Highly oxidized metals are constituents of oxidants, reactive intermediates, and materials with interesting conductive and magnetic properties. High-energy spectroscopies have played an important role in identifying and describing the bonding character of highly oxidized metals in these materials. A systematic study of Cu(III) K-edge X-ray absorption spectra was carried out to identify analytically useful signatures of Cu(III) in the K-edge, and to elucidate bonding descriptions for Cu(III)-containing complexes. K-edges for six Cu(III) complexes and their same-ligand Cu(II) counterparts are compared. Edges for the Cu(III) species generally appear at higher energies than their Cu(II) counterparts, though energy shifts between most individual edge features vary. However, for all Cu(III) compounds studied, the 1s f 3d transition in the preedge energy range exhibits a distinct, 2 eV shift to higher energy, relative to the known and relatively unvarying energy of the 1s f 3d transition in Cu(II) species. This energy shift provides a direct means of distinguishing Cu(III) from Cu(II). The K-edge for a complex containing Cu(II) coordinated to a 1e --oxidized ligand (phenoxyl) does not show such a change in the 1s f 3d transition energy. The analytical potential of the Cu K-edge was tested with good success using a mixed-valent trinuclear species. Cu(III) is detectable using the K-edge. The limitations of the K-edge as a Cu(III) analytical probe are discussed. An analysis applied to the 1s f 4p and 1s f 4p + shakedown transitions in the edge for a {Cu II 2 (µ-OH) 2 } 2+ dimer, using a configurational interaction (CI) model, predicted ∼75% d-character in the ground state. A similar analysis of the K-edge for {Cu III 2 (µ-O) 2 } 2+ indicates that the Cu in this complex has far more covalent bonds with the oxo bridging ligands (dcharacter ∼60%).

Chlorite dismutase catalyzes O2 release from chlorite with exquisite efficiency and specificity. The spectroscopic properties, ligand binding affinities, and steady state kinetics of chlorite dismutase from Dechloromonas aromatica were examined over pH 3–11.5 to gain insight into how the protonation state of the heme environment influences dioxygen formation. An acid/base transition was observed by UV/visible and resonance Raman spectroscopy with a pKa of 8.7, 2–3 pH units below analogous transitions observed in typical His-ligated peroxidases. This transition marks the conversion of a five coordinate high spin Fe(III) to a mixed high/low spin ferric-hydroxide, as confirmed by resonance Raman (rR) spectroscopy. The two Fe–OH stretching frequencies are quite low, consistent with a weak Fe–OH bond, despite the nearly neutral imidazole side chain of the proximal histidine ligand. The hydroxide is proposed to interact strongly with a distal H-bond donor, thereby weakening the Fe–OH bond. The rR spectra of Cld-CO as a function of pH reveal two forms of the complex, one in which there is minimal interaction of distal residues with the carbonyl oxygen and another, acidic form in which the oxygen is under the influence of positive charge. Recent crystallographic data reveal arginine 183 as the lone H-bond donating residue in the distal pocket. It is likely that this Arg is the strong, positively charged H-bond donor implicated by vibrational data to interact with exogenous axial heme ligands. The same Arg in its neutral (pKa ~ 6.5) form also appears to act as the active site base in binding reactions of protonated ligands, such as HCN, to ferric Cld. The steady state profile for the rate of chlorite decomposition is characterized by these same pKas. The 5 coordinate high spin acidic Cld is more active than the alkaline hydroxide-bound form. The acid form decomposes chlorite most efficiently when the distal Arg is protonated/cationic (maximum kcat = 2.0 (±0.6) × 105 s−1, kcat/KM = 3.2 (±0.4) × 107 M−1s−1, pH 5.2, 4 °C) and to a somewhat lesser extent when it acts as a H-bond donor to the axial hydroxide ligand under alkaline conditions.

Biomimetic functional models of the mononuclear copper enzyme galactose oxidase are presented that catalytically oxidize benzylic and allylic alcohols to aldehydes with O2 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.

