Experimental Definition of the = 1 π vs = 2 σ Reactivity and = 2 Character in the Ground State of an = 1 FeO Complex.

Iron(IV)-oxo intermediates found in iron enzymes and artificial catalysts are competent for H atom abstraction in catalytic cycles. For = 2 intermediates, both axial and equatorial approaches are well-established. The mechanism for = 1 sites is not as well understood: an equatorial approach is more energetically favorable, and an axial approach requires crossing from the = 1 to the = 2 surface.

Heterologous synthesis of the complex homometallic cores of nitrogenase P- and M-clusters in .

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 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 ([FeS]) and M-cluster core (or L-cluster; [FeSC]) 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 , , , and genes, and that of an L-cluster-containing NifB protein upon coexpression of , and genes alongside the gene, in .

X-ray Spectroscopic Study of the Electronic Structure of a Trigonal High-Spin Fe(IV)═O Complex Modeling Non-Heme Enzyme Intermediates and Their Reactivity.

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 3d orbital due to the short iron-oxo bond, the loss of inversion in the equatorial plane leads to 4p mixing into the 3d, providing structural insight and allowing the distinction of 6- vs 5-coordinate active sites as shown through application to the Fe(IV)═O intermediate of taurine dioxygenase.

Kβ X-ray Emission Spectroscopy of Cu(I)-Lytic Polysaccharide Monooxygenase: Direct Observation of the Frontier Molecular Orbital for HO Activation.

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 d Cu(I) active site in a LPMO using Kβ X-ray emission spectroscopy (XES).

Tuning the Type 1 Reduction Potential of Multicopper Oxidases: Uncoupling the Effects of Electrostatics and H-Bonding to Histidine Ligands.

In multicopper oxidases (MCOs), the type 1 (T1) Cu accepts electrons from the substrate and transfers these to the trinuclear Cu cluster (TNC) where O is reduced to HO. The T1 potential in MCOs varies from 340 to 780 mV, a range not explained by the existing literature. This study focused on the ∼350 mV difference in potential of the T1 center in Fet3p and laccase (TvL) that have the same 2His1Cys ligand set.

Methane Activation by a Mononuclear Copper Active Site in the Zeolite Mordenite: Effect of Metal Nuclearity on Reactivity.

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 O-activated [CuO] site. However, after autoreduction of the copper-containing zeolite mordenite and removal of the [CuO] active site, the zeolite is still methane reactive.

Incorporation of an Asymmetric Mo-Fe-S Cluster as an Artificial Cofactor into Nitrogenase.

Nitrogenase employs a sophisticated electron transfer system and a Mo-Fe-S-C cofactor, designated the M-cluster [(cit)MoFe S C]), to reduce atmospheric N to bioaccessible NH . 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 [Fe S (SEt) ] . Here, we demonstrate the utility of an asymmetric Mo-Fe-S cluster [Cp*MoFe S (SH)] as an alternative artificial cofactor upon incorporation into the cofactor-free nitrogenase scaffold.

Radical SAM-dependent formation of a nitrogenase cofactor core on NifB.

Nitrogenase is a versatile metalloenzyme that reduces N, CO and CO at its cofactor site. Designated the M-cluster, this complex cofactor has a composition of [(R-homocitrate)MoFeSC], and it is assembled through the generation of a unique [FeSC] 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.

Tracing the incorporation of the "ninth sulfur" into the nitrogenase cofactor precursor with selenite and tellurite.

Molybdenum nitrogenase catalyses the reduction of N to NH at its cofactor, an [(R-homocitrate)MoFeSC] cluster synthesized via the formation of a [FeSC] L-cluster prior to the insertion of molybdenum and homocitrate. We have previously identified a [FeSC] 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.

Effect of 3d/4p Mixing on 1s2p Resonant Inelastic X-ray Scattering: Electronic Structure of Oxo-Bridged Iron Dimers.

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.

A Thioether-Ligated Cupric Superoxide Model with Hydrogen Atom Abstraction Reactivity.

