RESUMO
Nitrogenase is an active target of heterologous expression because of its importance for areas related to agronomy, energy, and environment. One major hurdle for expressing an active Mo-nitrogenase in Escherichia coli is to generate the complex metalloclusters (P- and M-clusters) within this enzyme, which involves some highly unique bioinorganic chemistry/metalloenzyme biochemistry that is not generally dealt with in the heterologous expression of proteins via synthetic biology; in particular, the heterologous synthesis of the homometallic P-cluster ([Fe8S7]) and M-cluster core (or L-cluster; [Fe8S9C]) on their respective protein scaffolds, which represents two crucial checkpoints along the biosynthetic pathway of a complete nitrogenase, has yet to be demonstrated by biochemical and spectroscopic analyses of purified metalloproteins. Here, we report the heterologous formation of a P-cluster-containing NifDK protein upon coexpression of Azotobacter vinelandii nifD, nifK, nifH, nifM, and nifZ genes, and that of an L-cluster-containing NifB protein upon coexpression of Methanosarcina acetivorans nifB, nifS, and nifU genes alongside the A. vinelandii fdxN gene, in E. coli. Our metal content, activity, EPR, and XAS/EXAFS data provide conclusive evidence for the successful synthesis of P- and L-clusters in a nondiazotrophic host, thereby highlighting the effectiveness of our metallocentric, divide-and-conquer approach that individually tackles the key events of nitrogenase biosynthesis prior to piecing them together into a complete pathway for the heterologous expression of nitrogenase. As such, this work paves the way for the transgenic expression of an active nitrogenase while providing an effective tool for further tackling the biosynthetic mechanism of this important metalloenzyme.
Assuntos
Azotobacter vinelandii , Metaloproteínas , Nitrogenase/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fixação de Nitrogênio/genética , Oxirredutases/metabolismo , Metaloproteínas/metabolismo , Proteínas de Bactérias/metabolismoRESUMO
Fe K-edge X-ray absorption spectroscopy (XAS) has long been used for the study of high-valent iron intermediates in biological and artificial catalysts. 4p-mixing into the 3d orbitals complicates the pre-edge analysis but when correctly understood via 1s2p resonant inelastic X-ray scattering and Fe L-edge XAS, it enables deeper insight into the geometric structure and correlates with the electronic structure and reactivity. This study shows that in addition to the 4p-mixing into the 3dz2 orbital due to the short iron-oxo bond, the loss of inversion in the equatorial plane leads to 4p mixing into the 3dx2-y2,xy, providing structural insight and allowing the distinction of 6- vs 5-coordinate active sites as shown through application to the Fe(IV)âO intermediate of taurine dioxygenase. Combined with O K-edge XAS, this study gives an unprecedented experimental insight into the electronic structure of Fe(IV)âO active sites and their selectivity for reactivity enabled by the π-pathway involving the 3dxz/yz orbitals. Finally, the large effect of spin polarization is experimentally assigned in the pre-edge (i.e., the α/ß splitting) and found to be better modeled by multiplet simulations rather than by commonly used time-dependent density functional theory.
Assuntos
Eletrônica , Ferro , Raios X , Espectroscopia por Absorção de Raios X , Teoria da Densidade FuncionalRESUMO
Lytic polysaccharide monooxygenases (LPMOs) catalyze the degradation of recalcitrant carbohydrate polysaccharide substrates. These enzymes are characterized by a mononuclear Cu(I) active site with a three-coordinate T-shaped "His-brace" configuration including the N-terminal histidine and its amine group as ligands. This study explicitly investigates the electronic structure of the d10 Cu(I) active site in a LPMO using Kß X-ray emission spectroscopy (XES). The lack of inversion symmetry in the His-brace site enables the 3d/p mixing required for intensity in the Kß valence-to-core (VtC) XES spectrum of Cu(I)-LPMO. These Kß XES data are correlated to density functional theory (DFT) calculations to define the bonding, and in particular, the frontier molecular orbital (FMO) of the Cu(I) site. These experimentally validated DFT calculations are used to evaluate the reaction coordinate for homolytic cleavage of the H2O2 O-O bond and understand the contribution of this FMO to the low barrier of this reaction and how the geometric and electronic structure of the Cu(I)-LPMO site is activated for rapid reactivity with H2O2.