This PDF file includes:1. Complete experimental and computational details 2. Supplementary results 3. Supplementary discussion 4. List of supplementary figures 5. Supplementary Figure 1 to 26 6. Abbreviations list 7. Supplementary references 8. QM/MM optimized xyz coordinates of states 1-9 chromatography (HPAEC) coupled to pulsed amperometric detection (PAD) using a Dionex Bio-LC equipped with a CarboPac PA1 column as previously described. 3 To quantify A2 ox , a standard was produced in-house by treating chitobiose (Megazymes) with a chitooligosaccharide oxidase (ChitO) from Fusarium graminearum, which yields 100% conversion of chitobiose to chitobionic acid. 2,4 All chromatograms were recorded using Chromeleon 7.0 software.Chitin binding assay. The capacity of SmAA10A-WT and mutants thereof to bind β-chitin was tested by suspending 10 mg/mL of substrate in sodium phosphate buffer (50 mM, pH 7.0) in a total volume of 600 µL in 2 mL Eppendorf tubes. Reactions were started by the addition of SmAA10A (1 µM final concentration) and were incubated and stirred in an Eppendorf Comfort Thermomixer (at 40 °C, 1000 rpm). Samples were taken (100 µL) after 15, 30, 60, 120 and 240 min and immediately filtrated using a 96-well filter plate (Millipore) operated with a vacuum manifold to obtain the unbound protein fraction.In order to assess the percentage of bound proteins to the substrate, control samples with only enzyme and buffer were included, representing the maximum quantity of protein present in the samples (i.e. 100% unbound). The protein concentration in each sample was determined using the Bradford assay (Bio-Rad, Munich, Germany).H2O2 consumption experiments. H2O2 consumption by SmAA10A-WT and mutants thereof was measured according to a previously described protocol 5 using conditions that were slightly different from the standard reaction conditions described above: in order to be able to monitor the H2O2 consumption within a reasonable timescale the enzyme concentration had to be reduced and EDTA was added to reduce the background reaction of free metals-catalyzed H2O2 reduction (see Figure S6). After optimization, a standard reaction mixture contained the LPMO (50 nM) and H2O2 (100 µM) and EDTA (50 µM), without or with b-chitin (10 g.L -1 ), in sodium phosphate buffer (50 mM, pH 7.0), and the mixtures were incubated at 40 °C in a thermomixer (1000 rpm). The reactions were initiated by addition of AscA (20 µM final concentration). At regular intervals (t = 3, 6, 9, 12, 30 and 60 min), 70 µL of the reaction mixture was sampled, filtered as described above and 25 µL of the filtrate was mixed with 75 µL of a pre-mix of HRP (5 U.mL -1 final concentration) and Amplex® Red (ThermoFisher) (100 µM final concentration) in sodium phosphate buffer (50 mM pH 7.0). H2O2 concentrations waere then determined spectrophotometrically by measuring the absorbance at 540 nm in a microtiter plate reader.An H2O2 standard curve was prepared in the same conditions. Bioinformatics analysis. The sequence of the chitin-binding protein f...

Microbial aromatic catabolism offers a promising approach to convert lignin, a vast source of renewable carbon, into useful products. Aryl-O-demethylation is an essential biochemical reaction to ultimately catabolize coniferyl and sinapyl lignin-derived aromatic compounds, and is often a key bottleneck for both native and engineered bioconversion pathways. Here, we report the comprehensive characterization of a promiscuous P450 aryl-O-demethylase, consisting of a cytochrome P450 protein from the family CYP255A (GcoA) and a three-domain reductase (GcoB) that together represent a new two-component P450 class. Though originally described as converting guaiacol to catechol, we show that this system efficiently demethylates both guaiacol and an unexpectedly wide variety of lignin-relevant monomers. Structural, biochemical, and computational studies of this novel two-component system elucidate the mechanism of its broad substrate specificity, presenting it as a new tool for a critical step in biological lignin conversion.

No abstract

dioxygen ͉ compound I ͉ peroxidase ͉ peroxygenase ͉ chlorite

Chlorite dismutase (Cld) is a heme enzyme capable of rapidly and selectively decomposing chlorite (ClO2−) to Cl− and O2. The ability of Cld to promote O2 formation from ClO2− is unusual. Heme enzymes generally utilize ClO2− as an oxidant for reactions such as oxygen atom transfer to, or halogenation of, a second substrate. The X-ray crystal structure of Dechloromonas aromatica Cld co-crystallized with the substrate analogue nitrite (NO2−) was determined to investigate features responsible for this novel reactivity. The enzyme active site contains a single b-type heme coordinated by a proximal histidine residue. Structural analysis identified a glutamate residue hydrogen-bonded to the heme proximal histidine that may stabilize reactive heme species. A solvent-exposed arginine residue likely gates substrate entry to a tightly confined distal pocket. On the basis of the proposed mechanism of Cld, initial reaction of ClO2− within the distal pocket generates hypochlorite (ClO−) and a compound I intermediate. The sterically restrictive distal pocket probably facilitates the rapid rebound of ClO− with compound I forming the Cl− and O2 products. Common to other heme enzymes, Cld is inactivated after a finite number of turnovers, potentially via the observed formation of an off-pathway tryptophanyl radical species through electron migration to compound I. Three tryptophan residues of Cld have been identified as candidates for this off-pathway radical. Finally, a juxtaposition of hydrophobic residues between the distal pocket and the enzyme surface suggests O2 may have a preferential direction for exiting the active site.