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Oxygen-containing mononuclear iron species--iron(m)-peroxo, iron(III)--hydroperoxo and iron(IV)--oxo--are key intermediates in the catalytic activation of dioxygen by iron-containing metalloenzymes (1-7). It has been difficult to generate synthetic analogues of these three active iron-oxygen species in identical host complexes, which is necessary to elucidate changes to the structure of the iron centre during catalysis and the factors that control their chemical reactivities with substrates. Here we report the high-resolution crystal structure of a mononuclear non-haem side-on iron(III)-peroxo complex, [[Fe(III)(TMC)(OO)].sup.+]. We also report a series of chemical reactions in which this iron(III)-peroxo complex is cleanly converted to the iron(III)-hydroperoxo complex, [[Fe(III)(TMC)(OOH)].sup.2+], via a short-lived intermediate on protonation. This iron(III)-hydroperoxo complex then cleanly converts to the ferryl complex, [[Fe(IV)(TMC)(O)].sup.2+], via homolytic O-O bond cleavage of the iron(III)-hydroperoxo species. All three of these iron species--the three most biologically relevant iron-oxygen intermediates--have been spectroscopically characterized; we note that they have been obtained using a simple macrocyclic ligand. We have performed relative reactivity studies on these three iron species which reveal that the iron(III)-hydroperoxo complex is the most reactive of the three in the deformylation of aldehydes and that it has a similar reactivity to the iron(IV)-oxo complex in C-H bond activation of alkylaromatics. These reactivity results demonstrate that iron(III)-hydroperoxo species are viable oxidants in both nucleophilic and electrophilic reactions by iron-containing enzymes., TMC (1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane) is a macrocyclic ligand of remarkable versatility in the field of biomimetic chemistry of dioxygen activation by metal complexes. A variety of metal complexes of superoxo, peroxo and [...]

Bleomycin (BLM) is a glycopeptide anticancer drug capable of effecting single- and double-strand DNA cleavage. The last detectable intermediate prior to DNA cleavage is a low spin [Fe.sup.III] peroxy level species, termed activated bleomycin (ABLM). DNA strand scission is initiated through the abstraction of the C-4' hydrogen atom of the deoxyribose sugar unit. Nuclear resonance vibrational spectroscopy (NRVS) aided by extended X-ray absorption fine structure spectroscopy and density functional theory (DFT) calculations are applied to define the natures of [Fe.sup.III]BLM and ABLM as (BLM)[Fe.sup.III]--OH and (BLM/[Fe.sup.III]([[eta].sup.1]-OOH/species, respectively. The NRVS spectra of [Fe.sup.III]BLM and ABLM are strikingly different because in ABLM the [delta]Fe--O--O bending mode mixes with, and energetically splits, the doubly degenerate, intense O--Fe--[N.sub.ax] transaxial bends. DFT calculations of the reaction of ABLM with DNA, based on the species defined by the NRVS data, show that the direct H-atom abstraction by ABLM is thermodynamically favored over other proposed reaction pathways. nonheme iron | structure/reactivity doi/ 10.1073/pnas.1016323107

The P-cluster of nitrogenase is one of the most complex biological metallocenters known to date. Despite the recent advances in the chemical synthesis of P-cluster topologs, the biosynthetic mechanism of P-cluster has not been well defined. Here, we present a combined biochemical, electron paramagnetic resonance, and Xray absorption spectroscopy/extended X-ray absorption fine-structure investigation of the maturation process of P-clusters in [DELTA]nifH molybdenum-iron (MoFe) protein. Our data indicate that the previously identified, [[Fe.sub.4][S.sub.4]]-like cluster pairs in [DELTA]nifH MoFe protein are indeed the precursors to P-clusters, which can be reductively coupled into the mature [[Fe.sub.8][S.sub.7]] structures in the presence of Fe protein, MgATP, and dithionite. Moreover, our observation of a biphasic maturation pattern of P-clusters in [DELTA]nifH MoFe protein provides dynamic proof for the previously hypothesized, stepwise assembly mechanism of the two P-clusters in the [[alpha].sub.2]/[[beta].sub.2]- tetrameric MoFe protein, i.e., one P-cluster is formed in one [alpha][beta] dimer before the other in the second [alpha][beta] dimer. assembly | biosynthesis www.pnas.org/cgi/doi/10.1073/pnas.0909149106

Two terminal Au-oxo compounds are prepared and characterized by many physicochemical methods. These two compounds have established that both solids are diamagnetic and [super 31]P and [super 17]O NMR spectroscopy has confirmed that both have remained diamagnetic in solution.