Assuntos
Peróxido de Hidrogênio , Oxigenases de Função Mista , Oxigenases de Função Mista/química , Polissacarídeos/metabolismo , Domínio Catalítico , Espectrometria por Raios XRESUMO
In multicopper oxidases (MCOs), the type 1 (T1) Cu accepts electrons from the substrate and transfers these to the trinuclear Cu cluster (TNC) where O2 is reduced to H2O. The T1 potential in MCOs varies from 340 to 780 mV, a range not explained by the existing literature. This study focused on the â¼350 mV difference in potential of the T1 center in Fet3p and Trametes versicolor laccase (TvL) that have the same 2His1Cys ligand set. A range of spectroscopies performed on the oxidized and reduced T1 sites in these MCOs shows that they have equivalent geometric and electronic structures. However, the two His ligands of the T1 Cu in Fet3p are H-bonded to carboxylate residues, while in TvL they are H-bonded to noncharged groups. Electron spin echo envelope modulation spectroscopy shows that there are significant differences in the second-sphere H-bonding interactions in the two T1 centers. Redox titrations on type 2-depleted derivatives of Fet3p and its D409A and E185A variants reveal that the two carboxylates (D409 and E185) lower the T1 potential by 110 and 255-285 mV, respectively. Density functional theory calculations uncouple the effects of the charge of the carboxylates and their difference in H-bonding interactions with the His ligands on the T1 potential, indicating 90-150 mV for anionic charge and â¼100 mV for a strong H-bond. Finally, this study provides an explanation for the generally low potentials of metallooxidases relative to the wide range of potentials of the organic oxidases in terms of different oxidized states of their TNCs involved in catalytic turnover.
Assuntos
Ceruloplasmina , Histidina , Ceruloplasmina/química , Ligantes , Cobre/química , Trametes , Eletricidade Estática , Lacase/metabolismoRESUMO
The direct conversion of methane to methanol would have a wide reaching environmental and industrial impact. Copper-containing zeolites can perform this reaction at low temperatures and pressures at a previously defined O2-activated [Cu2O]2+ site. However, after autoreduction of the copper-containing zeolite mordenite and removal of the [Cu2O]2+ active site, the zeolite is still methane reactive. In this study, we use diffuse reflectance UV-vis spectroscopy, magnetic circular dichroism, resonance Raman spectroscopy, electron paramagnetic resonance, and X-ray absorption spectroscopy to unambiguously define a mononuclear [CuOH]+ as the CH4 reactive active site of the autoreduced zeolite. The rigorous identification of a mononuclear active site allows a reactivity comparison to the previously defined [Cu2O]2+ active site. We perform kinetic experiments to compare the reactivity of the [CuOH]+ and [Cu2O]2+ sites and find that the binuclear site is significantly more reactive. From the analysis of density functional theory calculations, we elucidate that this increased reactivity is a direct result of stabilization of the [Cu2OH]2+ H-atom abstraction product by electron delocalization over the two Cu cations via the bridging ligand. This significant increase in reactivity from electron delocalization over a binuclear active site provides new insights for the design of highly reactive oxidative catalysts.
