Nickel-Substituted Rubredoxin as a Minimal Enzyme Model for Hydrogenase

Abstract

A simple, functional mimic of [NiFe] hydrogenases based on a nickel-substituted rubredoxin (NiRd) protein is reported. NiRd is capable of light-initiated and solution-phase hydrogen production and demonstrates high electrocatalytic activity using protein film voltammetry. The catalytic voltammograms are modeled using analytical expressions developed for hydrogenase enzymes, revealing maximum turnover frequencies of approximately 20-100 s(-1) at 4 °C with an overpotential of 540 mV. These rates are directly comparable to those observed for [NiFe] hydrogenases under similar conditions. Like the native enzymes, the proton reduction activity of NiRd is strongly inhibited by carbon monoxide. This engineered rubredoxin-based enzyme is chemically and thermally robust, easily accessible, and highly tunable. These results have implications for understanding the enzymatic mechanisms of native hydrogenases, and, using NiRd as a scaffold, it will be possible to optimize this catalyst for application in sustainable fuel generation.

Experimental and DFT Investigations Reveal the Influence of the Outer Coordination Sphere on the Vibrational Spectra of Nickel-Substituted Rubredoxin, a Model Hydrogenase Enzyme

“…27 Briefly, an additional purification step was included. The protein was run on a size-exclusion column (5 mL Superdex-75, prep-grade, Sigma-Aldrich) in 50 mM Tris buffer, pH 8.0, following the trichloroacetic acid precipitation and metal exchange to ensure complete purity of all protein samples.…”

Photo‐Driven Hydrogen Evolution by an Artificial Hydrogenase Utilizing the Biotin‐Streptavidin Technology

“…Their oxygen sensitivity and production costs call for the development of artificial hydrogenases. Several artificial hydrogenases rely on the incorporation of artificial metal cofactors in host proteins (cytochrome c , rubredoxin, ferredoxin) or linking to a polypeptide . In the past decade, the biotin‐streptavidin technology has found widespread use for the assembly of artificial metalloenzymes (ArM) .…”

Ru–protein–Co biohybrids designed for solar hydrogen production: understanding electron transfer pathways related to photocatalytic function

“…12 More recent efforts to integrate catalysts into full protein architectures include incorporation of a synthetic diiron catalyst into apo-cyt c 13 and nitrobindin; 14 insertion of cobalt porphyrins 15,16 and cobaloximes 17 into apo-myoglobin; and a minimal hydrogenase model using a nickel substituted rubredoxin. 18 These protein-based systems operate similarly to multi-molecular synthetic systems requiring diffusional interaction of catalyst and PSs (typically [Ru(bpy) 3 ] 2+ (bpy = 2,2′-bipyridine) and related derivatives) and achieve up to 520 turnovers (TON) of H 2 with sodium ascorbate as a sacrificial electron donor. 19 To enable direct supramolecular-like charge separation, an 18-amino acid peptide of cyt c 556 has been modified by covalent binding of both a diiron catalyst and a [Ru(bpy)(tpy)(H 2 O)] 2+ (tpy = 2,2′:6′2′′-terpyridine) PS.…”