Multicopper oxidases (MCOs) catalyze the [4e.sup.-] reduction of [O.sub.2] to [H.sub.2]O. The reaction of the fully reduced enzyme with [O.sub.2] generates the native intermediate (NI), which undergoes a slow decay to the resting enzyme in the absence of substrate. NI is a fully oxidized form, but its spectral features are very different from those of the resting form (also fully oxidized), because the type 2 and the coupled-binuclear type 3 Cu centers in the [O.sub.2]-reducing trinuclear Cu cluster site are isolated in the resting enzyme, whereas these are all bridged by a [[micro].sub.3]-oxo ligand in NI. Notably, the one azide-bound NI ([NI.sub.Az]) exhibits spectral features very similar to those of NI, in which the [[micro].sub.3]-oxo ligand in NI has been replaced by a [[micro].sub.3]-bridged azide. Comparison of the spectral features of NI and [NI.sub.Az], combined with density functional theory (DFT) calculations, allows refinement of the NI structure. The decay of NI to the resting enzyme proceeds via successive proton-assisted steps, whereas the ratelimiting step involves structural rearrangement of the [[micro.sub.3]-oxo-bridge from inside to outside the cluster. This phenomenon is consistent with the slow rate of NI decay that uncouples the resting enzyme from the catalytic cycle, leaving NI as the catalytically relevant fully oxidized form of the MCO active site. The all-bridged structure of NI would facilitate electron transfer to all three Cu centers of the trinuclear cluster for rapid proton-coupled reduction of NI to the fully reduced form for catalytic turnover. laccase | superexchange | electron paramagnetic resonance | magnetic circular dicroism | density functional theory

The sulfur K-edge X-ray absorption spectroscopy (XAS) which was used to study a series of [Ni.sup.II] complexes based on the N,N'-bis(2-mercaptoethyl)-1,5-diazacyclootanato (DACO) ligand system revealed that RS[O.sub.2.sup.-] is comparable to R[S.sup.-] in [sigma] donation but is a worse [pi] donor.

A coalition of Cu L-edge and S K-edge X-ray absorption data and density functional theory calculations is correlated with [super 33]S electron paramagnetic resonance superhyperline results to obtain the dipole integral for the S 1s->3p transition for the dithiolene ligand maleonitriledithiolate (MNT). The conclusions are combined with the [l.sub.s] of sulfide to experimentally obtain a relation between the S 1s->4p transition energy and dipole integral over a large range of [Q.sup.S.sub.mol].

A detailed assessment of sulfur in native reticulated vitreous carbon (RVC) and in RVC following oxidative surface modification is reported using elemental analysis and sulfur K-edge X-ray absorption spectroscopy (XAS). It is observed that the detection of episulfide directly corroborates a proposed mechanism for the migration of elemental sulfur through carbon.

FeMo cofactor (FeMoco) biosynthesis is one of the most complicated processes in metalloprotein biochemistry. Here we show that Mo and homocitrate are incorporated into the Fe/S core of the FeMoco precursor while it is bound to NifEN and that the resulting fully complemented, FeMoco-like cluster is transformed into a mature FeMoco upon transfer from NifEN to MoFe protein through direct protein-protein interaction. Our findings not only clarify the process of FeMoco maturation, but also provide useful insights into the other facets of nitrogenase chemistry. biosynthesis | nitrogenase

