Enzymatic X-ray absorption spectroelectrochemistry.

The ability to observe the changes that occur at an enzyme active site during electrocatalysis can provide very valuable information for understanding the mechanism and ultimately aid in catalyst design. Herein, we discuss the development of X-ray absorption spectroscopy (XAS) in combination with electrochemistry for operando studies of enzymatic systems. XAS has had a long history of enabling geometric and electronic structural insights into the catalytic active sites of enzymes, however, XAS combined with electrochemistry (XA-SEC) has been exceedingly rare in bioinorganic applications. Herein, we discuss the challenges and opportunities of applying operando XAS to enzymatic electrocatalysts. The challenges due to the low concentration of the photoabsorber and the instability of the protein in the X-ray beam are discussed. Methods for immobilizing enzymes on the electrodes, while maintaining full redox control are highlighted. A case study of combined XAS and electrochemistry applied to a [NiFe] hydrogenase is presented. By entrapping the [NiFe] hydrogenase in a redox polymer, relatively high protein concentrations can be achieved on the electrode surface, while maintaining redox control. Overall, it is demonstrated that the experiments are feasible, but require precise redox control over the majority of the absorber atoms and careful controls to discriminate between electrochemically-driven changes and beam damage. Opportunities for future applications are discussed.

Reactivation of sulfide-protected [FeFe] hydrogenase in a redox-active hydrogel.

[FeFe] hydrogenases are highly active hydrogen conversion catalysts but are notoriously sensitive to oxidative damage. Redox hydrogels have been used for protecting hydrogenases from both high potential inactivation and oxygen inactivation under turnover conditions. However, [FeFe] hydrogenase containing redox hydrogels must be fabricated under strict anoxic conditions. Sulfide coordination at the active center of the [FeFe] hydrogenase from Desulfovibrio desulfuricans protects this enzyme from oxygen in an inactive state, which can be reactivated upon reduction. Here, we show that this oxygen-stable inactive form of the hydrogenase can be reactivated in a redox hydrogel enabling practical use of this highly O2 sensitive enzyme without the need for anoxic conditions.

Suppressing hydrogen peroxide generation to achieve oxygen-insensitivity of a [NiFe] hydrogenase in redox active films.

Redox-active films were proposed as protective matrices for preventing oxidative deactivation of oxygen-sensitive catalysts such as hydrogenases for their use in fuel cells. However, the theoretical models predict quasi-infinite protection from oxygen and the aerobic half-life for hydrogenase-catalyzed hydrogen oxidation within redox films lasts only about a day. Here, we employ operando confocal microscopy to elucidate the deactivation processes. The hydrogen peroxide generated from incomplete reduction of oxygen induces the decomposition of the redox matrix rather than deactivation of the biocatalyst. We show that efficient dismutation of hydrogen peroxide by iodide extends the aerobic half-life of the catalytic film containing an oxygen-sensitive [NiFe] hydrogenase to over one week, approaching the experimental anaerobic half-life. Altogether, our data support the theory that redox films make the hydrogenases immune against the direct deactivation by oxygen and highlight the importance of suppressing hydrogen peroxide production in order to reach complete protection from oxidative stress.

Viologen-modified electrodes for protection of hydrogenases from high potential inactivation while performing H2 oxidation at low overpotential.

In this work we present a viologen-modified electrode providing protection for hydrogenases against high potential inactivation. Hydrogenases, including O2-tolerant classes, suffer from reversible inactivation upon applying high potentials, which limits their use in biofuel cells to certain conditions. Our previously reported protection strategy based on the integration of hydrogenase into redox matrices enabled the use of these biocatalysts in biofuel cells even under anode limiting conditions. However, mediated catalysis required application of an overpotential to drive the reaction, and this translates into a power loss in a biofuel cell. In the present work, the enzyme is adsorbed on top of a covalently-attached viologen layer which leads to mixed, direct and mediated, electron transfer processes; at low overpotentials, the direct electron transfer process generates a catalytic current, while the mediated electron transfer through the viologens at higher potentials generates a redox buffer that prevents oxidative inactivation of the enzyme. Consequently, the enzyme starts the catalysis at no overpotential with viologen self-activated protection at high potentials.

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.