Electrostatic-Driven Activity, Loading, Dynamics, and Stability of a Redox Enzyme on Functionalized-Gold Electrodes for Bioelectrocatalysis

The oxygen reduction reaction is the limiting step in fuel cells, and many works are in progress to find efficient cathode catalysts. Among them, bilirubin oxidases are copper-based enzymes that reduce oxygen into water with low overpotentials. The factors that ensure electrocatalytic efficiency of the enzyme in the immobilized state are not well understood, however. In this work, we use a multiple methodological approach on a wide range of pH values for protein adsorption and electrocatalysis to demonstrate the effect of electrostatic interactions on the electrical wiring, dynamics, and stability of a bilirubin oxidase adsorbed on self-assembled-monolayers on gold. We show on one hand that the global charge of the enzyme controls the loading on the interface and that the specific activity of the immobilized enzyme decreases with the enzyme coverage. On the other hand, we show that the dipole moment of the protein and the charge in the vicinity of the Cu site acting as the entry point of electrons drive the enzyme orientation. In case of weak electrostatic interactions, we demonstrate that local pH variation affects the electron transfer rate as a result of protein mobility on the surface. On the contrary, stronger electrostatic interactions destabilize the protein structure and affect the stability of the catalytic signal. These data illustrate the interplay between immobilized protein dynamics and local environment that control the efficiency of bioelectrocatalysis

Wiring of Photosystem II to Hydrogenase for Photoelectrochemical Water Splitting.

In natural photosynthesis, light is used for the production of chemical energy carriers to fuel biological activity. The re-engineering of natural photosynthetic pathways can provide inspiration for sustainable fuel production and insights for understanding the process itself. Here, we employ a semiartificial approach to study photobiological water splitting via a pathway unavailable to nature: the direct coupling of the water oxidation enzyme, photosystem II, to the H2 evolving enzyme, hydrogenase. Essential to this approach is the integration of the isolated enzymes into the artificial circuit of a photoelectrochemical cell. We therefore developed a tailor-made hierarchically structured indium-tin oxide electrode that gives rise to the excellent integration of both photosystem II and hydrogenase for performing the anodic and cathodic half-reactions, respectively. When connected together with the aid of an applied bias, the semiartificial cell demonstrated quantitative electron flow from photosystem II to the hydrogenase with the production of H2 and O2 being in the expected two-to-one ratio and a light-to-hydrogen conversion efficiency of 5.4% under low-intensity red-light irradiation. We thereby demonstrate efficient light-driven water splitting using a pathway inaccessible to biology and report on a widely applicable in vitro platform for the controlled coupling of enzymatic redox processes to meaningfully study photocatalytic reactions.This work was supported by the U.K. Engineering and Physical Sciences Research Council (EP/H00338X/2 to E.R. and EP/G037221/1, nanoDTC, to D.M.), the UK Biology and Biotechnological Sciences Research Council (BB/K002627/1 to A.W.R. and BB/K010220/1 to E.R.), a Marie Curie Intra-European Fellowship (PIEF-GA-2013-625034 to C.Y.L), a Marie Curie International Incoming Fellowship (PIIF-GA-2012-328085 RPSII to J.J.Z) and the CEA and the CNRS (to J.C.F.C.). A.W.R. holds a Wolfson Merit Award from the Royal Society.This is the final version of the article. It first appeared from ACS Publications via http://dx.doi.org/10.1021/jacs.5b0373

Enhanced oxygen-tolerance of the full heterotrimeric membrane-bound [NiFe]-hydrogenase of ralstonia eutropha.

Hydrogenases are oxygen-sensitive enzymes that catalyze the conversion between protons and hydrogen. Water-soluble subcomplexes of membrane-bound [NiFe]-hydrogenases (MBH) have been extensively studied for applications in hydrogen-oxygen fuel cells as they are relatively tolerant to oxygen, although even these catalysts are still inactivated in oxidative conditions. Here, the full heterotrimeric MBH of Ralstonia eutropha, including the membrane-integral cytochrome b subunit, was investigated electrochemically using electrodes modified with planar tethered bilayer lipid membranes (tBLM). Cyclic voltammetry and chronoamperometry experiments show that MBH, in equilibrium with the quinone pool in the tBLM, does not anaerobically inactivate under oxidative redox conditions. In aerobic environments, the MBH is reversibly inactivated by O2, but reactivation was found to be fast even under oxidative redox conditions. This enhanced resistance to inactivation is ascribed to the oligomeric state of MBH in the lipid membrane

Le point central de la controverse sur la distinction de l'essence et de l'existence

de Poulpiquet Fr. A. Le point central de la controverse sur la distinction de l'essence et de l'existence. In: Revue néo-scolastique. 13ᵉ année, n°49, 1906. pp. 32-48