The Fe protein is indispensable for nitrogenase catalysis and biosynthesis. However, its function in iron-molybdenum cofactor (FeMoco) biosynthesis has not been clearly defined. Here we show that the Fe protein can act as a Mo/homocitrate insertase that mobilizes Mo/homocitrate for the maturation of FeMoco precursor on NifEN. Further, we establish that Mo/homocitrate mobilization by the Fe protein likely involves hydrolysis of MgATP and protein-protein interaction between the Fe protein and NifEN. Our findings not only clarify the role of the Fe protein in FeMoco assembly and assign another function to this multitask enzyme but also provide useful insights into a mechanism of metal trafficking required for the assembly of complex metalloproteins such as nitrogenase. biosynthesis

Distinct spectral features at the Fe L-edge of the two compounds K3[Fe(CN)6] and K4[Fe(CN)6] are identified and characterized as arising from contributions of the ligand pi* orbitals due to metal-to-ligand back-bonding. The analysis of L-edge spectral shape, energy shift, and total intensity demonstrates that Fe L-edge X-ray absorption spectroscopy provides a direct probe of metal-to-ligand back-bonding.

An attempt is made to evaluate the geometric and electronic structures of two mononuclear LCu[O.sub.2] systems using Cu K- and L-edge X-ray absorption spectroscopy (XAS) coupled with calculations. The XAS data, particularly the L-edge intensities, clearly demonstrates the Cu(II)-[O.sub.2-] and Cu(III)-[[O.sub.2].sup.2-] natures of [Cu[(O.sub.2)][HB[(3-Ad.5.Prpz).sub.3]]] and [Cu[(O.sub.2)]([beta]-diketiminate)] respectively.

A binuclear copper enzyme, a mu-eta(super 2):eta(super 2)-peroxodicopper (II) species is accepted generally to be the active oxidant, in tyrosinase. Reports reveal the characterization and reactivity of a mu-eta(super 2):eta(super 2)-peroxodicopper (II) complex synthesized by reacting the Cu(I) complex of the secondary diamine ligand N,N'-di-tert-butyl-ethylenediamine (DBED), ((DBED)Cu(MeCN))(X) with O2 at 193 K to give ({Cu(DBED)}2(O2))(X2).

The iron-molybdenum cofactor (FeMoco) of the nitrogenase MoFe protein is a highly complex metallocluster that provides the catalytically essential site for biological nitrogen fixation. FeMoco is assembled outside the MoFe protein in a stepwise process requiring several components, including NifB-co, an iron- and sulfur-containing FeMoco precursor, and NifEN, an intermediary assembly protein on which NifB-co is presumably converted to FeMoco. Through the comparison of Azotobacter vinelandii strains expressing the NifEN protein in the presence or absence of the nifB gene, the structure of a NifEN-bound FeMoco precursor has been analyzed by x-ray absorption spectroscopy. The results provide physical evidence to support a mechanism for FeMoco biosynthesis. The NifEN-bound precursor is found to be a molybdenum-free analog of FeMoco and not one of the more commonly suggested cluster types based on a standard [4Fe-4S] architecture. A facile scheme by which FeMoco and alternative, non-molybdenum-containing nitrogenase cofactors are constructed from this common precursor is presented that has important implications for the biosynthesis and biomimetic chemical synthesis of FeMoco. biosynthesis | extended x-ray absorption fine structure | nitrogenase | x-ray absorption spectroscopy

Sulfur K-edge X-ray absorption spectroscopy (XAS) and density functional theory (DFT) calculations are used to find the geometric and electronic structure of the active site of the non-heme iron enzyme nitrile hydratase (NHase). The pre-edge region of the X-ray absorption spectrum is sensitive to the Zeff of the Fe and discloses that the Fe in [FeNO](super 6) NHase species has a Zeff very similar to that of its photolyzed Fe(super III) counterpart.