Assuntos
Zeolitas , Zeolitas/química , Cobre/química , Metano/química , Domínio Catalítico , Metanol/química , Ligantes , Modelos Moleculares , Oxigênio/química , Espectroscopia de Ressonância de Spin Eletrônica , CátionsRESUMO
Nitrogenase employs a sophisticated electron transfer system and a Mo-Fe-S-C cofactor, designated the M-cluster [(cit)MoFe7 S9 C]), to reduce atmospheric N2 to bioaccessible NH3 . Previously, we have shown that the cofactor-free form of nitrogenase can be repurposed as a protein scaffold for the incorporation of a synthetic Fe-S cluster [Fe6 S9 (SEt)2 ]4- . Here, we demonstrate the utility of an asymmetric Mo-Fe-S cluster [Cp*MoFe5 S9 (SH)]3- as an alternative artificial cofactor upon incorporation into the cofactor-free nitrogenase scaffold. The resultant semi-artificial enzyme catalytically reduces C2 H2 to C2 H4 , and CN- into short-chain hydrocarbons, yet it is clearly distinct in activity from its [Fe6 S9 (SEt)2 ]4- -reconstituted counterpart, pointing to the possibility to employ molecular design and cluster synthesis strategies to further develop semi-artificial or artificial systems with desired catalytic activities.
Assuntos
Hidrocarbonetos , Nitrogenase , Hidrocarbonetos/metabolismo , Nitrogenase/metabolismo , OxirreduçãoRESUMO
Nitrogenase is a versatile metalloenzyme that reduces N2, CO and CO2 at its cofactor site. Designated the M-cluster, this complex cofactor has a composition of [(R-homocitrate)MoFe7S9C], and it is assembled through the generation of a unique [Fe8S9C] core prior to the insertion of Mo and homocitrate. NifB is a radical S-adenosyl-L-methionine (SAM) enzyme that is essential for nitrogenase cofactor assembly. This review focuses on the recent work that sheds light on the role of NifB in the formation of the [Fe8S9C] core of the nitrogenase cofactor, highlighting the structure, function and mechanism of this unique radical SAM methyltransferase.
Assuntos
Metaloproteínas , Nitrogenase , Metiltransferases , Molibdoferredoxina/química , Nitrogenase/química , S-Adenosilmetionina/químicaRESUMO
Reduced dithiolene ligands are bound to high valent Mo centers in the active site of the oxotransferase family of enzymes. Related model complexes have been studied with great insight by Prof. Holm and his colleagues. This study focuses on the other limit of dithiolene chemistry: an investigation of the 2-electron oxidized dithiolene bound to low-valent late transition metal (TM) ions (ZnII, CuI, and CuII). The bonding descriptions of the oxidized dithiolene [N,N-dimethyl piperazine 2,3-dithione (Me2Dt0)] complexes are probed using S K-edge X-ray absorption spectroscopy (XAS) and the results are correlated to density functional theory (DFT) calculations. These experimentally supported calculations are then extended to explain the different geometric structures of the three complexes. The ZnII(Me2Dt0)2 complex has only ligand-ligand repulsion so it is stabilized at the D2d symmetry limit. The CuI(Me2Dt0)2 complex has additional weak backbonding thus distorts somewhat from D2d toward D2h symmetry. The CuII(Me2Dt0)2 complex has a strong σ donor bond that leads to both a large Jahn-Teller stabilization to D2h and an additional covalent contribution to the geometry. The combined strong stabilization results in the square planar, D2h structure. This study quantifies the competition between the ligand-ligand repulsion and the change in electronic structures in determining the final geometric structures of the oxidized dithiolene complexes, and provides quantitative insights into the Jahn-Teller stabilization energy and its origin.
