Probing the Electrocatalytic Oxygen Reduction Reaction Reactivity of Immobilized Multicopper Oxidase CueO

The bioelectrocatalytic (oxygen reduction reaction, ORR) properties of the multicopper oxidase CueO immobilized on gold electrodes were investigated. Macroscopic electrochemical techniques were combined with in situ scanning tunneling microscopy (STM) and surface-enhanced Raman spectroscopy at the ensemble and at the single-molecule level. Self-assembled monolayer of mercaptopropionic acid, cysteamine, and p-aminothiophenol were chosen as redox mediators. The highest ORR activity was observed for the protein attached to amino-terminated adlayers. In situ STM experiments revealed that the presence of oxygen causes distinct structure and electronic changes in the metallic centers of the enzyme, which determine the rate of intramolecular electron transfer and, consequently, affect the rate of electron tunneling through the protein. Complementary Raman spectroscopy experiments provided access for monitoring structural changes in the redox state of the type 1 copper center of the immobilized enzyme during the CueO-catalyzed oxygen reduction cycle. These results unequivocally demonstrate the existence of a direct electronic communication between the electrode substrate and the type 1 copper center.

Electrochemical behaviour of different redox probes on single wall carbon nanotube buckypaper-modified electrodes

In the present work, the electrochemical properties of single-walled carbon nanotube buckypapers (BPs) were examined in terms of carbon nanotubes nature and preparation conditions. The performance of the different free-standing single wall carbon nanotube sheets was evaluated via cyclic voltammetry of several redox probes in aqueous electrolyte. Significant differences are observed in the electron transfer kinetics of the buckypaper-modified electrodes for both the outer- and inner-sphere redox systems. These differences can be ascribed to the nature of the carbon nanotubes (nanotube diameter, chirality and aspect ratio), surface oxidation degree and type of functionalities. In the case of dopamine, ferrocene/ferrocenium, and quinone/hydroquinone redox systems the voltammetric response should be thought as a complex contribution of different tips and sidewall domains which act as mediators for the electron transfer between the adsorbate species and the molecules in solution. In the other redox systems only nanotube ends are active sites for the electron transfer. It is also interesting to point out that a higher electroactive surface area not always lead to an improvement in the electron transfer rate of various redox systems. In addition, the current densities produced by the redox reactions studied here are high enough to ensure a proper electrochemical signal, which enables the use of BPs in sensing devices.

Enhancement of the Electrochemical Performance of SWCNT dispersed in a Silica Sol-gel Matrix by Reactive Insertion of a Conducting Polymer

The electroassisted encapsulation of Single-Walled Carbon Nanotubes was performed into silica matrices (SWCNT@SiO2). This material was used as the host for the potentiostatic growth of polyaniline (PANI) to yield a hybrid nanocomposite electrode, which was then characterized by both electrochemical and imaging techniques. The electrochemical properties of the SWCNT@SiO2-PANI composite material were tested against inorganic (Fe3+/Fe2+) and organic (dopamine) redox probes. It was observed that the electron transfer constants for the electrochemical reactions increased significantly when a dispersion of either SWCNT or PANI was carried out inside of the SiO2 matrix. However, the best results were obtained when polyaniline was grown through the pores of the SWCNT@SiO2 material. The enhanced reversibility of the redox reactions was ascribed to the synergy between the two electrocatalytic components (SWCNTs and PANI) of the composite material.

Electrochemical reactions of catechol, methylcatechol and dopamine at tetrahedral amorphous carbon (ta-C) thin film electrodes

The electrochemical reactions of dopamine, catechol and methylcatechol were investigated at tetrahedral amorphous carbon (ta-C) thin film electrodes. In order to better understand the reaction mechanisms of these molecules, cyclic voltammetry with varying scan rates was carried out at different pH values in H2SO4 and PBS solutions. The results were compared to the same redox reactions taking place at glassy carbon (GC) electrodes. All three catechols exhibited quasi-reversible behavior with sluggish electron transfer kinetics at the ta-C electrode. At neutral and alkaline pH, rapid coupled homogeneous reactions followed the oxidation of the catechols to the corresponding o-quinones and led to significant deterioration of the electrode response. At acidic pH, the extent of deterioration was considerably lower. All the redox reactions showed significantly faster electron transfer kinetics at the GC electrode and it was less susceptible toward surface passivation. An EC mechanism was observed for the oxidation of dopamine at both ta-C and GC electrodes and the formation of polydopamine was suspected to cause the passivation of the electrodes.

