A new electrochemical cell with a uniformly accessible electrode to study fast catalytic reactions

International audienceThe electrochemical study of fast catalytic reactions is limited by mass transport when using the conventional electrochemical cell with a rotating disk electrode (RDE). To overcome this issue, it is important to find a new device with improved transport properties that respects electrochemical constraints. We used numerical simulations of computational fluid dynamics to design a new electrochemical cell based on the so-called “jet flow” design for the kinetic studies of catalytic chemical reactions at the surface of an electrode. The new cell is characterized by a high, reliable and uniform mass transport over the electroactive part of its surface. We investigated the effects of the nozzle and the electrode diameters, the nozzle–electrode distance and the Reynolds number on the performance of the jet-electrode in the flow system. Through the optimization of the geometry of this jet electrode cell, we achieved a factor of 3 enhancement in transport compared to the rotating disk electrode. We succeeded in constructing the designed electrode, characterized it with electrochemical techniques, and found an excellent agreement between the transport properties deduced from the numerical simulations and those from the measurements

Oxidative inactivation of NiFeSe hydrogenase

International audienceWe propose a resolution to the paradox that spectroscopic studies of NiFeSe hydrogenase have not revealed any major signal attributable to NiIII states formed upon reaction with O2, despite the fact that two inactive states are formed upon either aerobic or anaerobic oxidation

Shewanella oneidensis: a new and efficient System for Expression and Maturation of heterologous [Fe-Fe] Hydrogenase from Chlamydomonas reinhardtii

<p>Abstract</p> <p>Background</p> <p>The eukaryotic green alga, <it>Chlamydomonas reinhardtii</it>, produces H₂ under anaerobic conditions, in a reaction catalysed by a [Fe-Fe] hydrogenase HydA1. For further biochemical and biophysical studies a suitable expression system of this enzyme should be found to overcome its weak expression in the host organism. Two heterologous expression systems used up to now have several advantages. However they are not free from some drawbacks. In this work we use bacterium <it>Shewanella oneidensis</it> as a new and efficient system for expression and maturation of HydA1 from <it>Chlamydomonas reinhardtii</it>.</p> <p>Results</p> <p>Based on codon usage bias and hydrogenase maturation ability, the bacterium <it>S. oneidensis</it>, which possesses putative [Fe-Fe] and [Ni-Fe] hydrogenase operons, was selected as the best potential host for <it>C. reinhardtii </it>[Fe-Fe] hydrogenase expression. Hydrogen formation by <it>S. oneidensis </it>strain AS52 (Δ<it>hydA</it>Δ<it>hyaB</it>) transformed with a plasmid bearing <it>Cr</it>HydA1 and grown in the presence of six different substrates for anaerobic respiration was determined. A significant increase in hydrogen evolution was observed for cells grown in the presence of trimethylamine oxide, dimethylsulfoxide and disodium thiosulfate, showing that the system of <it>S. oneidensis </it>is efficient for heterologous expression of algal [Fe-Fe] hydrogenase.</p> <p>Conclusion</p> <p>In the present work a new efficient system for heterologous expression and maturation of <it>C. reinhardtii </it>hydrogenase has been developed. HydA1 of <it>C. reinhardtii </it>was purified and shown to contain 6 Fe atoms/molecule of protein, as expected. Using DMSO, TMAO or thiosulfate as substrates for anaerobic respiration during the cell growth, 0.4 – 0.5 mg l^-1(OD₆₀₀= 1) of catalytically active HydA1 was obtained with hydrogen evolution rate of ~700 μmol H₂mg^-1min^-1.</p

A Hydrophilic Channel Is Involved in Oxidative Inactivation of a [NiFeSe] Hydrogenase

International audienceHydrogenases are metalloenzymes that catalyze the redox conversion between H2 and protons. The so-called [NiFeSe] hydrogenases are highly active for both H2 production and oxidation, but like all hydrogenases, they are inhibited by O2. In the [NiFeSe] enzyme from Desulfovibrio vulgaris Hildenborough this inhibition results from the oxidation of an active site cysteine ligand. We designed mutations that constrict a hydrophilic channel which connects the protein surface to this active site cysteine. Two of the variants show markedly increased tolerance to O2 inactivation, while they retain high catalytic activities in both directions of the reaction, and structural studies confirm that these mutations prevent the oxidation of the cysteine. Our results indicate that the diffusion of O2 or ROS to the active site can occur through a hydrophilic water channel, in contrast to the widely held assumption that only hydrophobic channels are involved in active site inactivation. This provides an original strategy for optimizing the enzyme by protein engineering

Electrochemical measurements of the kinetics of inhibition of two FeFe hydrogenases by O2 demonstrate that the reaction is partly reversible

International audienceThe mechanism of reaction of FeFe hydrogenases with oxygen has been debated. It is complex, apparently very dependent on the details of the protein structure, and difficult to study using conventional kinetic techniques. Here we build on our recent work on the anaerobic inactivation of the enzyme [Fourmond et al, Nat. Chem. 4 336 (2014)] to propose and apply a new method for studying this reaction. Using electrochemical measurements of the turnover rate of hydrogenase, we could resolve the first steps of the inhibition reaction and accurately determine their rates. We show that the two most studied FeFe hydrogenases, from Chlamydomonas reinhardtii and Clostridium acetobutylicum, react with O2 according to the same mechanism, despite the fact that the former is much more O2 sensitive than the latter. Unlike often assumed, both enzymes are reversibly inhibited by a short exposure to O2. This will have to be considered to elucidate the mechanism of inhibition, before any prediction can be made regarding which mutations will improve oxygen resistance. We hope that the approach described herein will prove useful in this respect

