subgroup representative AIM-1), and a combination of steady-state and pre-steady-state kinetic data demonstrate that the enzyme is likely to employ a metal ion-bridging hydroxide to initiate catalysis. The rate-limiting step is the decay of a chromophoric, tetrahedral intermediate, as is observed in various other MBLs. Thus, studying the properties of such “pristine” MBL-like proteins may provide insight into the structural plasticity of this family of enzymes that may facilitate functional promiscuity, while important insight into the evolution of MBLs may also be gained.

A new C-nucleoside structurally based on the hydroxyquinoline ligand was synthesized that is able to form stable pairs in DNA in both the absence and the presence of metal ions. The interactions between the metal centers in adjacent CuII-mediated base pairs in DNA were probed by electron paramagnetic resonance (EPR) spectroscopy. The metal-metal distance falls into the range of previously reported values. Fluorescence studies with a donor-DNA-acceptor system indicate that photoinduced charge-transfer processes across these metal-ion-mediated base pairs in DNA occur more efficiently than over natural base pairs. Ace of base: DNA duplex stability can be significantly increased by coordinating CuII ions to hydroxyquinoline base pairs (metal-mediated base pairing; see scheme). Fluorescence studies with a donor-DNA-acceptor system indicate that photoinduced charge-transfer processes across these metal-ion-mediated base pairs in DNA occur more efficiently than over natural base pairs.

An apitite for reduction: The diastereoisomers of the tetrachelating ligand 1,3-bis[(5H-dibenzo[a,d]cyclohepten-5-yl)phenylphosphanyl propane, bis(troppPh)propane, can be used to control the redox potentials of 16-electron rhodium(1) complexes. Whereas the R,S-isomer of the ligand enforces a more planar complex which is difficult to reduce, the R,R(S,S)-isomer favors a tetrahedrally distorted structure which is easily reduced to give a stable rhodium(0) complex (see structure, Rh silver, P yellow, C black, H white).

The initial steps of an enantioselective Diels-Alder reaction catalyzed by a CuII-bissulfoximine complex were followed by EXAFS (EXAFS=extended X-ray absorption fine structure), EPR (EPR=electron paramagnetic resonance) spectroscopy (CW-EPR, FID-detected EPR, pulse ENDOR, HYSCORE; CW=continuous wave; ENDOR=electron nuclear double resonance; HYSCORE=hyperfine sublevel correlation; FID=free induction decay), and UV-visible spectroscopy. The complexes formed between the parent CuX2 (X=Cl-, Br-, TfO-, SbF6-) salts, the chiral bissulfoximine ligand (S,S)-1, and N-(1-oxoprop-2-en-1-yl)oxazolidin-2-one (2) as the substrate in CH2Cl2 were investigated in frozen and fluid solution. In all cases, penta- or hexacoordinated CuII centers were established. The complexes with counterions indicating high stereoselectivity (TfO- and SbF6-) reveal one unique species in which substrate 2 binds to pseudoequatorial positions (via O atoms), shifting the counterions to axial locations. On the other hand, those lacking stereoselectivity (X=Cl- and Br-) form two species in which the parent halogen anions remain at equatorial positions preventing the formation of geometries compatible with those found for X=TfO- and SbF6-.

