Robust Carbon-Based Electrodes for Hydrogen Evolution through Site-Selective Covalent Attachment of an Artificial Metalloenzyme

Abstract

The use of biological systems for electrochemical energy conversion applications is often limited by instability of the protein or protein–electrode system. Here, we present a simple but efficient method for covalent attachment of nickel-substituted rubredoxin (NiRd), a model hydrogenase, to an unmodified graphite electrode based on amide bond formation. The resultant electrodes are shown to be highly active for H2 evolution over a period of several weeks. The effects of different attachment methods on interfacial electron transfer (ET) rates and catalysis are investigated, with decreased ET rates and increased background reactivity observed for surface-modified electrodes. Electrochemical simulations reveal that reduced protein dynamics of the attached NiRd enzyme are likely responsible for decreased catalytic rates by modulating the intramolecular proton transfer step. Ultimately, this straightforward approach can be broadly applied to diverse redox-active proteins and enzymes and will expand the utility of such systems by conferring increased stability over extended periods of time.

Reversible Electron Transfer and Substrate Binding Support [NiFe3S4] Ferredoxin as a Protein-Based Model for [NiFe] Carbon Monoxide Dehydrogenase

“…[NiFe 3 S 4 ] Fd was adsorbed to the surface of a pyrolytic graphite electrode (PGE). To mitigate the electrostatic repulsion expected between the negatively charged graphite surface and Fd (p I ∼ 3.6) and stabilize films, the coadsorbant neomycin was used. , Cyclic voltammetry revealed a reversible electrochemical transition with E 1/2 = +6 ± 4 mV [vs normal hydrogen electrode (NHE)] at pH 7 and a small peak separation of 31 mV between the anodic and cathodic waves (Figure A, inset). Analogous to [Fe 4 S 4 ] Fd, this midpoint potential is independent of the pH (Figure S2).…”

In Vivo Assembly of a Genetically Encoded Artificial Metalloenzyme for Hydrogen Production

“…Expression and purification of chemically reconstituted rubredoxin as well as construction of the N07P mutant were performed as described previously. ,,, All expression was carried out under aerobic growth conditions.…”

“…The NiRd enzyme generated by in vivo metalation is indistinguishable from NiRd prepared through the standard metal substitution procedure, as evidenced by optical spectroscopy and protein electrochemistry (Figure G). Electrocatalysis by NiRd shows a distinct voltammetric signature, with currents in the cathodic region of the voltammogram exhibiting a linear response with increasing applied potential, characteristic of slow electron transfer relative to chemical processes, and parallel waves in the cathodic and anodic directions, indicating an absence of low-potential inactivation processes. , The currents directly correspond to the catalytic H 2 evolution activity, while the position of the onset of the catalytic current indicates the overpotential for the reaction. In the purified wild-type (WT) NiRd, the onset potential for catalysis occurs at −800 mV vs NHE at pH 4.0 (Figure G).…”

Synthesis of [FeFe] Hydrogenase Mimics with Lipoic acid and its Selenium Analogue as Anchor Groups

“…[1,2] One significant issue in electrolysis and photocatalysis is the diffusion-limited transport of the catalytic centre to the electrode, that is necessary for an effective electron transfer. [3,4] Immobilizing [FeFe] H 2 ase enzymes or model complexes on the electrode surface to attain chemically modified electrodes were realized by electrostatic adsorption, [5] embedding in redoxactive hydrogels or polymers, [6] absorption into mesoporous electrodes, [7] reduction of diazonium salt spacers on carbon electrodes, [8,9] by carboxylic acid group on fluorine-doped tin oxide (FTO) or nickel oxide electrodes, [10,11] and thiol spacer groups on gold electrodes, with a functional moieties for further modification with the [FeFe] H 2 ase mimic or enzyme, amongst others. [4,12] However, [FeFe] H 2 ase mimics attached to electrodes lack consistency and high activity during the catalytic process, due to their sensitivity regarding high pH values or oxygen and irreversible electrocatalytic behaviour.…”