Catalytic Voltammetry of the Molybdoenzyme Sulfite Dehydrogenase from <i>Sinorhizobium meliloti</i>

Sulfite dehydrogenase from the soil bacterium <i>Sinorhizobium meliloti</i> (SorT) is a periplasmic, homodimeric molybdoenzyme with a molecular mass of 78 kDa. It differs from most other well studied sulfite oxidizing enzymes, as it bears no heme cofactor. SorT does not readily reduce ferrous horse heart cytochrome <i>c</i> which is the preferred electron acceptor for vertebrate sulfite oxidases. In the present study, ferrocene methanol (FM) (in its oxidized ferrocenium form) was utilized as an artificial electron acceptor for the catalytic SorT sulfite oxidation reaction. Cyclic voltammetry of FM was used to generate the active form of the mediator at the electrode surface. The FM-mediated catalytic sulfite oxidation by SorT was investigated by two different voltammetric methods, namely, (i) SorT freely diffusing in solution and (ii) SorT confined to a thin layer at the electrode surface by a semipermeable dialysis membrane. A single set of rate and equilibrium constants was used to simulate the catalytic voltammograms performed under different sweep rates and with various concentrations of sulfite and FM which provides new insights into the kinetics of the SorT catalytic mechanism. Further, we were able to model the role of the dialysis membrane in the kinetics of the overall catalytic system

Catalytic Electrochemistry of Xanthine Dehydrogenase

We report the mediated electrocatalytic voltammetry of the molybdoenzyme xanthine dehydrogenase (XDH) from <i>Rhodobacter capsulatus</i> at a thiol-modified Au electrode. The 2-electron acceptor <i>N</i>-methylphenazinium methanesulfonate (phenazine methosulfate, PMS) is an effective artificial electron transfer partner for XDH instead of its native electron acceptor NAD⁺. XDH catalyzes the oxidative hydroxylation of hypoxanthine to xanthine and xanthine to uric acid. Cyclic voltammetry was used to generate the active (oxidized) form of the mediator. Simulation of the catalytic voltammetry across a broad range of substrate and PMS concentrations at different sweep rates was achieved with the program DigiSim to yield a set of consistent rate and equilibrium constants that describe the catalytic system. This provides the first example of the mediated electrochemistry of a xanthine dehydrogenase (or oxidase) that is uncomplicated by interference from product oxidation. A remarkable two-step, sequential oxidation of hypoxanthine to uric acid via xanthine by XDH is observed

Low-Potential Amperometric Enzyme Biosensor for Xanthine and Hypoxanthine

The bacterial xanthine dehydrogenase (XDH) from Rhodobacter capsulatus was immobilized on an edge-plane pyrolytic graphite (EPG) electrode to construct a hypoxanthine/xanthine biosensor that functions at physiological pH. Phenazine methosulfate (PMS) was used as a mediator which acts as an artificial electron-transfer partner for XDH. The enzyme catalyzes the oxidation of hypoxanthine to xanthine and also xanthine to uric acid by an oxidative hydroxylation mechanism. The present electrochemical biosensor was optimized in terms of applied potential and pH. The electrocatalytic oxidation response showed a linear dependence on the xanthine concentration ranging from 1.0 × 10^–5 to 1.8 × 10^–3 M with a correlation coefficient of 0.994. The modified electrode shows a very low detection limit for xanthine of 0.25 nM (signal-to-noise ratio = 3) using controlled potential amperometry

A Kinetico-Mechanistic Study on Cu^II Deactivators Employed in Atom Transfer Radical Polymerization