The new tetradentate ligand 1,4-bis(5 H-dibenzo[a,d]cyclohepten-5-yl)-1,4-diazabuta-1,3-diene ((H)tropdad) allows the syntheses of the 16-electron cationic rhodium complexes [M((H)tropdad)](O(3)SCF(3)) (M=Rh, Ir). The structure of the rhodium complex was determined by X-ray analysis and points to a description of these as [M(+1)((H)tropdad)(0)] with short Cd-N bonds (av 1.285 A) and a long C-C bond (1.46 A) in the diazabutadiene (dad) moiety, that is the M-->dad charge-transfer is negligible. Both [Rh((H)tropdad)](+) and [Ir((H)tropdad)](+) are reduced at very low potentials (E(1) (1/2)= -0.56 V and E(1) (1/2)=-0.35 V, respectively) which allowed the quantitative synthesis of the neutral paramagnetic complexes [M((H)tropdad)](0) (M=Rh, Ir) by reacting the cationic precursor complexes simply with zinc powder. The [M((H)tropdad)](0) complexes are stable against protic reagents in organic solvents. Continuous wave and pulse EPR spectroscopy was used to characterize the paramagnetic species and the hyperfine coupling constants were determined: [Rh((H)tropdad)](0): A(iso)((14)N)=11.9 MHz, A(iso)((1)H)=14.3 MHz, A(iso)((103)Rh)= -5.3 MHz; [Ir((H)tropdad)](0): A(iso)((14)N)=11.9 MHz, A(iso)((1)H)=14.3 MHz. In combination with DFT calculations, the experimentally determined g and hyperfine matrices could be orientated within the molecular frame and the dominant spin density contributions were determined. These results clearly show that the complexes [M((H)tropdad)](0) are best described as [M(+1)((H)tropdad)(.-)] with a [16+1] electron configuration.

JBIC Journal of Biological Inorganic Chemistry, 13 (8), ISSN:0949-8257, ISSN:1432-1327

(Chemical Equation Presented) The spin reveals all: Electron paramagnetic resonance spectroscopy reveals the formation of a nickel-alkyl bond in the active site of methyl-coenzyme M reductase (MCR), the key enzyme of methanogenesis, when active MCRred1 reacts with the irreversible inhibitor 3-bromopropane sulfonate to form the MCRBPS species. © 2006 Wiley-VCH Verlag GmbH and Co. KGaA.

The unusual [4Fe-3S] cluster proximal to the active site plays a crucial role in allowing a class of [NiFe]-hydrogenases to function in the presence of O(2) through its unique ability to undergo two rapid, consecutive one-electron transfers. This property helps to neutralize reactive oxygen species. Mechanistic details and the role of the medial and distal clusters remain unresolved. To probe the Fe-S relay, continuous wave and pulse electron paramagnetic resonance (EPR) studies were conducted on the O(2)-tolerant hydrogenase from Escherichia coli (Hyd-1) and three variants with point mutations at the proximal and/or medial clusters. Reduction potentials of the proximal ([4Fe-3S](5+/4+/3+)) and medial ([3Fe-4S](+/0)) clusters were determined by potentiometry. The medial [3Fe-4S](+/0) reduction potential is exceptionally high, implicating a mechanistic role in O(2)-tolerance. Numerous experiments establish that the distal cluster has a ground state S > 1/2 in all three variants and indicate that this is also the case for native Hyd-1. Concurrent with the Hyd-1 crystal structure, EPR data for the 'superoxidized' P242C variant, in which the medial cluster is 'magnetically silenced', reveal two conformations of the proximal [4Fe-3S](5+) cluster, and X-band HYSCORE spectroscopy shows two (14)N hyperfine couplings attributed to one conformer. The largest, A((14)N) = [11.5,11.5,16.0] ± 1.5 MHz, characterizes the unusual bond between one Fe (Fe(4)) and the backbone amide-N of cysteine-20. The second, A((14)N) = [2.8,4.6,3.5] ± 0.3 MHz, is assigned to N(C19). The (14)N hyperfine couplings are conclusive evidence that Fe(4) is a valence-localized Fe(3+) in the superoxidized state, whose formation permits an additional electron to be transferred rapidly back to the active site during O(2) attack.

The Fe(IV)oxo complex of a coordinatively flexible multidentate mono-carboxylato ligand is obtained by the one electron oxidation of a low spin Fe(III) precursor in water.

