Olaf Rüdiger

Laccase electrode for direct electrocatalytic reduction of O2 to H2O with high-operational stability and resistance to chloride inhibition

Laccase from Trametes hirsuta basidiomycete has been covalently bound to graphite electrodes electrochemically modified with phenyl derivatives as a way to attach the enzyme molecules with an adequate orientation for direct electron transfer (DET). Current densities up to 0.5mA/cm(2) of electrocatalytic reduction of O(2) to H(2)O were obtained in absence of redox mediators, suggesting preferential orientation of the T1 Cu centre of the laccase towards the electrode. The covalent attachment of the laccase molecules to the functionalized electrodes permitted remarkable operational stability. Moreover, O(2) bioelectroreduction based on DET between the laccase and the electrode was not inhibited by chloride ions, whereas mediated bioelectrocatalysis was. In contrast, fluoride ions inhibited both direct and mediated electron transfers-based bioelectrocatalytic reduction of O(2). Thus, two different modes of laccase inhibition by halides are discussed.

A redox hydrogel protects hydrogenase from high-potential deactivation and oxygen damage

Hydrogenases are natures efficient catalysts for both the generation of energy via oxidation of molecular hydrogen and the production of hydrogen via the reduction of protons. However, their O2 sensitivity and deactivation at high potential limit their applications in practical devices, such as fuel cells. Here, we show that the integration of an O2-sensitive hydrogenase into a specifically designed viologen-based redox polymer protects the enzyme from O2 damage and high-potential deactivation. Electron transfer between the polymer-bound viologen moieties controls the potential applied to the active site of the hydrogenase and thus insulates the enzyme from excessive oxidative stress. Under catalytic turnover, electrons provided from the hydrogen oxidation reaction induce viologen-catalysed O2 reduction at the polymer surface, thus providing self-activated protection from O2. The advantages of this tandem protection are demonstrated using a single-compartment biofuel cell based on an O2-sensitive hydrogenase and H2/O2 mixed feed under anode-limiting conditions.

Mechanism of Protection of Catalysts Supported in Redox Hydrogel Films

The use of synthetic inorganic complexes as supported catalysts is a key route in energy production and in industrial synthesis. However, their intrinsic oxygen sensitivity is sometimes an issue. Some of us have recently demonstrated that hydrogenases, the fragile but very efficient biological catalysts of H2 oxidation, can be protected from O2 damage upon integration into a film of a specifically designed redox polymer. Catalytic oxidation of H2 produces electrons which reduce oxygen near the film/solution interface, thus providing a self-activated protection from oxygen [Plumeré et al., Nat Chem. 2014, 6, 822-827]. Here, we rationalize this protection mechanism by examining the time-dependent distribution of species in the hydrogenase/polymer film, using measured or estimated values of all relevant parameters and the numerical and analytical solutions of a realistic reaction-diffusion scheme. Our investigation sets the stage for optimizing the design of hydrogenase-polymer films, and for expanding this strategy to other fragile catalysts.

A redox hydrogel protects the O2 -sensitive [FeFe]-hydrogenase from Chlamydomonas reinhardtii from oxidative damage.

The integration of sensitive catalysts in redox matrices opens up the possibility for their protection from deactivating molecules such as O2 . [FeFe]-hydrogenases are enzymes catalyzing H2 oxidation/production which are irreversibly deactivated by O2 . Therefore, their use under aerobic conditions has never been achieved. Integration of such hydrogenases in viologen-modified hydrogel films allows the enzyme to maintain catalytic current for H2 oxidation in the presence of O2 , demonstrating a protection mechanism independent of reactivation processes. Within the hydrogel, electrons from the hydrogenase-catalyzed H2 oxidation are shuttled to the hydrogel-solution interface for O2 reduction. Hence, the harmful O2 molecules do not reach the hydrogenase. We illustrate the potential applications of this protection concept with a biofuel cell under H2 /O2 mixed feed.

Direct Comparison of the Performance of a Bio‐inspired Synthetic Nickel Catalyst and a [NiFe]‐Hydrogenase, Both Covalently Attached to Electrodes

The active site of hydrogenases has been a source of inspiration for the development of molecular catalysts. However, direct comparisons between molecular catalysts and enzymes have not been possible because different techniques are used to evaluate both types of catalysts, minimizing our ability to determine how far we have come in mimicking the enzymatic performance. The catalytic properties of the [Ni(P(Cy) 2 N(Gly) 2 )2 ](2+) complex with the [NiFe]-hydrogenase from Desulfovibrio vulgaris immobilized on a functionalized electrode were compared under identical conditions. At pH 7, the enzyme shows higher activity and lower overpotential with better stability, while at low pH, the molecular catalyst outperforms the enzyme in all respects. This is the first direct comparison of enzymes and molecular complexes, enabling a unique understanding of the benefits and detriments of both systems, and advancing our understanding of the utilization of these bio-inspired complexes in fuel cells.