Nitrogenase is a multicomponent metalloenzyme that catalyzes the conversion of atmospheric dinitrogen to ammonia. For decades, it has been generally believed that the [8Fe-7S] P-cluster of nitrogenase component I is indispensable for nitrogenase activity. In this study, we identified two catalytically active P-cluster variants by activity assays, metal analysis, and EPR spectroscopic studies. Further, we showed that both P-cluster variants resemble [4Fe-4S]-like centers based on x-ray absorption spectroscopic experiments. We believe that our findings challenge the dogma that the standard P-cluster is the only cluster species capable of supporting substrate reduction at the FeMo cofactor and provide important insights into the general mechanism of nitrogenase catalysis and assembly. MoFe protein | VFe protein

Sulfur K-edge X-ray absorption spectroscopy (XAS) is employed to directly probe ligand-metal bond covalency, where it is found that protein active sites are significantly less covalent than their related model complexes. Sulfur K-edge XAS data are reported on a series of P450 model complexes with increasing H-bonding to the ligated thiolate from its substituent.

The geometric and electronic structure of the untethered heme-peroxo-copper complex [(F8TPP)Fe(super III)-(O2(super 2))-Cu(super II)(TMPA)](CIO4) is investigated using Cu and K-edge EXAFS spectroscopy and density functional theory calculations in order to describe its geometric and electronic structure. Results reveal that the Fe and Cu K-edge EXAFS data were fit with a Cu...Fe distance of ~3.72 angstrom.

A series of metal-varied [ML(SC6F5)] model complexes (where L-hydrotris(3,5-diisopropyl-1-pyrazolyl)borate and M=Mn, Fe, Co, Ni, Cu, and Zn) related to blue copper proteins are studied. The results provide insight into the Irving-Williams series, where it is found that the bonding of the ligand being replaced by the thiolate makes a major contribution to the observed order of the stability constants over the series of metal ions.

Sulfur K-edge X-ray absorption spectroscopy (XAS) of the model complex [Fe(sub 4)S(sub 4)](super 1+, 2+, 3+) having both thiolate and sulfide ligands are studied and compared to results from DFT calculations on the resulting [Fe(sub 4)S(sub 4)](super 2+). The results reveal that the redox active molecular orbital (RAMO) of high potential proteins (HiPIPs) has 50% ligand character, and hence the HiPIP redox couple involves limited electronic relaxation.

A methodology for ascertaining the total covalency and the differential orbital covalency (DOC) that is, the differences in covalency in the different symmetry sets of the d orbitals is proposed. To account for the one-electron transition pathways available into the additional half- or unoccupied d orbitals in the 3d(super 5) or 3d(super 6) site, the intensity of the iron L-edge data is renormalized.

Detailed electronic and geometric structural descriptions of the blue copper sites in wild-type (WT) stellacyanin and its Q99M and Q99L axial mutants are obtained using a combination of XAS, resonance Raman, MCD, EPR, and DFT calculations. The results show that the origin of the short Cu-S(Cys) bond in blue copper proteins is weakened axial interaction, which leads to a shorter and more covalent Cu-S bond.

The extradiol dioxygenase, 2,3-dihydroxybiphenyl 1,2-dioxygenase is studied using magnetic circular dichroism (MCD), variable-temperature variable-field (VTVH) MCD, X-ray absorption (XAS) pre-edge, and extended X-ray absorption fine structure (EXAFS) spectroscopies. The electronic structure calculations provide insight into the relative lack of dioxygen reactivity for the resting enzyme and its activation upon substrate binding.

Magnetic circular dichroism (MCD) and X-ray absorption (XAS) spectroscopies are used to study the interaction of the biologically relevant Fe(super II)BLM complex with DNA. Parallel studies of BLM structural derivatives indicate that Fe(super II)iso-PEPLM, in which the carbonyl group is shifted on the mannose sugar, forms the same DNA-bound species as Fe(super II)BLM.

Phenylanine hydroxylase (PAH), a tetrahydrobioterin-dependent, nonheme iron enzyme, catalyzes the hydroxylation of L-Phe to L-Tyr in the rate-limiting step of phenylalanine catabolism. The results provide insight into the PAH reaction and disease mechanism at a molecular level, indicating that the first step of the mechanism is formation of a peroxy-pterin species.

The DNA binding properties of fused heterocycles imidazo[4,5-b]pyridine (Ip) and hydroxybenzimidazole (Hz) paired with pyrrole (Py) in eight-ring hairpin polyamides are reported. The recognition profile of Ip/Py and Hz/Py pairs were compared to the five-membered ring pairs Im/Py and Hp/Py on a DNA restriction fragment at four 6-base pair recognition sites.