We report the synthesis of a series of ruthenium complexes supported by the phosphine olefin ligand tropPPh2 (trop=5-H-dibenzo-[a,d]cyclohepten-5-yl) in the oxidation states 0, +I, and +II, formed via successive one-electron oxidization steps from Ru(0) (tropPPh2 )2 . The bidentate character of the tropPPh2 ligand and its steric hindrance force the complexes to adopt uncommon geometries, which were investigated by X-ray diffraction analysis. EPR data of the mononuclear Ru(I) complex reveal couplings of the unpaired spin with the ruthenium and two phosphorus nuclei, as well as the olefinic protons which show that the spin is mainly localized on the Ru(I) center.

A range of sol-gel synthesis conditions were used to prepare high surface area N-doped TiO(2) in the anatase phase. The N dopant was derived either from NH(3) in solution or from NH(3) gas bubbled through solution. Bulk N doping levels were determined by an inert gas fusion method and were compared with surface N doping levels determined by X-ray photoelectron spectroscopy. Comparison was also made with the concentration of paramagnetic species measured by electron spin resonance spectroscopy. It was found that both surface and bulk doping levels were typically around 3 orders of magnitude higher than the concentration of paramagnetic N-containing species. All N-doped samples showed higher visible region (lambda > 395 nm) photocatalytic activity than undoped anatase itself. It is argued that catalytic activity is associated with the presence of nitrogen bound to lattice oxygen to give (NO)'O which can be photoexcited to give (NO)(O)(X).

Methanogenic archaea utilize a specific pathway in their metabolism, converting C1 substrates (i.e., CO2) or acetate to methane and thereby providing energy for the cell. Methyl-coenzyme M reductase (MCR) catalyzes the key step in the process, namely methyl-coenzyme M (CH3-S-CoM) plus coenzyme B (HS-CoB) to methane and CoM-S-S-CoB. The active site of MCR contains the nickel porphinoid F430. We report here on the coordinated ligands of the two paramagnetic MCR red2 states, induced when HS-CoM (a reversible competitive inhibitor) and the second substrate HS-CoB or its analogue CH3-S-CoB are added to the enzyme in the active MCR red1 state (Ni(I)F430). Continuous wave and pulse EPR spectroscopy are used to show that the MCR red2a state exhibits a very large proton hyperfine interaction with principal values A((1)H) = [-43,-42,-5] MHz and thus represents formally a Ni(III)F430 hydride complex formed by oxidative addition to Ni(I). In view of the known ability of nickel hydrides to activate methane, and the growing body of evidence for the involvement of MCR in "reverse" methanogenesis (anaerobic oxidation of methane), we believe that the nickel hydride complex reported here could play a key role in helping to understand both the mechanism of "reverse" and "forward" methanogenesis.

(Chemical Equation Presented) Rh steps into the ring: The radical cation shown is best described as a rhodaazacyclopropane (A) rather than as a Rh I iminium ion complex (B). EPR and DFT data indicate that the delocalization of the unpaired electron into the ligand framework contributes to the stability of A (see plot of the singly occupied molecular orbital). © 2006 Wiley-VCH Verlag GmbH and Co. KGaA.

Methyl-coenzyme M reductase (MCR) catalyses the reduction of methyl-coenzyme M (CH3-S-CoM) with coenzyme B (H-S-CoB) to CH4 and CoM-S-S-CoB in methanogenic archaea. Here we present a pulse EPR study of the "ready" form MCR(ox1), providing a detailed description of the spin density and the coordination of coenzyme M (CoM) to the Ni cofactor F430. To achieve this, MCR was purified from cells grown in a 61Ni enriched medium and samples were prepared in D2O with the substrate analogue CoM either deuterated in the beta-position or with 33S in the thiol group. To obtain the magnetic parameters ENDOR and HYSCORE measurements were done at X- and Q-band, and CW EPR, at X- and W-band. The hyperfine couplings of the beta-protons of CoM indicate that the nickel to beta-proton distances in MCR(ox1) are very similar to those in Ni(II)-MCR(ox1-silent), and thus the position of CoM relative to F430 is very similar in both species. Our thiolate sulfur and nickel EPR data prove a Ni-S coordination, with an unpaired spin density on the sulfur of 7 +/- 3%. These results highlight the redox-active or noninnocent nature of the sulfur ligand on the oxidation state. Assuming that MCR(ox1) is oxidized relative to the Ni(II) species, the complex is formally best described as a Ni(III) (d7) thiolate in resonance with a thiyl radical/high-spin Ni(II) complex, Ni(III)-(-)SR Ni(II)-*SR.