Respiratory versatility in Desulfovibrio desulfuricans ATCC 27774 – a proteomic approach

Poster presented at the Bacterial Electron Transfer Processes and their Regulation Meeting, European Federation of Biotechnology Microbial Physiology Section, 15-18 March 2015, Vimeiro, Portugal.

Role of glutamic acid 216 in cytochrome P450 2D6 substrate binding and catalysis

Human cytochrome P450 (P450) 2D6 is an important enzyme involved in the metabolism of drugs, many of which are amines or contain other basic nitrogen atoms. Asp301 has generally been considered to be involved in electrostatic docking with the basic substrates, on the basis of previous modeling studies and site-directed mutagenesis. Substitution of Glu216 with a residue other than Asp strongly attenuated the binding of quinidine, bufuralol, and several other P450 2D6 ligands. Catalytic activity with the substrates bufuralol and 4-methoxyphenethylamine was strongly inhibited by neutral or basic mutations at Glu216 (>95%), to the same extent as the substitution of Asn at Asp301. Unlike the Asp301 mutants, the Gln216 mutant (E216Q) retained 40% enzyme efficiency with the substrate spirosulfonamide, devoid of basic nitrogen, suggesting that the substitutions at Glu216 affect binding of amine substrates more than other catalytic steps. Attempts to induce catalytic specificity toward new substrates by substitutions at Asp301 and Glu216 were unsuccessful. Collectively, the results provide evidence for electrostatic interaction of amine substrates with Glu216, and we propose that both of these acidic residues plus at least another residue(s) is (are) involved in binding the repertoire of P450 2D6 ligands.

Oxidation of mixed-valence CoIII/FeII complexes reversed at high pH: A kinetico-mechanistic study of water oxidation

The outer-sphere oxidation of Fell in the mixed-valence complex trans-[(LCoNCFeII)-Co-14S-N-III(CN)(6)](-), being L-14S an N3S2 macrocylic donor set on the cobalt(III) center, has been studied. The comparison with the known processes of N-5 macrocycle complexes has been carried out in view of the important differences occurring on the redox potential of the cobalt center. The results indicate that the outer-sphere oxidation reactions with S2O82- and [Co(ox)(3)](3-) involve a great amount of solvent-assisted hydrogen bonding that, as a consequence from the change from two amines to sulfur donors, are more restricted. This is shown by the more positive values found for DeltaS(double dagger) and DeltaV(double dagger). The X-ray structure of the oxidized complex has been determined, and it is clearly indicative of the above-mentioned solvent-assisted hydrogen bonding between nitrogen and cyanide donors on the cobalt and iron centers, respectively. trans-[(LCoNCFeIII)-Co-14S-N-III(CN)(6)], as well as the analogous N-5 systems trans-[(LCoNCFeIII)-Co-14-N-III(CN)(6)], trans-[(LCoNCFeIII)-Co-15-N-III-(CN)(6)], and cis-[(LCoNCFeIII)-Co-n-N-III(CN)(6)], Oxidize water to hydrogen peroxide at pH > 10 with a rather simple stoichiometry, i.e., [(LCoNCFeIII)-Co-n-N-III(CN)(5)] + OH- - [(LCoNCFeII)-Co-n-N-III(CN)(5)](-) + 1/2H(2)O(2). In this way, the reversibility of the iron oxidation process is achieved. The determination of kinetic and thermal and pressure activation parameters for this water to hydrogen peroxide oxidation leads to the kinetic determination of a cyanide based OH- adduct of the complex. A second-order dependence on the base concentration is associated with deprotonation of this adduct to produce the final inner-sphere reduction process. The activation enthalpies are found to be extremely low (15 to 35 kJ mol(-1)) and responsible for the very fast reaction observed. The values of DeltaS(double dagger) and DeltaV(double dagger) (-76 to -113 J K-1 mol(-1) and -5.5 to -8.9 cm(3) mol(-1), respectively) indicate a highly organized but not very compressed transition state in agreement with the inner-sphere one-electron transfer from O2- to Fe-III.