Assuntos
Zinco , Ligantes , Modelos Moleculares , Oxirredução , Espectroscopia por Absorção de Raios XRESUMO
The Fe protein of nitrogenase plays multiple roles in substrate reduction and cluster maturation via its redox-active [Fe4 S4 ] cluster. Here we report the synthesis and characterization of a water-soluble [Fe4 Se4 ] cluster that is used to substitute the [Fe4 S4 ] cluster of the Azotobacter vinelandii Fe protein (AvNifH). Biochemical, EPR and XAS/EXAFS analyses demonstrate the ability of the [Fe4 Se4 ] cluster to adopt the super-reduced, all-ferrous state upon its incorporation into AvNifH. Moreover, these studies reveal that the [Fe4 Se4 ] cluster in AvNifH already assumes a partial all-ferrous state ([Fe4 Se4 ]0 ) in the presence of dithionite, where its [Fe4 S4 ] counterpart in AvNifH exists solely in the reduced state ([Fe4 S4 ]1+ ). Such a discrepancy in the redox properties of the AvNifH-associated [Fe4 Se4 ] and [Fe4 S4 ] clusters can be used to distinguish the differential redox requirements for the substrate reduction and cluster maturation of nitrogenase, pointing to the utility of chalcogen-substituted FeS clusters in future mechanistic studies of nitrogenase catalysis and assembly.
Assuntos
Azotobacter vinelandii , Proteínas Ferro-Enxofre , Proteínas Ferro-Enxofre/química , Nitrogenase/química , Oxirredução , Oxirredutases/químicaRESUMO
Molybdenum nitrogenase catalyses the reduction of N2 to NH3 at its cofactor, an [(R-homocitrate)MoFe7S9C] cluster synthesized via the formation of a [Fe8S9C] L-cluster prior to the insertion of molybdenum and homocitrate. We have previously identified a [Fe8S8C] L*-cluster, which is homologous to the core structure of the L-cluster but lacks the 'ninth sulfur' in the belt region. However, direct evidence and mechanistic details of the L*- to L-cluster conversion upon 'ninth sulfur' insertion remain elusive. Here we trace the 'ninth sulfur' insertion using SeO32- and TeO32- as 'labelled' SO32-. Biochemical, electron paramagnetic resonance and X-ray absorption spectroscopy/extended X-ray absorption fine structure studies suggest a role of the 'ninth sulfur' in cluster transfer during cofactor biosynthesis while revealing the incorporation of Se2-- and Te2--like species into the L-cluster. Density functional theory calculations further point to a plausible mechanism involving in situ reduction of SO32- to S2-, thereby suggesting the utility of this reaction to label the catalytically important belt region for mechanistic investigations of nitrogenase.
Assuntos
Coenzimas/química , Proteínas Ferro-Enxofre/química , Nitrogenase/química , Ácido Selenioso/química , Enxofre/química , Telúrio/química , Proteínas Arqueais/química , Teoria da Densidade Funcional , Espectroscopia de Ressonância de Spin Eletrônica , Methanosarcina/enzimologia , Modelos Químicos , Espectroscopia por Absorção de Raios XRESUMO
The central role of cupric superoxide intermediates proposed in hormone and neurotransmitter biosynthesis by noncoupled binuclear copper monooxygenases like dopamine-ß-monooxygenase has drawn significant attention to the unusual methionine ligation of the CuM ("CuB") active site characteristic of this class of enzymes. The copper-sulfur interaction has proven critical for turnover, raising still-unresolved questions concerning Nature's selection of an oxidizable Met residue to facilitate C-H oxygenation. We describe herein a model for CuM, [(TMGN3S)CuI]+ ([1]+), and its O2-bound analog [(TMGN3S)CuII(O2â¢-)]+ ([1·O2]+). The latter is the first reported cupric superoxide with an experimentally proven Cu-S bond which also possesses demonstrated hydrogen atom abstraction (HAA) reactivity. Introduction of O2 to a precooled solution of the cuprous precursor [1]B(C6F5)4 (-135 °C, 2-methyltetrahydrofuran (2-MeTHF)) reversibly forms [1·O2]B(C6F5)4 (UV/vis spectroscopy: λmax 442, 642, 742 nm). Resonance Raman studies (413 nm) using 16O2 [18O2] corroborated the identity of [1·O2]+ by revealing Cu-O (446 [425] cm-1) and O-O (1105 [1042] cm-1) stretches, and extended X-ray absorption fine structure (EXAFS) spectroscopy showed a Cu-S interatomic distance of 2.55 Å. HAA reactivity between [1·O2]+ and TEMPO-H proceeds rapidly (1.28 × 10-1 M-1 s-1, -135 °C, 2-MeTHF) with a primary kinetic isotope effect of kH/kD = 5.4. Comparisons of the O2-binding behavior and redox activity of [1]+ vs [2]+, the latter a close analog of [1]+ but with all N atom ligation (i.e., N3S vs N4), are presented.