Encapsulation of the Na+ ions of the [Na2(P 4R4)] ion triple with a R,R or S,S configuration at the inner phosphorus atoms by cryptands has enabled cation-separated dianions to be obtained in a crystalline form. Fragmentation of the {Na([2.2.2]cryptand)} 2(P4R4) into radical anions (P 2R2).- occurs in solution (see scheme), as determined by EPR spectroscopy.

Evidence is presented of significant covalency in the ytterbium 4f shell of tris-cyclopentadienyl ytterbium (YbCp(3)) in its electronic ground state, that can be represented by the superposition of an ionic configuration Yb(III):4f(13)(Cp(3)) and a charge-transfer configuration Yb(II):4f(14)(Cp(3))(-1). The relative weights of these configurations were determined from (i) the difference in their 4f photoionization cross sections, (ii) the accumulation of spin-density centered on the (13)C atoms of the Cp ring, as measured by a pulsed EPR (HYSCORE) experiment, (iii) the reduction in the spin-density in the 4f shell, manifest in the (171)Yb hyperfine interaction, and (iv) the principal values of the g-tensor, obtained from the EPR spectrum of a frozen glass solution at 5.4 K. Each of these methods finds that the spin density attributable to the charge transfer configuration is in the range 12-17%. The presence of configuration interaction (CI) also accounts for the highly anomalous energies, intensities, and vibronic structure in the "f-f" region of the optical spectrum, as well as the strict adherence of the magnetic susceptibility to the Curie law in the range 30-300 K.

The structural, chemical and electronic changes of ceria as a function of decreasing particle size have been studied: at sizes below 5 nm the total amount of reducible oxygen dramatically increases due to superoxide formation on the ceria surface, a result indicative of a size dependent oxygen buffering capacity. © The Royal Society of Chemistry 2010.

"Chemical Equation Presented" Radical changes: One single P-Rh-P angle determines whether the odd electron in the paramagnetic complex [Rh(trop2PPh)(PPh3)] is delocalized over the whole molecule (see picture, blue) or is localized on the P - Rh unit (red). The two energetically almost degenerate electromers exist in a fast equilibrium, and the "red" complex has the highest spin density at Rh of all low valent Rh complexes observed to date. © 2010 Wiley-VCH Verlag GmbH and Co. KGaA.

Methyl-coenzyme M reductase (MCR) is the key enzyme in methane formation by methanogenic Archaea. It converts the thioether methyl-coenzyme M and the thiol coenzyme B into methane and the heterodisulfide of coenzyme M and coenzyme B. The catalytic mechanism of MCR and the role of its prosthetic group, the nickel hydrocorphin coenzyme F(430), is still disputed, and no intermediates have been observed so far by fast spectroscopic techniques when the enzyme was incubated with the natural substrates. In the presence of the competitive inhibitor coenzyme M instead of methyl-coenzyme M, addition of coenzyme B to the active Ni(I) state MCR(red1) induces two new species called MCR(red2a) and MCR(red2r) which have been characterized by pulse EPR spectroscopy. Here we show that the two MCR(red2) signals can also be induced by the S-methyl- and the S-trifluoromethyl analogs of coenzyme B. (19)F-ENDOR data for MCR(red2a) and MCR(red2r) induced by S-CF(3)-coenzyme B show that, upon binding of the coenzyme B analog, the end of the 7-thioheptanoyl chain of coenzyme B moves closer to the nickel center of F(430) by more than 2 A as compared to its position in both, the Ni(I) MCR(red1) form and the X-ray structure of the inactive Ni(II) MCR(ox1-silent) form. The finding that the protein is able to undergo a conformational change upon binding of the second substrate helps to explain the dramatic change in the coordination environment induced in the transition from MCR(red1) to MCR(red2) forms and opens the possibility that nickel coordination geometries other than square planar, tetragonal pyramidal, or elongated octahedral might occur in intermediates of the catalytic cycle.