Proton Coupled Electronic Rearrangement within the H-Cluster as an Essential Step in the Catalytic Cycle of [FeFe] Hydrogenases

The active site of [FeFe] hydrogenases, the H-cluster, consists of a [4Fe-4S] cluster connected via a bridging cysteine to a [2Fe] complex carrying CO and CN- ligands as well as a bridging aza-dithiolate ligand (ADT) of which the amine moiety serves as a proton shuttle between the protein and the H-cluster. During the catalytic cycle, the two subclusters change oxidation states: [4Fe-4S]H2+ ⇔ [4Fe-4S]H+ and [Fe(I)Fe(II)]H ⇔ [Fe(I)Fe(I)]H thereby enabling the storage of the two electrons needed for the catalyzed reaction 2H+ + 2e- ⇄ H2. Using FTIR spectro-electrochemistry on the [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1) at different pH values, we resolve the redox and protonation events in the catalytic cycle and determine their intrinsic thermodynamic parameters. We show that the singly reduced state Hred of the H-cluster actually consists of two species: Hred = [4Fe-4S]H+ - [Fe(I)Fe(II)]H and HredH+ = [4Fe-4S]H2+ - [Fe(I)Fe(I)]H (H+) related by proton coupled electronic rearrangement. The two redox events in the catalytic cycle occur on the [4Fe-4S]H subcluster at similar midpoint-potentials (-375 vs -418 mV); the protonation event (Hred/HredH+) has a pKa ≈ 7.2.

Interaction of the active site of the Ni–Fe–Se hydrogenase from Desulfovibrio vulgaris Hildenborough with carbon monoxide and oxygen inhibitors

The study of Ni–Fe–Se hydrogenases is interesting from the basic research point of view because their active site is a clear example of how nature regulates the catalytic function of an enzyme by the change of a single residue, in this case a cysteine, which is replaced by a selenocysteine. Most hydrogenases are inhibited by CO and O2. In this work we studied these inhibition processes for the Ni–Fe–Se hydrogenase from Desulfovibrio vulgaris Hildenborough by combining catalytic activity measurements, followed by mass spectrometry or chronoamperometry, with Fourier transform IR spectroscopy experiments. The results show that the CO inhibitor binds to Ni in both conformations of the active site of this hydrogenase in a way similar to that in standard Ni–Fe hydrogenases, although in one of the CO-inhibited conformations the active site of the Ni–Fe–Se hydrogenase is more protected against the attack by O2. The inhibition of the Ni–Fe–Se hydrogenase activity by O2 could be explained by oxidation of the terminal cysteine ligand of the active-site Ni, instead of the direct attack of O2 on the bridging site between Ni and Fe.

Impact of alterations near the [NiFe] active site on the function of the H2 sensor from Ralstonia eutropha

In proteobacteria capable of H2 oxidation under (micro)aerobic conditions, hydrogenase gene expression is often controlled in response to the availability of H2. The H2‐sensing signal transduction pathway consists of a heterodimeric regulatory [NiFe]‐hydrogenase (RH), a histidine protein kinase and a response regulator. To gain insights into the signal transmission from the Ni–Fe active site in the RH to the histidine protein kinase, conserved amino acid residues in the L0 motif near the active site of the RH large subunit of Ralstonia eutropha H16 were exchanged. Replacement of the strictly conserved Glu13 (E13N, E13L) resulted in loss of the regulatory, H2‐oxidizing and D2/H+ exchange activities of the RH. According to EPR and FTIR analysis, these RH derivatives contained fully assembled [NiFe] active sites, and para‐/ortho‐H2 conversion activity showed that these centres were still able to bind H2. This indicates that H2 binding at the active site is not sufficient for the regulatory function of H2 sensors. Replacement of His15, a residue unique in RHs, by Asp restored the consensus of energy‐linked [NiFe]‐hydrogenases. The respective RH mutant protein showed only traces of H2‐oxidizing activity, whereas its D2/H+‐exchange activity and H2‐sensing function were almost unaffected. H2‐dependent signal transduction in this mutant was less sensitive to oxygen than in the wild‐type strain. These results suggest that H2 turnover is not crucial for H2 sensing. It may even be detrimental for the function of the H2 sensor under high O2 concentrations.

Spectroscopic and Electrochemical Characterization of the [NiFeSe] Hydrogenase from Desulfovibrio vulgaris Miyazaki F: Reversible Redox Behavior and Interactions between Electron Transfer Centers

Characterizing a new hydrogenase: The newly isolated [NiFeSe] hydrogenase from Desulfovibrio vulgaris Miyazaki F displays catalytic properties distinct from other hydrogenase proteins. Here we apply site-specific spectroscopic and electrochemical techniques to characterize these unique features at the molecular level.

Dual properties of a hydrogen oxidation Ni-catalyst entrapped within a polymer promote self-defense against oxygen

The Ni(P2N2)2 catalysts are among the most efficient non-noble-metal based molecular catalysts for H2 cycling. However, these catalysts are O2 sensitive and lack long term stability under operating conditions. Here, we show that in a redox silent polymer matrix the catalyst is dispersed into two functionally different reaction layers. Close to the electrode surface is the “active” layer where the catalyst oxidizes H2 and exchanges electrons with the electrode generating a current. At the outer film boundary, insulation of the catalyst from the electrode forms a “protection” layer in which H2 is used by the catalyst to convert O2 to H2O, thereby providing the “active” layer with a barrier against O2. This simple but efficient polymer-based electrode design solves one of the biggest limitations of these otherwise very efficient catalysts enhancing its stability for catalytic H2 oxidation as well as O2 tolerance.Bio-inspired Ni-based molecular catalysts are efficient for H2 oxidation, but are suffering from the poor stability in the presence of O2. Here, the authors develop a strategy to boost greatly their stability by dispersing them in a hydrophobic and redox-silent polymer matrix.