José Luis Olloqui-Sariego

Interprotein Coupling Enhances the Electrocatalytic Efficiency of Tobacco Peroxidase Immobilized at a Graphite Electrode.

Covalent immobilization of enzymes at electrodes via amide bond formation is usually carried out by a two-step protocol, in which surface carboxylic groups are first activated with the corresponding cross-coupling reagents and then reacted with protein amine groups. Herein, it is shown that a modification of the above protocol, involving the simultaneous incubation of tobacco peroxidase and the pyrolytic graphite electrode with the cross-coupling reagents produces higher and more stable electrocatalytic currents than those obtained with either physically adsorbed enzymes or covalently immobilized enzymes according to the usual immobilization protocol. The remarkably improved electrocatalytic properties of the present peroxidase biosensor that operates in the 0.3 V ≤ E ≤ 0.8 V (vs SHE) potential range can be attributed to both an efficient electronic coupling between tobacco peroxidase and graphite and to the formation of intra- and intermolecular amide bonds that stabilize the protein structure and improve the percentage of anchoring groups that provide an adequate orientation for electron exchange with the electrode. The optimized tobacco peroxidase sensor exhibits a working concentration range of 10-900 μM, a sensitivity of 0.08 A M(-1) cm(-2) (RSD 0.05), a detection limit of 2 μM (RSD 0.09), and a good long-term stability, as long as it operates at low temperature. These parameter values are among the best reported so far for a peroxidase biosensor operating under simple direct electron transfer conditions.

Analytical Expressions for Proton Transfer Voltammetry: Analogy to Surface Redox Voltammetry with Frumkin Interactions

Theory for interfacial proton transfer voltammetry of a molecular film containing any acid/base loading has been developed under equilibrium conditions. Diagnostic criteria to disentangle the interplay between diffuse layer and ionization effects are outlined. Easy-to-use analytical expressions for the voltammetric features are derived for the particular case of an invariant diffuse layer effect, which turn out to be entirely analogous to those for a surface redox conversion with Frumkin interactions. It is demonstrated that, regardless of the electrolyte concentration, significant ionization of the external acid groups located nearby the diffuse layer is sufficient for the fulfillment of this relevant particular case. A strategy is outlined to determine the amount, the intrinsic pKa, and the burial depth of the voltammetrically active groups from the surface concentration dependence of the main voltammetric features. Self-assembled monolayers of 11-mercaptoundecanoic acid deposited on Au(111), containing higher amounts of buried carboxylic groups than previously reported, have been studied to assess more critically the influence of electrostatic effects on the ionization process. Preliminary evidence suggests that the protonation/deprotonation voltammetric wave involves physisorbed rather than chemisorbed thiol molecules. Application of the present theoretical approach to this system reveals that the voltammetrically active carboxylic groups are located close to the electrode surface and become more acidic upon increasing their surface concentration.

Temperature-Driven Changeover in the Electron-Transfer Mechanism of a Thermophilic Plastocyanin.

Electron-transfer kinetics of the thermophilic protein Plastocyanin from Phormidium laminosum adsorbed on 1,ω-alkanedithiol self-assembled monolayers (SAMs) deposited on gold have been investigated. The standard electron-transfer rate constant has been determined as a function of electrode-protein distance and solution viscosity over a broad temperature range (0-90 °C). For either thin or thick SAMs, the electron-transfer regime remains invariant with temperature, whereas for the 1,11-undecanethiol SAM of intermediate chain length, a kinetic regime changeover from a gated or friction-controlled mechanism at low temperature (0-30 °C) to a nonadiabatic mechanism above 40 °C is observed. To the best of our knowledge, this is the first time a thermal-induced transition between these two kinetic regimes is reported for a metalloprotein.

Electrosynthesis of Trichloroacetic Acid by Electrochemical Carboxylation of Carbon Tetrachloride

The electrochemical carboxylation of carbon tetrachloride (CT) has been investigated to obtain trichloroacetic acid, providing a strategy for the elimination/conversion of this substance. The electrochemical behavior of CT in the presence of CO 2 has been studied by means of cyclic voltammetry in acetonitrile using tetrabutylammonium perchlorate as supporting electrolyte. Ag as working electrode exhibits electrocatalytic activity toward the electroreduction of CT in the presence of CO 2 , shifting reduction potentials to more positive values compared to other inert electrodes such as the glassy carbon electrode and graphite. The influence of the cathode and anode material has also been investigated by preparative electrolysis. The electrocarboxylation of the CT is remarkably more efficient if Ag is used as cathode and Zn as anode, giving high current efficiency, excellent trichloroacetate yield, and minimal subproducts formation.