A numerical approach to modeling the catalytic voltammetry of surface-confined redox enzymes

A finite difference method for simulating voltammograms of electrochemically driven enzyme catalysis is presented. The method enables any enzyme mechanism to be simulated. The finite difference equations can be represented as a matrix equation containing a nonlinear sparse matrix. This equation has been solved using the software package Mathematica. Our focus is on the use of cyclic voltammetry since this is the most commonly employed electrochemical method used to elucidate mechanisms. The use of cyclic voltammetry to obtain data from systems obeying Michaelis-Menten kinetics is discussed, and we then verify our observations on the Michaelis-Menten system using the finite difference simulation. Finally, we demonstrate how the method can be used to obtain mechanistic information on a real redox enzyme system, the complex bacterial molybdoenzyme xanthine dehydrogenase.

A PbS quantum-cube: conducting polymer composite for photovoltaic applications

We have developed a new non-polar synthesis for lead sulfide (PbS) quantum-cubes in the conjugated polymer poly-2-methoxy, 5-(2-ethyl-hexyloxy-p-phenylenevinylene) MEH-PPV. The conducting polymer acts to template and control the quantum-cube growth. Transmission electron microscopy of the composites has shown a bimodal distribution of cube sizes between 5 and 15 nm is produced with broad optical absorption from 300 to 650 nm. Photoluminescence suggests electronic coupling between the cubes and the conducting polymer matrix. The synthesis and initial characterization are presented in this paper. (C) 2003 Elsevier B.V. All rights reserved.

The acidic nature of the CcmG redox-active center is important for cytochrome c maturation in Escherichia coli

Cytochrome c biogenesis in Escherichia coli is a complex process requiring at least eight genes (ccmABC DEFGH). One of these genes, ccmG, encodes a thioredoxin-like protein with unusually specific redox activity. Here, we investigate the basis for CcmG function and demonstrate the importance of acidic residues surrounding the redox-active center.

A new approach to the synthesis of nanocrystal conjugated polymer composites

A novel one pot process has been developed for the preparation of PbS nanocrystals in the conjugated polymer poly 2-methoxy,5-(2 ethyl-hexyloxy-p-phenylenevinylene) (MEH-PPV). Current techniques for making such composite materials rely upon synthesizing the nanocrystals and conducting polymer separately, and subsequently mixing them. This multi-step technique has two serious drawbacks: templating surfactant must be removed before mixing, and co-solvent incompatibility causes aggregation. In our method, we eliminate the need for an initial surfactant by using the conducting polymer to terminate and template nanocrystal growth. Additionally, the final product is soluble in a single solvent. We present materials analysis which shows PbS nanocrystals can be grown directly in a conducting polymer, the resulting composite is highly ordered and nanocrystal size can be controlled.

Molecular mixed-valence cyanide bridged Co-III-Fe-II complexes

Cyano-bridged mixed-valence compounds have been known for a long time, i.e., Prussian Blue polymeric solids. Nevertheless, the interest in discrete complexes having a well-defined molecular nuclearity has emerged more recently. There are numerous examples of cyano-bridged mixed-valence complexes in the recent literature, as they show promising and useful applications in electrochromism, molecular magnetism and molecular electronics. In this paper, the reactivity, synthetic and structural chemistry, as well as some physical and chemical properties, of a series of discrete dinuclear mixed-valence cyano-bridged complexes of general formulae [LnCoIII(mu NC)Fe-II(CN)(5)](-) (L = pentadentate macrocyclic ligand) are reviewed. Special emphasis is given to the synthetic strategy, redox properties and metal-to-metal-charge-transfer (MMCT) band energy. Tuning the MMCT transition energy has been possible by changing the redox potential of the metal centers, both through structural and outer-sphere changes. The redox processes that involve the appearance and disappearance of these MMCT bands in the visible region have been dealt with in relation to the possible uses of the complexes. (c) 2004 Elsevier B.V. All rights reserved.