Assuntos
Cobre/química , Hidrogênio/química , Sulfetos/química , Superóxidos/química , Teoria da Densidade Funcional , Cinética , Conformação Molecular , Oxirredução , Espectrofotometria UltravioletaRESUMO
1s2p resonant inelastic X-ray scattering (1s2p RIXS) has proven successful in the determination of the differential orbital covalency (DOC, the amount of metal vs ligand character in each d molecular orbital) of highly covalent centrosymmetric iron environments including heme models and enzymes. However, many reactive intermediates have noncentrosymmetric environments, e.g., the presence of strong metal-oxo bonds, which results in the mixing of metal 4p character into the 3d orbitals. This leads to significant intensity enhancement in the metal K-pre-edge and as shown here, the associated 1s2p RIXS features, which impact their insight into electronic structure. Binuclear oxo bridged high spin Fe(III) complexes are used to determine the effects of 4p mixing on 1s2p RIXS spectra. In addition to developing the analysis of 4p mixing on K-edge XAS and 1s2p RIXS data, this study explains the selective nature of the 4p mixing that also enhances the analysis of L-edge XAS intensity in terms of DOC. These 1s2p RIXS biferric model studies enable new structural insight from related data on peroxo bridged biferric enzyme intermediates. The dimeric nature of the oxo bridged Fe(III) complexes further results in ligand-to-ligand interactions between the Fe(III) sites and angle dependent features just above the pre-edge that reflect the superexchange pathway of the oxo bridge. Finally, we present a methodology that enables DOC to be obtained when L-edge XAS is inaccessible and only 1s2p RIXS experiments can be performed as in many metalloenzyme intermediates in solution.
Assuntos
Compostos Férricos/química , Teoria Quântica , Eletrônica , Estrutura Molecular , Espalhamento de Radiação , Raios XRESUMO
The dynamics of photodissociation and recombination in heme proteins represent an archetypical photochemical reaction widely used to understand the interplay between chemical dynamics and reaction environment. We report a study of the photodissociation mechanism for the Fe(II)-S bond between the heme iron and methionine sulfur of ferrous cytochrome c. This bond dissociation is an essential step in the conversion of cytochrome c from an electron transfer protein to a peroxidase enzyme. We use ultrafast X-ray solution scattering to follow the dynamics of Fe(II)-S bond dissociation and 1s3p (Kß) X-ray emission spectroscopy to follow the dynamics of the iron charge and spin multiplicity during bond dissociation. From these measurements, we conclude that the formation of a triplet metal-centered excited state with anti-bonding Fe(II)-S interactions triggers the bond dissociation and precedes the formation of the metastable Fe high-spin quintet state.