Copper complexes of tertiary amine ligands have emerged as the catalysts of choice in the extensively employed atom transfer radical polymerization (ATRP) protocol. The halide ligand substitution reactions of five-coordinate copper­(II) complexes of tris­[2-(dimethylamino)­ethyl]­amine (Me₆tren), one of the most active ATRP catalysts, has been studied in a range of organic solvents using stopped-flow techniques. The kinetic and activation parameters indicate that substitution reactions on [Cu^II(Me₆tren)­X]⁺ (X^– = Cl^– and Br^–) and [Cu^II(Me₆tren)­(Solv)]²⁺ (Solv = MeCN, DMF, DMSO, MeOH, EtOH) are dissociatively activated; this behavior is independent of the solvent used. Adjusting the effective concentration of the solvent by addition of an olefinic monomer to the solution does not affect the kinetics of the halide binding (<i>k</i>_on) but can alter the outer-sphere association equilibrium constant (<i>K</i>_OS) between reactants prior to the formal ligand substitution. Halide (X^–/Y^–) exchange reactions (X = Br and Y = Cl) involving the complex [Cu­(Me₆tren)­X]⁺ and Y^– reveal that the substitution is thermodynamically favored. The influence of solvent on the substitution reactions of [Cu­(Me₆tren)­X]⁺ is complex; the more polar DMF confers a greater entropic driving force but larger enthalpic demands than MeCN. These substitution reactions are compared with those for copper­(II) complexes bearing the tris­[2-(diethylamino)­ethyl]­amine (Et₆tren) and tris­[2-(pyridyl)­methyl]­amine (tpa) ligands, which have also been used as catalysts for ATRP. Changing the ligand has a significant impact on the kinetics of X^–/Y^– exchange. These correlations are discussed in relation to the ability of five-coordinate [CuLX]⁺ complexes to deactivate radicals in ATRP

Computational Insights on the Geometrical Arrangements of Cu(II) with a Mixed-Donor N₃S₃ Macrobicyclic Ligand

The macrobicyclic mixed-donor N₃S₃ cage ligand AMME-N₃S₃sar (1-methyl-8-amino-3,13,16-trithia-6,10,19-triazabicyclo[6.6.6]­eicosane) can form complexes with Cu­(II) in which it acts as hexadentate (N₃S₃) or tetradentate (N₂S₂) donor. These two complexes are in equilibrium that is strongly influenced by the presence of halide ions (Br^– and Cl^–) and the nature of the solvent (DMSO, MeCN, and H₂O). In the absence of halides the hexadentate coordination mode of the ligand is preferred and the encapsulated complex (“Cu-in²⁺”) is formed. Addition of halide ions in organic solvents (DMSO or MeCN) leads to the tetradentate complex (“Cu-out⁺”) in a polyphasic kinetic process, but no Cu-out⁺ complex is formed when the reaction is performed in water. Here we applied density functional theory calculations to study the mechanism of this interconversion as well as to understand the changes in the reactivity associated with the presence of water. Calculations were performed at the B3LYP/(SDD,6-31G**) level, in combination with continuum (MeCN) or discrete-continuum (H₂O) solvent models. Our results show that formation of Cu-out⁺ in organic media is exergonic and involves sequential halide-catalyzed inversion of the configuration of a N-donor of the macrocycle, rapid halide coordination, and inversion of the configuration of a S-donor. In aqueous solution the solvent is found to have an effect on both the thermodynamics and the kinetics of the reaction. Thermodynamically, the process becomes endergonic mainly due to the preferential solvation of halide ions by water, while the kinetics is influenced by formation of a network of H-bonded water molecules that surrounds the complex

Mediated Electrochemistry of Nitrate Reductase from <i>Arabidopsis thaliana</i>

Herein we report the mediated electrocatalytic voltammetry of the plant molybdoenzyme nitrate reductase (NR) from <i>Arabidopsis thaliana</i> using the established truncated molybdenum-heme fragment at a glassy carbon (GC) electrode. Methyl viologen (MV), benzyl viologen (BV), and anthraquinone-2-sulfonic acid (AQ) are employed as effective artificial electron transfer partners for NR, differing in redox potential over a range of about 220 mV and delivering different reductive driving forces to the enzyme. Nitrate is reduced at the Mo active site of NR, yielding the oxidized form of the enzyme, which is reactivated by the electro-reduced form of the mediator. Digital simulation was performed using a single set of enzyme dependent parameters for all catalytic voltammetry obtained under different sweep rates and various substrate or mediator concentrations. The kinetic constants from digital simulation provide new insight into the kinetics of the NR catalytic mechanism

The Reversible Electrochemical Interconversion of Formate and CO₂ by Formate Dehydrogenase from <i>Cupriavidus necator</i>