The enterobacterium Escherichia coli synthesizes two H(2) uptake enzymes, Hyd-1 and Hyd-2. We show using precise electrochemical kinetic measurements that the properties of Hyd-1 and Hyd-2 contrast strikingly, and may be individually optimized to function under distinct environmental conditions. Hyd-2 is well suited for fast and efficient catalysis in more reducing environments, to the extent that in vitro it behaves as a bidirectional hydrogenase. In contrast, Hyd-1 is active for H(2) oxidation under more oxidizing conditions and cannot function in reverse. Importantly, Hyd-1 is O(2) tolerant and can oxidize H(2) in the presence of air, whereas Hyd-2 is ineffective for H(2) oxidation under aerobic conditions. The results have direct relevance for physiological roles of Hyd-1 and Hyd-2, which are expressed in different phases of growth. The properties that we report suggest distinct technological applications of these contrasting enzymes.

By deprotonation of the corresponding tris(pyrazolyl)methane ((Me)Tpm) precursor complexes [M((Me)Tpm)(2)][(OTf)(2)] (1 and 2), the zwitterionic tris(pyrazolyl)methanide iron(II) (3) and cobalt(II) (4) "sandwich" complexes of the general formula [M((Me)Tpmd)(2)] are easily accessible. The structurally characterised complexes 3 and 4 are the first such homoleptic 3d transition metal species to feature two "naked", outward pointing pyramidal carbanions. Density functional theory calculations show metal-centred HOMOs and LUMOs with the destabilised carbanion orbitals in the energy region of the filled transition-metal-centred frontier orbitals. The electronic structures of these complexes have been investigated in detail by various spectroscopic techniques such as NMR, EPR, SQUID, Mössbauer, etc. Both complexes adopt a high-spin (HS) configuration at room temperature in solution and in the solid state. A thermally induced high-spin to low-spin (HS-LS) transition is observed for the iron(II) complex 3. The HS-LS transition temperature of 3 in solution differs from that in the solid state, which in turn strongly depends on the amount of solvent molecules in the crystal lattice. Electrochemical studies on the corresponding cobalt(II) complex 4 provide evidence for a HS-Co(II) LS-Co(III) transition upon oxidation, which was confirmed by preliminary synthetic oxidation reactions. Overall, it can be concluded that the related kappa(3)N-donor ligands tris(pyrazolyl)hydroborates (Tp(R)) and (R)Tpmd ligands have similar bonding properties and that the metal cations experience more or less the same ligand environment.

(Chemical Equation Presented) A little goes a long way: An iridium-nitrogen-radical complex is a highly active and selective catalyst for the dehydrogenation (oxidation) of primary alcohols to aldehydes in the presence of the oxidant benzoquinone (see simplified scheme). With only 0.01 mol% of the complex, turnover frequencies of up to 150 000 s-1 are reached. © 2007 Wiley-VCH Verlag GmbH and Co. KGaA.

An organometallic methyl-nickel species was detected in the enzyme methyl-coenzyme M reductase (MCR). This is the key enzyme in microbial methane production and is probably also involved in anaerobic methane oxidation. Incubation of MCR with 13C-bromomethane results in the formation of an electron paramagnetic resonance (EPR) active nickel-methyl species in the active site of this enzyme. High-resolution pulse electron nuclear double resonance and hyperfine sublevel correlation investigations showed the presence of 13C hyperfine couplings of 18-44 MHz that are associated with the nickel-based EPR signal. The large 13C hyperfine interaction shows unambiguously that the methyl group from bromomethane is directly coordinated to the nickel ion. The EPR structure and parameters are fully supported by density functional theory calculations. Copyright © 2007 American Chemical Society.