Tuning the photophysical behavior of luminescent cyclam derivatives by cation binding and excited state redox potential

The emission from two photoactive 14-membered macrocyclic ligands, 6-((naphthalen-1-ylmethyl)-amino)trans-6,13-dimethyl- 13-amino- 1,4,8,11 -tetraaza-cyclotetradecane (L-1) and 6-((anthracen-9-ylmethyl)-amino)trans-6,13 -dimethyl - 13 -amino- 1,4,8, 1 1-tetraaza-cyclotetradecane (L-2) is strongly quenched by a photoinduced electron transfer (PET) mechanism involving amine lone pairs as electron donors. Time-correlated single photon counting (TCSPC), multiplex transient grating (TG), and fluorescence upconversion (FU) measurements were performed to characterize this quenching mechanism. Upon complexation with the redox inactive metal ion, Zn(II), the emission of the ligands is dramatically altered, with a significant increase in the fluorescence quantum yields due to coordination-induced deactivation of the macrocyclic amine lone pair electron donors. For [ZnL2](2+), the substituted exocyclic amine nitrogen, which is not coordinated to the metal ion, does not quench the fluorescence due to an inductive effect of the proximal divalent metal ion that raises the ionization potential. However, for [ZnL1](2+), the naphthalene chromophore is a sufficiently strong excited-state oxidant for PET quenching to occur.

Direct electrochemistry of enzymes from the cytochrome P4502C family

The human cytochromes P450 are responsible for the clearance of similar to 90% of xenobiotics yet comparatively little is known about their electrochemistry. Here we report the first direct electrochemistry of P450s from the 2C subfamily; one of the major groups of enzymes from this family. Specifically, the proteins that we have examined are recombinant human P450s 2C9, 2C 18 and 2C 19 and reversible Fe-III/II couples are seen in the absence of dioxygen. Even in the presence of trace amounts of dioxygen, a pronounced cathodic response is seen which is assigned to catalytic reduction of the bound dioxygen ligand by the ferrous P450. (c) 2005 Elsevier B.V. All rights reserved.

Respiratory gene clusters of Metallosphaera sedula - differential expression and transcriptional organization

Metallosphaera sedula is a thermoacidophilic Crenarchaeon which is capable of leaching metals from sulfidic ores. The authors have investigated the presence and expression of genes encoding respiratory complexes in this organism when grown heterotrophically or chemolithotrophically on either sulfur or pyrite. The presence of three gene clusters, encoding two terminal oxidase complexes, the quinol oxidase SoxABCD and the SoxM oxidase supercomplex, and a gene cluster encoding a high-potential cytochrome b and components of a bc(1) complex analogue (cbsBA-soxL2N gene cluster) was established. Expression studies showed that the soxM gene was expressed to high levels during heterotrophic growth of M. sedula on yeast extract, while the soxABCD mRNA was most abundant in cells grown on sulfur. Reduced-minus-oxidized difference spectra of cell membranes showed cytochrome-related peaks that correspond to published spectra of Sulfolobus-type terminal oxidase complexes. In pyrite-grown cells, expression levels of the two monitored oxidase gene clusters were reduced by a factor of 10-12 relative to maximal expression levels, although spectra of membranes clearly contained oxidase-associated haems, suggesting the presence of additional gene clusters encoding terminal oxidases in M. sedula. Pyrite- and sulfur-grown cells contained high levels of the cbsA transcript, which encodes a membrane-bound cytochrome b with a possible role in iron oxidation or chemolithotrophy. The cbsA gene is not co-transcribed with the soxL2N genes, and therefore does not appear to be an integral part of this bc(1) complex analogue. The data show for the first time the differential expression of the Sulfolobus-type terminal oxidase gene clusters in a Crenarchaeon in response to changing growth modes.