Tommaso Ferri

Bioelectrochemical reduction of CO2 to CH4 via direct and indirect extracellular electron transfer by a hydrogenophilic methanogenic culture

This study describes the performance of a microbial biocathode, based on a hydrogenophilic methanogenic culture, capable of reducing carbon dioxide to methane, at high rates (up to 0.055 + or - 0.002 mmol d(-1) mgVSS(-1)) and electron capture efficiencies (over 80%). Methane was produced, at potentials more negative than -650 mV vs. SHE, both via abiotically produced hydrogen gas (i.e., via hydrogenophilic methanogenesis) and via direct extracellular electron transfer. The relative contribution of these two mechanisms was highly dependent on the set cathode potential. Both cyclic voltammetry tests and batch potentiostatic experiments indicated that the capacity for extracellular electron transfer was a constitutive trait of the hydrogenophilic methanogenic culture. In principle, both electrons and carbon dioxide required for methane production could be obtained from a bioanode carrying out the oxidation of waste organic substrates.

Insights into Cytochrome c−Cardiolipin Interaction. Role Played by Ionic Strength†

The finding that cytochrome c (cyt c) plays a role in programmed cell death after its release from the mitochondrion has recently renewed interest in this protein. The structural changes in cytochrome c observed at early stages of the apoptotic process have been related to changes occurring in the protein when it forms a complex with phospholipid vesicles. Among the lipids constituting the membrane, cardiolipin is the one thought to bind to cyt c. In this paper, we have investigated the influence exerted by ionic strength on cytochrome c-cardiolipin interaction and found that formation of the cytochrome c-cardiolipin complex occurs via two distinct transitions, implying a high-affinity site and a low-affinity site. Ionic strength significantly influences complex stability; sodium chloride dissociates the complex through two distinct transitions, the second of which occurs at a very high anion concentration. ATP also dissociates the complex, but under the conditions that were investigated, its action is limited to the high-affinity site. The dissociation process is characterized by a very slow kinetic rate constant ( k obs = 4.2 x 10 (-3) s (-1)) and requires several minutes to be completed. We ascribe it to the high activation barrier met by the protein when restoring the native Fe(III)-M80 axial bond. The peroxidase activity shown by cardiolipin-bound cytochrome c is indicative of a less packed protein tertiary conformation in the complex. In line with earlier reports, these data highlight the manifold functions of cytochrome c besides the well-known role it plays in oxidative phosphorylation, shedding more light on the properties of the cytochrome c-cardiolipin complex, involved in the progression of early stages of apoptosis.

Protein immobilization at gold–thiol surfaces and potential for biosensing

AbstractsSelf-assembled monolayers (SAMs) provide a convenient, flexible and simple system to tailor the interfacial properties of metals, metal oxides and semiconductors. Monomolecular films prepared by self-assembly are attractive for several exciting applications because of the unique possibility of making the selection of different types of terminal functional groups and as emerging tools for nanoscale observation of biological interactions. The tenability of SAMs as platforms for preparing biosurfaces is reviewed and critically discussed. The different immobilization approaches used for anchoring proteins to SAMs are considered as well as the nature of SAMs; particular emphasis is placed on the chemical specificity of protein attachment in view of preserving protein native structure necessary for its functionality. Regarding this aspect, particular attention is devoted to the relation between the immobilization process and the electrochemical response (i.e. electron transfer) of redox proteins, a field where SAMs have attracted remarkable attention as model systems for the design of electronic devices. Strategies for creating protein patterns on SAMs are also outlined, with an outlook on promising and challenging future directions for protein biochip research and applications.

Direct electrochemistry of membrane-entrapped horseradish peroxidase.: Part II: Amperometric detection of hydrogen peroxide

Abstract Direct (unmediated) electrochemistry of horseradish peroxidase (HRP) immobilized within a polymeric film is investigated at a pyrolytic graphite electrode by dc cyclic voltammetry, in the absence and in the presence of hydrogen peroxide. Under the latter condition, a reduction wave centered at approx. −280 mV (vs. SCE) is observed, the intensity of which is strictly dependent on the hydrogen peroxide concentration. This permits a voltammetric investigation of the electrocatalytic reduction of hydrogen peroxide. Flow and flow–injection measurements carried out at constant potential under the same conditions, support voltammetric data. The suitability of the immobilized HRP-based electrodic system to monitor the presence of important analytes, such as glucose or choline, in solution, is also discussed. To this issue, suitable amounts of HRP and glucose oxidase (or, in turn, choline oxidase) were simultaneously entrapped in the polymer. The results obtained are of potential value for basic and applied biochemistry and represent a first step for construction of a mediator-free (third-generation) biosensor which may find application in the biosensoristic area.

Extended cardiolipin anchorage to cytochrome c: a model for protein-mitochondrial membrane binding.