Assuntos
Citocromos c/metabolismo , Compostos Ferrosos/metabolismo , Ferro/metabolismo , Metais/metabolismo , Metionina/metabolismo , Citocromos c/química , Transporte de Elétrons/efeitos da radiação , Compostos Ferrosos/química , Heme/química , Heme/metabolismo , Ferro/química , Metais/química , Metionina/química , Simulação de Dinâmica Molecular , Fotólise , Espectrometria por Raios XRESUMO
Cu(I) active sites in metalloproteins are involved in O2 activation, but their O2 reactivity is difficult to study due to the Cu(I) d10 closed shell which precludes the use of conventional spectroscopic methods. Kß X-ray emission spectroscopy (XES) is a promising technique for investigating Cu(I) sites as it detects photons emitted by electronic transitions from occupied orbitals. Here, we demonstrate the utility of Kß XES in probing Cu(I) sites in model complexes and a metalloprotein. Using Cu(I)Cl, emission features from double-ionization (DI) states are identified using varying incident X-ray photon energies, and a reasonable method to correct the data to remove DI contributions is presented. Kß XES spectra of Cu(I) model complexes, having biologically relevant N/S ligands and different coordination numbers, are compared and analyzed, with the aid of density functional theory (DFT) calculations, to evaluate the sensitivity of the spectral features to the ligand environment. While the low-energy Kß2,5 emission feature reflects the ionization energy of ligand np valence orbitals, the high-energy Kß2,5 emission feature corresponds to transitions from molecular orbitals (MOs) having mainly Cu 3d character with the intensities determined by ligand-mediated d-p mixing. A Kß XES spectrum of the Cu(I) site in preprocessed galactose oxidase (GOpre) supports the 1Tyr/2His structural model that was determined by our previous X-ray absorption spectroscopy and DFT study. The high-energy Kß2,5 emission feature in the Cu(I)-GOpre data has information about the MO containing mostly Cu 3dx2-y2 character that is the frontier molecular orbital (FMO) for O2 activation, which shows the potential of Kß XES in probing the Cu(I) FMO associated with small-molecule activation in metalloproteins.
Assuntos
Cobre/metabolismo , Galactose Oxidase/metabolismo , Cobre/química , Teoria da Densidade Funcional , Galactose Oxidase/química , Oxigênio/química , Oxigênio/metabolismo , Espectrometria por Raios XRESUMO
Determining the requirements for efficient oxygen (O2) activation is key to understanding how enzymes maintain efficacy and mitigate unproductive, often detrimental reactivity. For the α-ketoglutarate (αKG)-dependent nonheme iron enzymes, both a concerted mechanism (both cofactor and substrate binding prior to reaction with O2) and a sequential mechanism (cofactor binding and reaction with O2 precede substrate binding) have been proposed. Deacetoxycephalosporin C synthase (DAOCS) is an αKG-dependent nonheme iron enzyme for which both of these mechanisms have been invoked to generate an intermediate that catalyzes oxidative ring expansion of penicillin substrates in cephalosporin biosynthesis. Spectroscopy shows that, in contrast to other αKG-dependent enzymes (which are six coordinate when only αKG is bound to the FeII), αKG binding to FeII-DAOCS results in â¼45% five-coordinate sites that selectively react with O2 relative to the remaining six-coordinate sites. However, this reaction produces an FeIII species that does not catalyze productive ring expansion. Alternatively, simultaneous αKG and substrate binding to FeII-DAOCS produces five-coordinate sites that rapidly react with O2 to form an FeIV=O intermediate that then reacts with substrate to produce cephalosporin product. These results demonstrate that the concerted mechanism is operative in DAOCS and by extension, other nonheme iron enzymes.