Combining experimental and theoretical methods to learn about the reactivity of gas-processing metalloenzymes

International audienceAfter enzymes were first discovered in the late XIX century, and for the first seventy years of enzymology, kinetic experiments were the only source of information about enzyme mechanisms. Over the following fifty years, these studies were taken over by approaches that give information at the molecular level, such as crystallography, spectroscopy and theoretical chemistry (as emphasized by the Nobel Prize in Chemistry awarded last year to M. Karplus, M. Levitt and A. Warshel). In this review, we thoroughly discuss the interplay between the information obtained from theoretical and experimental methods, by focussing on enzymes that process small molecules such as H 2 or CO 2 (hydrogenases, CO-dehydrogenase and carbonic anhydrase), and that are therefore relevant in the context of energy and environment. We argue that combining theoretical chemistry (DFT, MD, QM/MM) and detailed investigations that make use of modern kinetic methods, such as protein film voltammetry, is an innovative way of learning about individual steps and/or complex reactions that are part of the catalytic cycles. We illustrate this with recent results from our labs and others, including studies of gas transport along substrate channels, long range proton transfer, and mechanisms of catalysis, inhibition or inactivation. Broader context Some reactions which are very important in the context of energy and environment, such as the conversion between CO and CO2 , or H+ and H2 , are catalyzed in living organisms by large and complex enzymes that use inorganic active sites to transform substrates, chains of redox centers to transfer electrons, ionizable amino acids to transfer protons, and networks of hydrophobic cavities to guide the diffusion of substrates and products within the protein. This highly sophisticated biological plumbing and wiring makes turnover frequencies of thousands of substrate molecules per second possible. Understanding the molecular details of catalysis is still a challenge. We explain in this review how a great deal of information can be obtained using an interdisciplinary approach that combines state-of-the art kinetics and computational chemistry. This differs from—and complements—the more traditional strategies that consist in trying to see the catalytic intermediates using methods that rely on the interaction between light and matter, such as X-ray diffraction and spectroscopic techniques

Reversible H2 oxidation and evolution by hydrogenase embedded in a redox polymer film

Efficient electrocatalytic energy conversion requires devices to function reversibly, that is, to deliver a substantial current at a minimal overpotential. Redox-active films can effectively embed and stabilize molecular electrocatalysts, but mediated electron transfer through the film typically makes the catalytic response irreversible. Here we describe a redox-active film for bidirectional (oxidation or reduction) and reversible hydrogen conversion, which consists of [FeFe] hydrogenase embedded in a low-potential, 2,2′-viologen-modified hydrogel. When this catalytic film served as the anode material in a H2/O2 biofuel cell, an open circuit voltage of 1.16 V was obtained—a benchmark value near the thermodynamic limit. The same film also acted as a highly energy efficient cathode material for H2 evolution. We explained the catalytic properties using a kinetic model, which shows that reversibility can be achieved even though intermolecular electron transfer is slower than catalysis. This understanding of reversibility simplifies the design principles of highly efficient and stable bioelectrocatalytic films, advancing their implementation in energy conversion. [Figure not available: see fulltext.]

Reactivation from the Ni-B state in [NiFe] hydrogenase of Ralstonia eutropha is controlled by reduction of the superoxidised proximal cluster

The tolerance towards oxic conditions of O2-tolerant [NiFe] hydrogenases has been attributed to an unusual [4Fe–3S] cluster that lies proximal to the [NiFe] active site. Upon exposure to oxygen, this cluster converts to a superoxidised (5+) state, which is believed to secure the formation of the so-called Ni–B state that is rapidly reactivated under reducing conditions. Here, the reductive reactivation of the membrane-bound [NiFe]-hydrogenase (MBH) from Ralstonia eutropha in a native-like lipid membrane was characterised and compared to a variant that instead carries a typical [4Fe–4S] proximal cluster. Reactivation from the Ni–B state was faster in the [4Fe–4S] variant, suggesting that the reactivation rate in MBH is limited by the reduction of the superoxidised [4Fe–3S] cluster. We propose that the [4Fe–3S] cluster plays a major role in protecting MBH by blocking the reversal of electron transfer to the [NiFe] active site, which would produce damaging radical oxygen species

Electrochemical insights into the mechanism of NiFe membrane-bound hydrogenases

Hydrogenases are enzymes of great biotechnological relevance because they catalyse the interconversion of H2, water (protons) and electricity using non-precious metal catalytic active sites. Electrochemical studies into the reactivity of NiFe membrane-bound hydrogenases (MBH) have provided a particularly detailed insight into the reactivity and mechanism of this group of enzymes. Significantly, the control centre for enabling O2 tolerance has been revealed as the electron-transfer relay of FeS clusters, rather than the NiFe bimetallic active site. The present review paper will discuss how electrochemistry results have complemented those obtained from structural and spectroscopic studies, to present a complete picture of our current understanding of NiFe MBH