A [16 + 1] valence electron configured rhodium aminyl radical complex could be synthesized and characterized in detail by pulse EPR spectroscopy and DFT calculations. The unpaired electron is delocalized over the metal center and two adjacent nitrogens. H-abstraction reactions from thiols and triethylsilane show that the spin density is predominantly localized on both nitrogens.

This review discusses the application of pulse EPR to the characterization of disordered systems, with an emphasis on samples containing transition metals. Electron nuclear double-resonance (ENDOR), electron-spin-echo envelope-modulation (ESEEM), and double electron-electron resonance (DEER) methodologies are outlined. The theory of field modulation is outlined, and its application is illustrated with DEER experiments. The simulation of powder spectra in EPR is discussed, and strategies for optimization are given. The implementation of this armory of techniques is demonstrated on a rich variety of chemical systems: several porphyrin derivatives that are found in proteins and used as model systems, otherwise highly reactive aminyl radicals stabilized with electron-rich transition metals, and nitroxide-copper-nitroxide clusters. These examples show that multi-frequency continuous-wave (CW) and pulse EPR provides detailed information about disordered systems. © 2006 Verlag Helvetica Chimica Acta AG.

Metal-stabilized phenoxyl radicals appear to be important intermediates in a variety of enzymatic oxidations. We report that transition metal coordination also supports an aminyl radical, resulting in a stable crystalline complex: [Rh(I)(trop 2 N . )(bipy)] + OTf – (where trop is 5-H-dibenzo[a,d]cycloheptene-5-yl, bipy is 2,2′-bipyridyl, OTf – is trifluorosulfonate). It is accessible under mild conditions by one-electron oxidation of the amide complex [Rh(I)(trop 2 N)(bipy)], at a potential of –0.55 volt versus ferrocene/ferrocenium. Both electron paramagnetic resonance spectroscopy and density functional theory support 57% localization of the unpaired spin at N. In reactions with H-atom donors, the Rh-coordinated aminyl behaves as a nucleophilic radical.

The structure of Cu(II) complex 3 formed within the course of a stereoselective Diels-Alder reaction was investigated by EXAFS, CW-EPR at X- and W-band, HYSCORE, pulsed ENDOR, and UV-vis spectroscopy. The experimental techniques indicate that the chiral bis(sulfoximine) ligand (S,S)-1 and the dienophile form a tetragonally distorted complex in CH(2)Cl(2). The ligand binds to the Cu(II) center via the imine nitrogens, whereas the dienophile interacts via the carbonyl oxygen atoms. The additional sites of the first coordination sphere are occupied by counterions and, presumably, solvent molecules. At the axial position, a triflate anion binds via an oxygen atom.