The central role of cupric superoxide intermediates proposed in hormone and neurotransmitter biosynthesis by noncoupled binuclear copper monooxygenases like dopamine-β-monooxygenase has drawn significant attention to the unusual methionine ligation of the Cu ("Cu") 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 Cu, [(NS)Cu] ([]), and its O-bound analog [(NS)Cu(O)] ([·O]).

Short-lived metal-centered excited state initiates iron-methionine photodissociation in ferrous cytochrome c.

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.

Kβ X-ray Emission Spectroscopy as a Probe of Cu(I) Sites: Application to the Cu(I) Site in Preprocessed Galactose Oxidase.

Cu(I) active sites in metalloproteins are involved in O activation, but their O reactivity is difficult to study due to the Cu(I) d closed shell which precludes the use of conventional spectroscopic methods. Kβ 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 Kβ XES in probing Cu(I) sites in model complexes and a metalloprotein.

Evaluation of a concerted vs. sequential oxygen activation mechanism in α-ketoglutarate-dependent nonheme ferrous enzymes.

Determining the requirements for efficient oxygen (O) 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 O) and a sequential mechanism (cofactor binding and reaction with O 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.

Biological sulfur in the blood cells of Ascidia ceratodes: XAS spectroscopy and a cellular-enzymatic hypothesis for vanadium reduction in the ascidians.

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.

The Electronic Structure of the Metal Active Site Determines the Geometric Structure and Function of the Metalloregulator NikR.

NikR is a nickel-responsive metalloregulator protein that controls the level of Ni ions in living cells. Previous studies have shown that NikR can bind a series of first-row transition metal ions but binds to DNA with high affinity only as a Ni complex. To understand this metal selectivity, S K-edge X-ray absorption spectroscopy of NikR bound to different metal ions was used to evaluate the different electronic structures.

X-ray Absorption Spectroscopy as a Probe of Ligand Noninnocence in Metallocorroles: The Case of Copper Corroles.

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[BrTPC], and Cu[(CF)TPC] (TPC = meso-triphenylcorrole), and the analogous Cu(II) porphyrins, Cu[TPP], Cu[BrTPP], and Cu[(CF)TPP] (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.

Synchrotron X-radiolysis of l-cysteine at the sulfur K-edge: Sulfurous products, experimental surprises, and dioxygen as an oxidoreductant.

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.

Tuning the Geometric and Electronic Structure of Synthetic High-Valent Heme Iron(IV)-Oxo Models in the Presence of a Lewis Acid and Various Axial Ligands.

High-valent ferryl species (e.g., (Por)Fe═O, 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.

Formylglycine-generating enzyme binds substrate directly at a mononuclear Cu(I) center to initiate O activation.

The formylglycine-generating enzyme (FGE) is required for the posttranslational activation of type I sulfatases by oxidation of an active-site cysteine to C-formylglycine. FGE has emerged as an enabling biotechnology tool due to the robust utility of the aldehyde product as a bioconjugation handle in recombinant proteins. Here, we show that Cu(I)-FGE is functional in O activation and reveal a high-resolution X-ray crystal structure of FGE in complex with its catalytic copper cofactor.

Resonant inelastic X-ray scattering determination of the electronic structure of oxyhemoglobin and its model complex.

Hemoglobin and myoglobin are oxygen-binding proteins with S = 0 heme {FeO} active sites. The electronic structure of these sites has been the subject of much debate. This study utilizes Fe K-edge X-ray absorption spectroscopy (XAS) and 1s2p resonant inelastic X-ray scattering (RIXS) to study oxyhemoglobin and a related heme {FeO} model compound, [(pfp)Fe(1-MeIm)(O)] (pfp = meso-tetra(α,α,α,α--pivalamido-phenyl)porphyrin, or TpivPP, 1-MeIm = 1-methylimidazole) (pfpO), which was previously analyzed using L-edge XAS.

Mechanism of selective benzene hydroxylation catalyzed by iron-containing zeolites.

A direct, catalytic conversion of benzene to phenol would have wide-reaching economic impacts. Fe zeolites exhibit a remarkable combination of high activity and selectivity in this conversion, leading to their past implementation at the pilot plant level. There were, however, issues related to catalyst deactivation for this process.