Development of Solid-Binding Peptide (SBP)-Fused Formate Dehydrogenase (FDH) for Direct Electrical Contact to NADH Regeneration-Enabled Electroenzymatic CO2 Conversion System

Abstract

With the launch of a new international climate regime, efforts to reduce greenhouse gases (GHGs) causing global warming and climate change have been bolstered. Especially since carbon dioxide (CO2) accounts for about 80 percent of total GHG emission, carbon capture and utilization (CCU) technology that mitigates greenhouse effect and converts CO2 into useful compounds, has been attracting attention as one of the major climate change solutions. CO2 utilization aims to sequestrate CO2 in the atmosphere and convert CO2 into useful chemicals to finally close the carbon cycle. CO2 is an attractive feedstock in terms that it is abundant, inexpensive, and renewable, but this remains challenging because it is a kinetically and thermodynamically stable molecule. Therefore, an efficient conversion catalyst is required to chemically facilitate CO2 conversion. Recently, electroenzymatic CO2 reduction has received much interest as a promising approach. This study focused on CO2 reducing enzyme, formate dehydrogenase (EC 1.17.1.9) which is capable of reversibly catalyzing CO2 reduction and formate oxidation. FDHs existed in a diverse array of organisms can be divided into NAD+/NADH-dependent FDHs and metal-independent FDHs. One of them, the NAD+/NADH-dependent FDH, which does not include a metal cofactor at the active site and has high oxygen tolerance, was selected as an enzyme catalyst. However, there are challenges to achieve the high reaction rate and continuous NADH regeneration because CO2 reduction activity is much slower than formate oxidation and the inherent complexity due to their reliance on the diffusional nature of these soluble cofactors limiting their application. Herein, gold-binding peptide (GBP) is genetically introduced at the NAD+/NADH-dependent formate dehydrogenase (FDH) to add a non-native gold-binding activity for directly wiring the enzyme to the electrode surface. Our gold-binding kinetics studies on the native and synthetic FDHs reveal that the gold-binding properties of the fused enzymes are highly dependent on the fusion site as well as the tertiary structure of the fusion enzyme that controls the efficiency of gold-binding domain display fusion. As such, the fusion of GBP at C-terminus (FDHgbpC) shows the highest gold-binding activity, followed by GBP at both termini FDHgbpNC and FDHgbpN. More importantly, the presence of GBP yields an integrated stable enzyme-electrode for the bioelectrocatalyzed reaction at the enzyme-electrode interface. Direct electrochemical NAD+ reduction and NADH oxidation produced by the enzymatic reaction in the presence of CO2 and formate, respectively, was observed at low overpotential for all types of enzyme-electrodes indicates that direct-electrical contact of the cofactor binding site to the electrode surface was achieved. The enhanced electron transfer kinetics can be explained by the short distance (proximity) between the cofactor binding site and the electrode surface. In terms of the CO2 reduction reaction, the reductive catalytic current was not observed immediately upon the addition of both NADH and CO2, suggesting a slow CO2 reduction activity to enzymatically generate NAD+ and the subsequent sluggish electron transfer from the electrode to NAD+. Nevertheless, direct electrical contacting of NAD+/NADH-dependent FDH was demonstrated in this study which could be useful for electroenzymatic system for electrocatalysis system and NAD+/NADH-dependent redox enzyme system.