A sensitive 3D NMR pulse scheme, (H)C(CA)NH-COSY is presented for the assignment of 13C(super delta1) IIE chemical shifts in large perdeuterated, methyl protonated proteins. The experiment presented utilizes COZY-based transfer steps and refocuses undesirable 13C-13C scalar couplings that degrade the efficiency of TOCSY transfers.

The reaction of trans-4-hydroxynonenal (HNE) with DNA gives four diastereomeric 1,N(super 2)-gamma-hydroxypropano adducts of deoxyguanosine. Stereospecific syntheses of the four adducts at the nucleoside level are accomplished.

Research has been conducted on the valence-delocalized diiron(II,III) complexes. The spectroscopic properties, molecular structure and synthesis of these complexes are reported.

Research has been conducted on the ferrous wild-type phenylalanine hydroxylase. Results indicate that ligand loss at the Fe(II) active site of this hydroxylase.

The collective XAS results for HC4H2-[Fe4S4]-Ni and HC5H-[Fe4S4]-M demonstrate the presence of an Fe4S4 cluster and support the existence of the distorted square-planar coordination units [Ni (super II) (S.Cys)(N.His)(Sub 3)] and [Ni(super II)(S.Cys)(Sub 2)(N.His)(sub 2)] in the HC4H2 and HC5H metallopeptides, respectively. First XAS study of a de novo designed metallopeptide intended to stabilize a bridged biological assembly, and one of a few XAS analyses of metal derivatives of designed peptides.

The physical processes and the chemical reactions involved in magnetization transfer between water and large proteins, such as collagen, in bovine Achilles tendon are studied. The acquisition of water signal, which originates from the protein magnetization in the DQF-magnetization transfer (MT) technique, give rise to the possibility of creating images that are related directly to the amount of protein and its intramolecular dipolar interactions.

Research has been conducted on the dioxygen-binding non-heme diiron protein hemerythrin. The preparation of the dinucleating bis(carboxylate) ligand dibenzofuran-4,6-bis(diphenylacetate) as a synthetic model for the dioxygen-binding non-heme diiron protein hemerythrin is discussed.

The protonation steps of N-(phosphonomethyl)glycine and structures of its copper(II) complexes in aqueous solution are described. The infrared analyses distinguish between the varying degrees of protonation in both N-(phosphonomethyl)glycine and in the 1:1 copper(II)-N-(phosphonomethyl)glycine complexes. Data indicate that CuL(sup)- and CuHL structures are the same differing only in protonation of the phosphonate group.

The sulfur K-edge x-ray absorption spectroscopy of 2Fe-2S ferredoxin is discussed with regard to covalency of oxidized and reduced forms.

The blue copper sites in Cucumis sativus stellacyanin and Rhus vernicifera stellacyanin are investigated by X-ray absorption spectroscopy (XAS) in order to probe the effects of axial amide coordination. Blue copper proteins exhibit high-redox potentials and rapid electron-transfer rates compared to normal tetragonal copper complexes.

A study that expands upon X-ray absorption spectroscopy (XAS) studies by including a larger number of molecules with variant and physiologically relevant terminal ligands is presented. It provides unique XAS data and characteristics that will prove useful in characterizing metric features and structures of enzyme sites.

The equilibrium geometries of the molbdenum oxo/peroxo compounds MoO(sub n)(O2)(sub 3-n) and the related complexes [MoO(sub n)(O2)(sub 3-n)(OPH3)] and [MoO(sub n)(O2)(sub 3-n)(OPH3)(H2O)] (n =0-3) is calculated using gradient-corrected density-functional theory at theB3LYP level. Where ethylene is directly bonded to the metal, energy minimum structures are found for the molybdenum species.

Complexes between Fe(II), Co(II) and NI(II) and the peptide with a 1:1 stoichiometry were established by spectrophotomeric titrations. Absorption spectra, K-edge X-ray absorption data and EXAFS analysis show the coordination structure of three complexes, which are among the first characterized complexes of a cysteinyl cyclopeptide.