The 17-electron complexes [M(tropp(ph))2] (M=Co0, Rh0, Ir0) were prepared and isolated (tropp = tropylidene phosphane). A structural analysis of [Co(tropp(ph))2] revealed this complex to be almost tetrahedral, while the heavier homologues have more planar structures. Partially deuterated tropp complexes [D6][M(tropp(ph))2] were synthesised for M = Rh and Ir in order to enhance the resolution in the EPR spectra. This synthesis involves a four-fold intramolecular C-H activation reaction, whereby alkyl groups are transformed into olefins. Dihydrides were observed as intermediates for M = Ir. The electronic and geometric structures of all complexes [M(tropp(ph))2] (M = Co, Rh, Ir) and [D6][M(tropp(ph))2] (M = Rh, Ir) were investigated by continuous wave (CW) and echo-detected EPR in combination with pulse ENDOR and ESEEM techniques. In accord with their planar structures, cis and trans isomers were detected for [M(tropp(ph))2] (M = Rh0, Ir0) for which a dynamic equilibrium was established. The thermodynamic data show that the cis isomer is slightly preferred by deltaH(o) = -4.7 +/- 0.3 kJ mol(-1) (M = Rh) and delta H(o) = -5.1 +/- 0.5 kJ mol(-1); (M = Ir). The entropies for the process trans-[M(tropp(ph))2] cis-[M(tropp(ph))2] are also negative [deltaS(o) = -5 +/- 1.5 J mol(-1) (M = Rh); deltaS(o) = -17 +/- 3.7 J mol(-1) (M = Rh)], indicating higher steric congestion in the cis isomers. The cobalt(0) and irdium(0) complexes show rather large g anisotropies, while that of the rhodium(0) complex is small (Co: g(parallel) = 2.320, g(perpendicular) = 2.080; cis-Rh: g(parallel) = 2.030, g(perpendicular) = 2.0135; trans-Rh: g(parallel) = 2.050, g(perpendicular) = 2.030; cis-Ir: g(parallel) = 2.030, g(perpendicular) = 2.060; trans-Ir: g(parallel) = 1.980, g(perpendicular) = 2.150). The g matrices of [M(tropp(ph))2] (M = Co, Rh) are axially symmetric with g(parallel) > g(perpendicular), indicating either a distorted square planar structure (SOMO essentially d(x2 - y2) or a compressed tetrahedron (SOMO essentially d(xy)). Interestingly, for [Ir(tropp(ph))2] the inverse ordering, g(perpendicular) > g(parallel) is found; this cannot be explained by simple ligand field arguments and must await a more sophisticated analysis. The hyperfine interactions of the unpaired electron with the metal nuclei, phosphorus nuclei, protons, deuterons and carbon nuclei were determined. By comparison with atomic constants, the spin densities on these centres were estimated and found to be small. However, the good agreement of the distance between the olefinic protons and the metal centres determined from the dipolar coupling parameter indicates that the unpaired electron is primarily located at the metal centre.

The interaction of Cu(II) with the ligand tdci (1,3,5-trideoxy-1,3,5-tris(dimethylamino)-cis-inositol) was studied both in the solid state and in solution. The complexes that were formed were also tested for phosphoesterase activity. The pentanuclear complex [Cu(5)(tdciH(-2))(tdci)(2)(OH)(2)(NO(3))(2)](NO(3))(4).6H(2)O consists of two dinuclear units and one trinuclear unit, having two shared copper(II) ions. The metal centers within the pentanuclear structure have three distinct coordination environments. All five copper(II) ions are linked by hydroxo/alkoxo bridges forming a Cu(5)O(6) cage. The Cu-Cu separations of the bridged centers are between 2.916 and 3.782 A, while those of the nonbridged metal ions are 5.455-5.712 A. The solution equilibria in the Cu(II)-tdci system proved to be extremely complicated. Depending on the pH and metal-to-ligand ratio, several differently deprotonated mono-, di-, and trinuclear complexes are formed. Their presence in solution was supported by mass, CW, and pulse EPR spectroscopic study, too. In these complexes, the metal ions are presumed to occupy tridentate [O(ax),N(eq),O(ax)] coordination sites and the O-donors of tdci may serve as bridging units between two metal ions. Additionally, deprotonation of the metal-bound water molecules may occur. The dinuclear Cu(2)LH(-3) species, formed around pH 8.5, provides outstanding rate acceleration for the hydrolysis of the activated phosphodiester bis(4-nitrophenyl)phosphate (BNPP). The second-order rate constant of BNPP hydrolysis promoted by the dinuclear complex (T = 298 K) is 0.95 M(-1) s(-1), which is ca. 47600-fold higher than that of the hydroxide ion catalyzed hydrolysis (k(OH)). Its activity is selective for the phosphodiester, and the hydrolysis was proved to be catalytic. The proposed bifunctional mechanism of the hydrolysis includes double Lewis acid activation and intramolecular nucleophilic catalysis.