Maximizing (Electro)catalytic CO₂ Reduction with a Ferredoxin-Based Reduction Potential Gradient

Abstract

Tuning the electron-transfer (ET) steps in redox reactions has proven to be an effective strategy to modulate enzyme catalysis. Here, we use 2-oxoacid:ferredoxin oxidoreductase (OFOR) as a model system to study the two-electron reduction of CO 2 and to evaluate the effect of intermolecular ET on catalysis by systematically engineering the reduction potential (E m ) of the electron donor, ferredoxin (Fd), aiming to maximize catalytic CO 2 reduction. Based on the newly determined crystal structure for Fd1 from Hydrogenobacter thermophilus (HtFd1), we expanded the E m of HtFd1 (−485 mV, vs the standard hydrogen electrode) to a range from −440 to −555 mV through changes in the hydrogen bonding network and solvent exposure of the [4Fe− 4S] cluster, with −555 mV being the lowest potential observed for a monocluster [4Fe−4S] Fd. Specifically, replacement of a polar residue (serine) with a non-polar residue (alanine or valine) at the +2 position with respect to the C4 of the C1XXC2XXC3X n C4 [4Fe−4S] cluster-ligating motif abolished an extensive hydrogen bonding network and decreased E m by 40 mV; the introduction of a non-ligating "fifth cysteine" at the +4 position of C4 decreased E m by 60 mV; and increasing the solvent exposure of the [4Fe−4S] cluster by replacement of a bulky residue (leucine) with glycine at the −1 position of C3 increased E m by 45 mV. We constructed an Fd library of 24 Fds with a "potential gradient", ranging from −378 to −555 mV, and evaluated the impact of the Fd potential on OFOR activity. Here, we found that decreasing the Fd potential and therefore providing a larger driving force are beneficial for CO 2 reduction, but only to a point. With the HtFd1_T13I_S64A variant (−515 mV), we were able to achieve a maximum turnover frequency of 81.3 min −1 (or a specific activity of 625 nmol min −1 mg −1 ) in CO 2 reduction.

Evolving a New Electron Transfer Pathway for Nitrogen Fixation Uncovers an Electron Bifurcating-Like Enzyme Involved in Anaerobic Aromatic Compound Degradation

“…The threonine residue at position 11 in Fer1 is adjacent to a cysteine predicted to coordinate one of the [4Fe-4S] clusters in Fer1. Many other low-potential 2[4Fe-4S] Fds encode isoleucine at this position and similar threonine to isoleucine substitutions in other Fds have been shown to lower the reduction potential of Fds ( 5 , 34 , 42 ). This suggests that the T11I substitution in Fer1 lowers its reduction potential, although it is unclear why this would facilitate interaction with AadN.…”

“…In particular, Fds have specialized over evolutionary time to associate with specific partner proteins, allowing Fds to selectively shuttle electrons to specific pathways ( 1 , 2 ). Key factors such as the structure and charge of the Fd binding surface ( 3 ), regulation of Fd abundance ( 4 ), and the reduction potential of the Fd ( 5 – 7 ) affect which partner proteins interact with Fd. In theory, these properties can be altered to enable an Fd to interact with a new partner protein(s) to reroute electron flow, but this remains a challenge for rational design since these properties are ill-defined for many Fds.…”

Toward an Understanding of Bimetallic MXene Solid‐Solution in Binder‐Free Electrocatalyst Cathode for Advanced Li–CO2 Batteries

“…[1,2] At present, there are many ways to achieve "carbon neutrality" by enriching, capturing, and converting carbon dioxide (CO 2 )-based greenhouse gases into carbon products, such as adsorption, chemical synthesis, thermos-catalysis, electrocatalysis, photocatalysis, and so on. [3][4][5][6] Among them, electrocatalysis is considered as an effective method to achieve "carbon neutralization" because it can convert renewable electric energy into chemical energy and realize a closed carbon cycle. However, the cost of electrocatalyst is not commercialize; when reducing CO 2 , high overpotential is required, and the side reactions are inevitable, resulting in high energy consumption and low CO 2 reduction efficiency.…”

Selecting a new electron transfer pathway for nitrogen fixation uncovers an electron bifurcating-like enzyme involved in anaerobic aromatic compound degradation

“…In particular, Fds have specialized over evolutionary time to associate with specific partner proteins, allowing Fds to selectively shuttle electrons to specific pathways (1,2). Key factors such as the structure and charge of the Fd binding surface (3), regulation of Fd abundance (4), and the reduction potential of the Fd (5)(6)(7) all affect which partner proteins interact with a Fd. In theory, these properties can be altered to enable a Fd to interact with a new partner protein(s) to re-route electron flow, but this remains a challenge for rational design since these properties are ill-defined for many Fds.…”