Reconstitution of supramolecular organization involved in energy metabolism at electrochemical interfaces for biosensing and bioenergy production.

How the redox proteins and enzymes involved in bioenergetic pathways are organized is a relevant fundamental question, but our understanding of this is still incomplete. This review provides a critical examination of the electrochemical tools developed in recent years to obtain knowledge of the intramolecular and intermolecular electron transfer processes involved in metabolic pathways. Furthermore, better understanding of the electron transfer processes associated with energy metabolism will provide the basis for the rational design of biotechnological devices such as electrochemical biosensors, enzymatic and microbial fuel cells, and hydrogen production factories. Starting from the redox complexes involved in two relevant bacterial chains, i.e., from the hyperthermophile Aquifex aeolicus and the acidophile Acidithiobacillus ferrooxidans, examination of protein-protein interactions using electrochemistry is first reviewed, with a focus on the orientation of a protein on an electrochemical interface mimic of a physiological interaction between two partners. Special attention is paid to current research in the electrochemistry of essential membrane proteins, which is one mandatory step toward the understanding of energy metabolic pathways. The complex and challenging architectures built to reconstitute a membrane-like environment at an electrode are especially considered. The role played by electrochemistry in the attempt to consider full bacterial metabolism is finally emphasized through the study of whole cells immobilized at electrodes as suspensions or biofilms. Before the performances of biotechnological devices can be further improved to make them really attractive, questions remain to be addressed in this particular field of research. We discuss the bottlenecks that need to be overcome in the future.

High electrolyte concentration effect on enzymatic oxygen reduction.

The nature, the composition and the concentration of electrolytes is essential for electrocatalysis involving redox enzymes. Here, we discuss the effect of various electrolyte compositions with increasing ionic strengths on the stability and activity towards O2 reduction of the bilirubin oxidase from Myrothecium verrucaria (Mv BOD). Different salts, Na2SO4, (NH4)2SO4, NaCl, NaClO4, added to a phosphate buffer (PB) were evaluated with concentrations ranging from 100 mM up to 1.7 M. On functionalized carbon nanotube-modified electrodes, it was shown that the catalytic current progressively decreased with increasing salt concentrations. The process was reversible suggesting it was not related to enzyme leakage. The enzyme was then immobilized on gold electrodes modified by self-assembling of thiols. When the enzyme was simply adsorbed, the catalytic current decreased in a reversible way, thus behaving similarly as on carbon nanotubes. Enzyme mobility at the interface induced by a modification in the interactions between the protein and the electrode upon salt addition may account for this behavior. When the enzyme was covalently attached, the catalytic current increased. Enzyme compaction is proposed to be at the origin of such catalytic current increase because of shorter distances between the first copper site electron acceptor and the electrode.

Biocatalysts for fuel cells: efficient hydrogenase orientation for H2 oxidation at electrodes modified with carbon nanotubes.

We report the modification of gold and graphite electrodes with commercially available carbon nanotubes for immobilization of Desulfovibrio fructosovorans [NiFe] hydrogenase, for hydrogen evolution or consumption. Multiwalled carbon nanotubes, single-walled carbon nanotubes (SWCNs), and amine-modified and carboxyl-functionalized SWCNs were used and compared throughout. Two separate methods were performed: covalent attachment of oriented hydrogenase by controlled architecture of carbon nanotubes at gold electrodes, and adsorption of hydrogenase at carbon-nanotube-coated pyrolytic graphite electrodes. In the case of self-assembled carbon nanotubes at gold electrodes, hydrogenase orientation based on electrostatic interaction with the electrode surface was found to control the electrocatalytic process for H(2) oxidation. In the case of carbon nanotube coatings on pyrolytic graphite electrodes, catalysis was controlled more by the geometry of the nanotubes than by the orientation of the enzyme. Noticeably, shortened SWCNs were demonstrated to allow direct electron transfer and generate high and quite stable current densities for H(2) oxidation via adsorbed hydrogenase, despite having many carboxylic surface functions that could yield unfavorable hydrogenase orientation for direct electron transfer. This result is attributable to the high degree of oxygenated surface functions in addition to the length of shortened SWCNs that yields highly divided materials.

Electron transfer in an acidophilic bacterium: interaction between a diheme cytochrome and a cupredoxin.

Acidithiobacillus ferrooxidans, a chemolithoautotrophic Gram-negative bacterium, has a remarkable ability to obtain energy from ferrous iron oxidation at pH 2. Several metalloproteins have been described as being involved in this respiratory chain coupling iron oxidation with oxygen reduction. However, their properties and physiological functions remain largely unknown, preventing a clear understanding of the global mechanism. In this work, we focus on two metalloproteins of this respiratory pathway, a diheme cytochrome c4 (Cyt c4) and a green copper protein (AcoP) of unknown function. We first demonstrate the formation of a complex between these two purified proteins, which allows homogeneous intermolecular electron-transfer in solution. We then mimic the physiological interaction between the two partners by replacing one at a time with electrodes displaying different chemical functionalities. From the electrochemical behavior of individual proteins, we show that, while electron transfer on AcoP requires weak electrostatic interaction, electron transfer on Cyt c4 tolerates different charge and hydrophobicity conditions, suggesting a pivotal role of this protein in the metabolic chain. The electrochemical study of the proteins incubated together demonstrates an intermolecular electron transfer involving the protein complex, in which AcoP is reduced through the high potential heme of Cyt c4. Modelling of the electrochemical signals at different scan rates allows us to estimate the rate constant of this intermolecular electron transfer in the range of a few s-1. Possible routes for electron transfer in the acidophilic bacterium are deduced.