The article presents analysis of electric dipole contributions to pre-edge intensities in ligand K-edge X-ray absorption spectra based on metal-ligand covalent-bonding contributions for a prototype system. Identification of one- and two-center contributions to the intensity is presented.

Research has been conducted on the oxo-sulfido functional group, essential to the activity of the xanthine oxidase family of the enzymes. A route to the molecules that contain this group is described.

Research was conducted to examine the geometric and electronic structure of the reduced iron center. The methodologies used were applied circular dichroism, magnetic circular dichroism, variable-temperature-variable-field molecular circular dichroism, X-ray absorption pre-edge and extended X-ray absorption fine structure spectroscopies. Results indicate that the coordination number increases upon reduction which is attributed to the coordination of a second solvent ligand.

The solid form of the copper complex [(Cu(MePY2))2O2]2+ has been investigated using resonance Raman spectroscopy and X-ray absorption spectroscopy. The results showed that the intermediate geometry of the crystal structure is not representative of the true structure. The crystalline material is shown to be a solid solution composed of 20% of the bis-mu-oxo core and 80% of the side-on peroxide isomer. Molecular orbital and thermodynamic calculations were used to determine the relative reactivities of the two isomers with respect to H-atom abstraction via a sigma* mechanism.

A mutant of a Pseudomonas aeruginosa azurin is studied in reduced and oxidized forms using X-ray absorption spectroscopy. The mutant azurin studied, C112D, acts as a direct probe of the copper-thiolate bond's significance in the electron transfer function of azurin since C112D's native blue copper site has been destroyed. Results also provide insight into the spectroscopy and active site structure of C112D.

Spectroscopic and magnetic data on human ceruloplasmin (Cp) are reconciled with the stoichiometry of six coppers per molecule determined by copper quantitation and X-ray crystallography and to clarify the possible functions of Cp's additional copper sites. The resting and peroxide-oxidized forms of the Cp enzyme were targeted. Among other results derived, it appears that of three Type 1 copper sites found in Cp, one of them, which is redox-inactive and permanently reduced, cannot be catalytically relevant in terms of physiological function in the peroxide-oxidized form studied.

Biomimetic functional models of the mononuclear copper enzyme galactose oxidase are presented that catalytically oxidize benzylic and allylic alcohols to aldehydes with [O.sub.2] under mild conditions. The mechanistic fidelity between the models and the natural system is pronounced. Modest structural mimicry proves sufficient to transfer an unusual ligand-based radical mechanism, previously unprecedented outside the protein matrix, to a simple chemical system., An important goal of bioinorganic chemistry is the development of small inorganic complexes that not only reproduce structural and spectroscopic features, but also function in a manner similar to their [...]

A detailed description of the active site in laccase, a multicopper oxidase containing four coppers, and ascorbate oxidase, has been obtained using spectroscopy linked with crystallography. It has been established that peroxide binds with a low-affinity to the fully oxidized trinuclear site. The binding produces an adduct which is geometrically similar to the oxygen intermediate of T1Hg Lc. In both the peroxide adduct and the oxygen intermediate of T1Hg Lc, the bound oxygen species is not reduced beyond the peroxo level.

The energy, intensity, and splitting of the 1s -> 3d pre-edge feature and its sensitivity to the electronic and geometric structure of the iron site was measured. This was based on the XAS Fe-K edge data on high- and low-spin ferrous and ferric inorganic model complexes. Results reveal that the energy splitting and intensity distribution of the pre-edge features of these complexes vary with spin state, oxidation state, geometry and bridging ligation.

X-ray absorption spectroscopy (XAS) has been used to calculate four-body multiple scattering (MS)signals for the investigation of two cyanide-bridged iron-copper molecular assemblies pertinent to cyanide-inhibited heme-copper oxidases. These complexes differ significantly in the CU-N-C angle and the Cu-N distance. GNXAS analysis shows that long-range interaction of Fe-Cu of the former complex is attributed to the strong MS effects in the linear bridge. Calculations show that MS effects are sensitive to the Cu-N-C angle thus a large MS amplitude enhancement occurs for a linear four-body geometry