Two models have been proposed to explain the interaction of cytochrome c with cardiolipin (CL) vesicles. In one case, an acyl chain of the phospholipid accommodates into a hydrophobic channel of the protein located close the Asn52 residue, whereas the alternative model considers the insertion of the acyl chain in the region of the Met80-containing loop. In an attempt to clarify which proposal offers a more appropriate explanation of cytochrome c–CL binding, we have undertaken a spectroscopic and kinetic study of the wild type and the Asn52Ile mutant of iso-1-cytochrome c from yeast to investigate the interaction of cytochrome c with CL vesicles, considered here a model for the CL-containing mitochondrial membrane. Replacement of Asn52, an invariant residue located in a small helix segment of the protein, may provide data useful to gain novel information on which region of cytochrome c is involved in the binding reaction with CL vesicles. In agreement with our recent results revealing that two distinct transitions take place in the cytochrome c–CL binding reaction, data obtained here support a model in which two (instead of one, as considered so far) adjacent acyl chains of the liposome are inserted, one at each of the hydrophobic sites, into the same cytochrome c molecule to form the cytochrome c–CL complex.

Determination of selenium speciation in river waters by adsorption on iron(III)-Chelex-100 resin and differential pulse cathodic stripping voltammetry

Abstract This paper describes the preconcentration of Se(IV) from polluted waters by iron(III)-loaded Chelex-100 resin and its determination by differential pulse cathodic stripping voltammetry. The method is used to study selenium speciation in waters, and a speciation method, suitable for concentration levels of environmental significance (ng/l), is defined and applied to a real polluted system (Tevere river). Besides the determination of Se(IV) and Se(VI), the method allows discrimination between “free” and “bound” dissolved Se(IV). As regards bound Se(IV), a non-labile interaction with an “unidentified species” is hypothesised and the conditional formation constant of the hypothesised process evaluated as well as the concentration of “unidentified species”. Finally, the distribution of Se(VI) and -(IV) added to untreated water samples was studied.

The humic acid analogue antraquinone-2,6-disulfonate (AQDS) serves as an electron shuttle in the electricity-driven microbial dechlorination of trichloroethene to cis-dichloroethene

Quinone moieties in humic substances have previously been shown to serve as extracellular electron acceptors in different metabolic pathways. Here we show that the humic acid analogue antraquinone-2,6-disulfonate (AQDS) can also serve as an electron donor in the microbial reductive dechlorination of TCE to cis-DCE. In a bioelectrochemical system (BES), equipped with a glassy carbon electrode (cathode) polarized at -250mV vs. SHE, electrically reduced AQDS served as the shuttle of electrons between the electrode surface and the dechlorinating bacteria. Interestingly, AQDS selectively stimulated only the first step of the TCE dechlorination sequence, leading to the formation of cis-DCE. Bioelectrochemical experiments carried out using a dechlorinating culture, highly enriched in the cis-DCE dechlorinating microorganism Dehalococcoides spp., confirmed the inability of reduced AQDS to serve as an electron donor for cis-DCE dechlorination. The results of this study have implications for the development of bioelectrochemical systems for groundwater remediation, as well as for the biogeochemical fate of chlorinated solvents in humic substances-rich subsurface environments.

An electrochemical multienzymatic biosensor for determination of cholesterol.

This paper describes an electrochemical biosensor for free cholesterol monitoring. The sensor is a multienzymatic electrodic system in which horseradish peroxidase and cholesterol oxidase are simultaneously immobilized within a polymeric film, on the surface of a pyrolitic graphite electrode. From voltammetric and amperometric (flow-injection) data obtained, the efficiency, reproducibility and stability of the system are discussed. Results obtained, of interest for basic and applied biochemistry, represent a first step for construction of a mediator-free biosensor with potentialities for a successful application in the biosensor area.

Membrane supported bilayer lipid membranes array: preparation, stability and ion-channel insertion

Abstract In the present paper we describe the preparation and characterization of reconstituted bilayer lipid membranes (BLM) supported on a polycarbonate membrane. BLMs self-assemble on the holes of the membrane, which was previously covered with Au and an octadecanethiol (ODT) self-assembled monolayer (SAM). The obtained mixed hybrid bilayer lipid membranes (MHBLM) array allows recording of relatively intense signals. The steps leading to formation of the system have been investigated, its stability determined and reproducibility evaluated. The insertion of an ion channel (Gramicidin D) and a K + carrier (Valinomycine) was also evaluated.

Direct electrochemical evidence for an equilibrium intermediate in the guanidine-induced unfolding of cytochrome c.

This paper reports a voltammetric and spectroscopic investigation of the guanidine-induced unfolding of cytochrome c at neutral pH and 25 degrees C. Electrochemical data provide direct evidence for the presence of an equilibrium intermediate (form I) strictly dependent on the denaturant concentration. The midpoint potential of form I has been determined (E1/2 = +0.010 V vs. NHE) and its structural features defined from analysis of the circular dichroism and absorbance spectroscopy data obtained under the same experimental conditions. From the correlation of electrochemical and spectroscopic data, we propose that the features detected by the intermediate conform to the molten globule state.