New trends in enzyme immobilization at nanostructured interfaces for efficient electrocatalysis in biofuel cells

International audienceBiofuel cells, and among them enzymatic biofuel cells, are expected to take part in a sustainable economy in a next future. The development of such biodevices requires significant improvements in terms of efficiency of enzyme immobilization at the electrodes, so as to enable direct electron transfer, and to increase and stabilize the current densities. Many works during the last years aimed at reaching higher current densities, thus power densities, while increasing the long term stability of the enzymatic bioelectrodes. Search for new enzymes, wild type or mutants, new entrapment procedures, but also new electrode architectures, have been targeted. This review focuses on the materials developed and involved during the last few years to meet these demands via nanostructuration of electrode interfaces. Discussion is essentially focused on cases where direct electron transfer between enzymes and electrochemical interfaces are involved. After having introduced the main reasons for the need of nanostructuration, the materials and methods that are newly developed are described. The consequences on improved performances for enzymatic bioelectrodes are discussed, and finally major challenges for future research are addressed

Recent developments in high surface area bioelectrodes for enzymatic fuel cells

International audienceEnzymatic fuel cells (EFC) function in a similar way to low temperature proton exchange membrane fuel cells (PEMFC) but use enzymes instead of noble metals as catalysts for fuel and oxidant transformation. The need for EFCs that deliver enhanced power has resulted in high surface/volume ratio conductive materials being considered as enzyme host matrices. While the enhanced surface has effectively led to higher catalytic currents, the use of high surface area nanomaterials (HSM) has also induced new issues related to the wiring of enzymes, the role of porosity, and the effect on stability. This review discusses the most important features reported in this area over the past three years, and proposes future directions concerning EFC applications. Recent developments in high surface area bioelectrodes for enzymatic fuel cells. Available from: https://www.researchgate.net/publication/318305120_Recent_developments_in_high_surface_area_bioelectrodes_for_enzymatic_fuel_cells [accessed Jul 19, 2017]

H 2 /O 2 enzymatic fuel cells: from proof-of-concept to powerful devices

International audienceIntensive research during the last 15 years on mechanistic understanding of hydrogenases, the key enzyme for H2 transformation in many microorganisms, has authorized the concept of green energy production through H2/O2 enzymatic fuel cells (EFCs), in which enzymes are used as biodegradable and bioavailable biocatalysts. More recently, great effort has been put in the improvement of the interfacial electron transfer process between the enzymes and high surface area conductive materials in order to shift from a proof-of-concept to a usable power device. Herein, we analyze the main issues that have been addressed during the last 5 years to make this breakthrough. After a brief introduction on the structure of hydrogenases and bilirubin oxidases, a widely used enzyme for O2 reduction, we compare their activity with that of platinum. We introduce the first H2/O2 EFCs and discuss their main limitations mainly related to the sensitivity of hydrogenases to O2 and oxidative potentials. We then review the discovery of new enzymes in the biodiversity and the advances in the control of the functional immobilization of these enzymes on electrodes that have permitted to overcome these limitations. We finally present all the reported H2/O2 EFCs, with a critical discussion on the perspectives of such devices

How to advance the frontiers of current biofuel cells: design of a H2/O2 biofuel cell based on thermostable enzymes

International audienceA new generation of mediatorless H2/O2 biofuel cells was designed based on a hyperthermophilic O2-tolerant hydrogenase and a thermostable bilirubin oxidase both immobilized on carbon nanofibers. A power density up to 1.5 ± 0.2 mW.cm-2 at 60°C was reached. This first demonstration of a H2/O2 biofuel cell able to deliver electricity over a wide range of temperatures, from 30°C up to 80°C, and over a large pH window, allows considering this device as an alternative power supply for small portable applications in various environments, including extreme ones

Pore size effect of MgO-templated carbon on enzymatic H 2 oxidation by the hyperthermophilic hydrogenase from Aquifex aeolicus

International audienceHydrogenase from the hyperthermophilic bacterium Aquifex aeolicus was immobilized in MgO-templated carbon (MgOC). Two different pore sizes were investigated, large pore size of 150 nm (MgOC150) and smaller pore size of 35 nm (MgOC35). Direct H2 oxidation proceeded in both MgOC150 and MgOC35. Hydrogenase embedded in the carbon material exhibited the expected properties in terms of onset for H2 oxidation and kinetics of formation of the inactive state at high potentials, whatever the size of the pores. Pore size much larger than the size of the enzyme favored the loading of the enzyme, yielding to high catalytic current reported to the capacitance. Pore size closer to the enzyme diameter, as determined by DLS, enhanced the stability of the enzyme at high temperature

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

International audienceHow 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