The bacterial molybdenum (Mo)-containing formate dehydrogenase (FdsDABG) from Cupriavidus necator is a soluble NAD+-dependent enzyme belonging to the DMSO reductase family. The holoenzyme is complex and possesses nine redox-active cofactors including a bis(molybdopterin guanine dinucleotide) (bis-MGD) active site, seven iron–sulfur clusters, and 1 equiv of flavin mononucleotide (FMN). FdsDABG catalyzes the two-electron oxidation of HCOO– (formate) to CO2 and reversibly reduces CO2 to HCOO– under physiological conditions close to its thermodynamic redox potential. Here we develop an electrocatalytically active formate oxidation/CO2 reduction system by immobilizing FdsDABG on a glassy carbon electrode in the presence of coadsorbents such as chitosan and glutaraldehyde. The reversible enzymatic interconversion between HCOO– and CO2 by FdsDABG has been realized with cyclic voltammetry using a range of artificial electron transfer mediators, with methylene blue (MB) and phenazine methosulfate (PMS) being particularly effective as electron acceptors for FdsDABG in formate oxidation. Methyl viologen (MV) acts as both an electron acceptor (MV2+) in formate oxidation and an electron donor (MV+•) for CO2 reduction. The catalytic voltammetry was reproduced by electrochemical simulation across a range of sweep rates and concentrations of formate and mediators to provide new insights into the kinetics of the FdsDABG catalytic mechanism

Reversible Rearrangements of Cu(II) Cage Complexes: Solvent and Anion Influences

The macrobicyclic mixed donor cage ligand AMME-N₃S₃sar (1-methyl-8-amino-3,13,16-trithia-6,10,19-triazabicyclo[6.6.6]­eicosane) is capable of binding to Cu­(II) as either a hexadentate (N₃S₃) or tetradentate (N₂S₂) ligand. The “Cu-in” (hexadentate)/“Cu-out” (tetradendate) equilibrium for the {Cu­(AMME-N₃S₃sar)}²⁺ units is strongly influenced by both solvent (DMSO, MeCN, and water) and halide ions (Br^– and Cl^–). We have established a crucial role of the solvent in these processes through the formation of intermediate solvato complexes, which are substituted by incoming halide ions triggering a final isomerization reaction. Surprisingly, for reactions carried out in the usually strongly coordinating solvent water, the completely encapsulated N₃S₃-bound “Cu-in” form is dominant. Furthermore, the small amounts of the “Cu-out” form present in equilibrated DMSO or MeCN solutions revert entirely to the “Cu-in” form in aqueous media, thus preventing reaction with halide anions which otherwise lead to partial or even complete decomposition of the complex. From the kinetic, electrochemical, and EPR results, the existence of an outer-sphere H-bonded network of water molecules interacting with the complex inhibits egress of the Cu­(II) ion from the cage ligand. This is extremely relevant in view of outer sphere interactions present in strongly hydrogen bonding solvents and their effects on Cu­(II) complexation

Afurika nanbu ni okeru gengo no kiki - Matthias Brenzinger-shi ni kiku [Language Endangerment ion Southern Africa - Interview with Matthias Brenzinger]

Viridicatumtoxins, which belong to a rare class of fungal tetracycline-like mycotoxins, were subjected to comprehensive spectroscopic and chemical analysis, leading to reassignment/assignment of absolute configurations and characterization of a remarkably acid-stable antibiotic scaffold. Structure activity relationship studies revealed exceptional growth inhibitory activity against vancomycin-resistant Enterococci (IC₅₀ 40 nM), >270-fold more potent than the commercial antibiotic oxytetracycline

Structure and Absolute Configuration of Methyl (3<i>R</i>)‑Malonyl-(13<i>S</i>)‑hydroxycheilanth-17-en-19-oate, a Sesterterpene Derivative from the Roots of <i>Aletris farinosa</i>

We report the isolation and structure elucidation of a new cheilanthane sesterterpene from the roots of <i>Aletris farinosa</i> that possesses an unusual malonate half-ester functional group. The structure of <b>1</b> was determined via mass spectrometry and 1D and 2D NMR spectroscopy, while its absolute configuration was determined via X-ray crystallographic analysis performed on its methyl ester derivative <b>2</b>