Stereoselective Electrochemical Reduction of Imazapyr in Aqueous Media Without Chiral Auxiliaries

The electrochemical reduction of imazapyr at the static mercury drop electrode was studied by cyclic voltammetry as a function of pH in aqueous buffered media. The process leads to the 2,3-C=N double bond reduction in the imidazoline moiety in all media. The products have been isolated by controlled-potential electrolyses and identified by high performance liquid chromatography-tandem mass spectrometry measurements and 1 H-NMR, 13 C-NMR, and IR spectra. Although no chiral auxiliary was used, a moderate diasteroisomeric excess was observed. The diasteromeric ratio depends on pH of the electrolyses.

An Efficient Electrochemical Carboxylation of Polychloromethanes at Zinc Cathode in Acetonitrile

An efficient and selective electrochemical carboxylation process of some polychloromethanes was developed to synthesize chloroacetic acids using a zinc cathode and sacrificial anodes under a galvanostatic regime. The electrochemical behavior of carbon tetrachloride (CT), chloroform (CF), and its carboxylated products was studied by cyclic voltammetry in acetonitrile using tetrabutylammonium perchlorate as a supporting electrolyte in the presence and absence of CO 2 . Galvanostatic electrolyses of CT and CF in the presence of CO 2 were carried out in an undivided cell. The influence of some operative parameters on the performance of the process was studied. The electrochemical carboxylation of CT resulted in high faradaic yields and in the selective formation of trichloroacetate (TCA). The electrocarboxylation of CF led to high current efficiencies and a good dichloroacetate yield. TCA and dichloromethane (DCM) were obtained as by-products in this process. The formation of TCA and DCM suggests that the electrocarboxylation reaction of CF competes with the self-protonation of the carbanion CHCl - 2 generated in its reduction process.

Protein crosslinking improves the thermal resistance of plastocyanin immobilized on a modified gold electrode

Increasing the thermal stability of immobilized proteins is a motivating goal for improving the performance of electrochemical biodevices. In this work, we propose the immobilization of crosslinked plastocyanin from the thermophilic cyanobacterium Phormidium laminosum by simultaneous incubation of a mixture of plastocyanin and the coupling reagents. The thermal stability of the so built covalently immobilized protein films has been assessed by cyclic voltammetry in the 0-90 °C temperature range and has been compared to that of physisorbed films. It is shown that the protein loss along a thermal cycle is significantly reduced in the case of the crosslinked films, whose redox properties remain unaltered along a cyclic heating-cooling thermal scan, and can withstand the contact with 70 °C solutions for four hours. Comparison of thermal unfolding curves obtained by circular dichroism spectroscopy of both free and crosslinked protein confirms the improved thermic resistance of the crosslinked plastocyanin. Notably, the electron transfer thermodynamics of physisorbed and crosslinked plastocyanin films are quite similar, suggesting that the formation of intra- and inter-protein amide bonds do not affect the integrity and functionality of the copper redox centers. UV-Vis absorption and circular dichroism measurements corroborate that protein crosslinking does not alter the coordination geometry of the metal center.

Key Role of the Local Hydrophobicity in the East Patch of Plastocyanins on Their Thermal Stability and Redox Properties

Understanding the molecular basis of the thermal stability and functionality of redox proteins has important practical applications. Here, we show a distinct thermal dependence of the spectroscopic and electrochemical properties of two plastocyanins from the thermophilic cyanobacterium Phormidium laminosum and their mesophilic counterpart from Synechocystis sp. PCC 6803, despite the similarity of their molecular structures. To explore the origin of these differences, we have mimicked the local hydrophobicity in the east patch of the thermophilic protein by replacing a valine of the mesophilic plastocyanin by isoleucine. Interestingly, the resulting mutant approaches the thermal stability, redox thermodynamics, and dynamic coupling of the flexible site motions of the thermophilic protein, indicating the existence of a close connection between the hydrophobic packing of the east patch region of plastocyanin and the functional control and stability of the oxidized and reduced forms of the protein.