Assuntos
Transferases Intramoleculares/química , Ferro/química , Ácidos Cetoglutáricos/química , Ferroproteínas não Heme/química , Proteínas de Ligação às Penicilinas/química , Espécies Reativas de Oxigênio/química , Ativação Enzimática , Oxirredução , Penicilina G/química , Especificidade por SubstratoRESUMO
Two samples of living blood cells and of cleared blood plasma from the Phlebobranch tunicate Ascidia ceratodes from Bodega Bay, California, and one of fresh Henze solution from A. ceratodes of Monterey Bay, California, have been examined using sulfur K-edge x-ray absorption spectroscopy (XAS). Biological sulfur included sulfate esters, sulfate and bisulfate ions, benzothiazole, thianthrene, epi-sulfide, thiol and disulfide. Glutathione dominated reduced sulfur, from which an average intracellular Voltage of -0.21 V was calculated. Sulfate-bisulfate ratios yielded blood cell pH values of 2.0 and 2.8. Total blood cell [sulfur] was 373±9 mM or 296±73 mM from BaSO4 gravimetry. Two plasma samples (pH 6.9 or 7.0; [S] = 33±6 mM or 26±4 mM) were dominated by sulfate and disulfide. Fresh Henze solution evidenced a sulfur inventory similar to blood cells, with calculated pH = 2.7. A V(III)-sulfonate fraction varied systematically with intracellular pH across six independent blood cell samples, implying a vanadium mobilization pathway. Bodega Bay and Monterey Bay A. ceratodes appear to maintain alternative suites of low-valent sulfur. The significance of the vanabins to vanadium metabolism is critically examined in terms of known protein - V(IV) biochemistry. Finally, a detailed hypothesis for the reduction of [VO4]3- to V(III) in ascidians is introduced. A vanadium oxido-reductase is proposed to span the signet ring membrane and to release V(III) into the inner acidic vacuole. The V(V) to V(III) reduction is predicted require an inner-sphere mechanism, a thiol reductant, 7-coordinate V(III), a biologically accessible Voltage, and proton-facilitated release of V(III).
Assuntos
Células Sanguíneas/metabolismo , Enxofre/metabolismo , Urocordados/metabolismo , Vanádio/metabolismo , Espectroscopia por Absorção de Raios X , Animais , OxirreduçãoRESUMO
Nitrogenases catalyze the reduction of N2 to NH4+ at its cofactor site. Designated the M-cluster, this [MoFe7 S9 C(R-homocitrate)] cofactor is synthesized via the transformation of a [Fe4 S4 ] cluster pair into an [Fe8 S9 C] precursor (designated the L-cluster) prior to insertion of Mo and homocitrate. We report the characterization of an eight-iron cofactor precursor (designated the L*-cluster), which is proposed to have the composition [Fe8 S8 C] and lack the "9th sulfur" in the belt region of the L-cluster. Our X-ray absorption and electron spin echo envelope modulation (ESEEM) analyses strongly suggest that the L*-cluster represents a structural homologue to the l-cluster except for the missing belt sulfur. The absence of a belt sulfur from the L*-cluster may prove beneficial for labeling the catalytically important belt region, which could in turn facilitate investigations into the reaction mechanism of nitrogenases.
Assuntos
Coenzimas/química , Nitrogenase/metabolismo , Análise Espectral/métodos , Enxofre/química , Modelos Moleculares , Estrutura Molecular , Nitrogenase/química , Espectroscopia por Absorção de Raios XRESUMO
The question of ligand noninnocence in Cu corroles has long been a topic of discussion. Presented herein is a Cu K-edge X-ray absorption spectroscopy (XAS) study, which provides a direct probe of the metal oxidation state, of three Cu corroles, Cu[TPC], Cu[Br8TPC], and Cu[(CF3)8TPC] (TPC = meso-triphenylcorrole), and the analogous Cu(II) porphyrins, Cu[TPP], Cu[Br8TPP], and Cu[(CF3)8TPP] (TPP = meso-tetraphenylporphyrin). The Cu K rising-edges of the Cu corroles were found to be about 0-1 eV upshifted relative to the analogous porphyrins, which is substantially lower than the 1-2 eV shifts typically exhibited by authentic Cu(II)/Cu(III) model complex pairs. In an unusual twist, the Cu K pre-edge regions of both the Cu corroles and the Cu porphyrins exhibit two peaks split by 0.8-1.3 eV. Based on time-dependent density functional theory calculations, the lower- and higher-energy peaks were assigned to a Cu 1s â 3d x2- y2 transition and a Cu 1s â corrole/porphyrin π* transition, respectively. From the Cu(II) porphyrins to the corresponding Cu corroles, the energy of the Cu 1s â 3d x2- y2 transition peak was found to upshift by 0.6-0.8 eV. This shift is approximately half that observed between Cu(II) to Cu(III) states for well-defined complexes. The Cu K-edge XAS spectra thus show that although the metal sites in the Cu corroles are more oxidized relative to those in their Cu(II) porphyrin analogues, they are not oxidized to the Cu(III) level, consistent with the notion of a noninnocent corrole. The relative importance of σ-donation versus corrole π-radical character is discussed.