Assemblies of dendrimers and proteins on carbon and gold electrodes.

Dendritic macromolecules of two adjacent (G3.5 and G4) generations have been used to modify gold or carbon electrodes. The structure and stability of deposited films have been explored by quartz crystal microbalance (QCM), Surface Plasma Resonance (SPR) and electrochemistry. Dendrimers have been shown to adsorb spontaneously on electrode materials as compressed macromolecular films. They are able to inhibit (G3.5) or promote (G4) electroactive anionic species such as Fe(CN)(6)(3-/4-) used as a probe system. Mixed protein/dendrimer assemblies have been constructed with proteins differing in charge, nature of the prosthetic groups and sizes such as lysozyme, cytochrome c, polyhemic cytochrome c(3) or glucose oxidase. Generally, the stability of adsorbed films seems to be limited to one dendrimer/protein bilayer. Owing to the satisfactory stability of composite cytochrome c(3)/G3.5 or glucose oxidase/G4 films, biosensing applications are described for metal bioremediation and glucose detection, respectively.

Synthesis and enzymatic photo-activity of an O2 tolerant hydrogenase-CdSe@CdS quantum rod bioconjugate.

This communication reports on the preparation of stable and photo-active nano-heterostructures composed of O2 tolerant [NiFe] hydrogenase extracted from the Aquifex aeolicus bacterium grafted onto hydrophilic CdSe/CdS quantum rods in view of the development of H2/O2 biofuel cells. The resulting complex is efficient towards H2 oxidation, displays good stability and new photosensitive properties.

Electroanalysis of cationic species at membrane-carbon electrodes modified by polysaccharides. Bioaccumulation at microorganism-modified electrodes.

Membrane-carbon electrodes modified with polysaccharides suspensions entrapped between a dialysis membrane and the carbon surface were used for electroanalysis of various cationic species. Cationic complexes of ruthenium and cobalt, metallic cations (Cu(2+), Fe(3+), UO(2)(2+)) as well as methylviologen were considered. By investigating various parameters (concentration of the suspension, pH) binding of the cations by the polysaccharides was demonstrated. Comparison of cations uptake by different kinds of polysaccharides such as alginic acid, polygalacturonic acid, pectin, dextran and agar was performed. This study has been extended to natural biomaterials, alga and lichen, which are known to contain polysaccharides. The interest of the membrane-electrode strategy is described.

Biochemical and spectroscopic characterization of two new cytochromes isolated from Desulfuromonas acetoxidans.

The multimeric cytochromes described to date in sulfate- and sulfur-reducing bacteria are associated with diverse respiratory modes involving the use of elemental sulfur or oxidized sulfur compounds as terminal acceptors. They exhibit no structural similarity with the other cytochrome c classes and are characterized by a bis-histidinyl axial iron coordination and low redox potentials. We have purified two new cytochromes c with markedly different molecular masses (10 000 and 50 000) from the bacterium Desulfuromonas acetoxidans, which uses anaerobic sulfur respiration as its sole energy source. The characterization by electrochemistry and optical and EPR spectroscopies revealed the cytochrome c (Mr = 10 000) to be the first monohemic cytochrome c exhibiting a bis-histidinyl axial coordination and a low redox potential (-220 mV). The cytochrome c (Mr = 50 000) contains four hemes of low potential (-200, -210, -370, and -380 mV) with the same axial coordination. The N-terminal amino acid sequences were compared with that of the trihemic cytochrome c7, previously described in D. acetoxidans and which is related to tetrahemic cytochrome c3 from sulfate reducing bacteria. Some homology was found between cytochrome c (Mr = 10 000) and cytochrome c7. Both D. acetoxidans cytochromes c are located in the periplasmic space and their biochemical and spectroscopic properties indicate that they belong to the class III cytochromes.

The Desulfuromonas acetoxidans triheme cytochrome c7 produced in Desulfovibrio desulfuricans retains its metal reductase activity.

Multiheme cytochrome c proteins that belong to class III have been recently shown to exhibit a metal reductase activity, which could be of great environmental interest, especially in metal bioremediation. To get a better understanding of these activities, the gene encoding cytochrome c7 from the sulfur-reducing bacterium Desulfuromonas acetoxidans was cloned from genomic DNA by PCR and expressed in Desulfovibrio desulfuricans G201. The expression system was based on the cyc transcription unit from Desulfovibrio vulgaris Hildenborough and led to the synthesis of holocytochrome c7 when transferred by electrotransformation into the sulfate reducer Desulfovibrio desulfuricans G201. The produced cytochrome was indistinguishable from the protein purified from Desulfuromonas acetoxidans cells with respect to several biochemical and biophysical criteria and exhibited the same metal reductase activities as determined from electrochemical experiments. This suggests that the molecule was correctly folded in the host organism. Desulfovibrio desulfuricans produces functional multiheme c-type cytochromes from bacteria belonging to a different genus and may be considered a suitable host for the heterologous biogenesis of multiheme c-type cytochromes for either structural or engineering studies. This report, which presents the first example of the transformation of a Desulfovibrio desulfuricans strain by electrotransformation, describes work that is the first necessary step of a protein engineering program that aims to specify the structural features that are responsible for the metal reductase activities of multiheme cytochrome c7.