RESUMO
In situ inventory of sulfurous products from the sulfur K-edge synchrotron X-radiolysis of l-cysteine in solid-phase and anaerobic (pH 5) and air-saturated (pH 5, 7, and 9) solutions without and with 40% glycerol is reported. Sequential K-edge X-ray Absorption Spectroscopic (XAS) spectra were acquired. l-cysteine degraded systematically in the X-ray beam. Radiolytic products were inventoried by fits using the XAS spectra of sulfur model compounds. Solid l-cysteine declined to 92% fraction after a single K-edge XAS scan. After six scans, 60% remained, accompanied by 14% cystine, 16% thioether, 5.4% elemental sulfur, and smaller fractions of more highly oxidized products. In air-saturated pH 5 solution, 73% of l-cysteine remained after ten scans, with 2% cystine and 19% elemental sulfur. Oxidation increased with 40% glycerol, yielding 67%, 5%, and 23% fractions, respectively, after ten scans. Higher pH solutions exhibited less radiolytic chemistry. All the reactivity followed first-order kinetics. The anaerobic experiment displayed two reaction phases, with sharp changes in kinetics and radiolytic chemistry. Unexpectedly, the radiolytic oxidation of l-cysteine was increased in anaerobic solution. After ten scans, only 60% of the l-cysteine remained, along with 17% cystine, 22% elemental sulfur, and traces of more highly oxidized products. A new aerobic reaction cycle is hypothesized, wherein dissolved dioxygen captures radiolytic H⢠or eaq -, enters HO2 â¢/O2 â¢-, reductively quenches cysteine thiyl radicals, and cycles back to O2. This cycle is suggested to suppress the radiolytic production of cystine in aerobic solution.
RESUMO
High-valent ferryl species (e.g., (Por)FeIVâO, Cmpd-II) are observed or proposed key oxidizing intermediates in the catalytic cycles of heme-containing enzymes (P-450s, peroxidases, catalases, and cytochrome c oxidase) involved in biological respiration and oxidative metabolism. Herein, various axially ligated iron(IV)-oxo complexes were prepared to examine the influence of the identity of the base. These were generated by addition of various axial ligands (1,5-dicyclohexylimidazole (DCHIm), a tethered-imidazole system, and sodium derivatives of 3,5-dimethoxyphenolate and imidazolate). Characterization was carried out via UV-vis, electron paramagnetic resonance (EPR), 57Fe Mössbauer, Fe X-ray absorption (XAS), and 54/57Fe resonance Raman (rR) spectroscopies to confirm their formation and compare the axial ligand perturbation on the electronic and geometric structures of these heme iron(IV)-oxo species. Mössbauer studies confirmed that the axially ligated derivatives were iron(IV) and six-coordinate complexes. XAS and 54/57Fe rR data correlated with slight elongation of the iron-oxo bond with increasing donation from the axial ligands. The first reported synthetic H-bonded iron(IV)-oxo heme systems were made in the presence of the protic Lewis acid, 2,6-lutidinium triflate (LutH+), with (or without) DCHIm. Mössbauer, rR, and XAS spectroscopic data indicated the formation of molecular Lewis acid ferryl adducts (rather than full protonation). The reduction potentials of these novel Lewis acid adducts were bracketed through addition of outer-sphere reductants. The oxidizing capabilities of the ferryl species with or without Lewis acid vary drastically; addition of LutH+ to F8Cmpd-II (F8 = tetrakis(2,6-difluorophenyl)porphyrinate) increased its reduction potential by more than 890 mV, experimentally confirming that H-bonding interactions can increase the reactivity of ferryl species.