Transmembrane NADH Oxidation with Tetracyanoquinodimethane

Abstract

The design of efficient schemes for nicotinamide adenine dinucleotide (NAD) regeneration is essential for the development of enzymatic biotechnological processes in order to sustain continuous production. In line with our motivation for the encapsulation of redox cascades in liposomes to serve as microbioreactors, we developed a straightforward strategy for the interfacial oxidation of entrapped NADH by ferricyanide as an external electron acceptor. Instead of the commonly applied enzymatic regeneration methods, we employed a hydrophobic redox shuttle embedded in the liposome bilayer. Tetracyanoquinodimethane (TCNQ) mediated electron transfer across the membrane and thus allowed us to shortcut and emulate part of the electron transfer chain functionality without the involvement of membrane proteins. To describe the experimental system, we developed a mathematical model which allowed for the determination of rate constants and exhibited handy predictive utility.

Artificial Organelles for Energy Regeneration

“…Also there is a possibility that other mentioned NAD + regeneration systems, e.g. light‐ or chemically‐driven, are also capable of establishing PMF. Finally the straightforward disadvantages of most reported systems is absence of recycling of sacrificial electron donors/acceptors, which has to be taken into account in order to design truly sustainable systems.…”

“…Current motivation to study NAD + regeneration in closed compartments includes higher spatial and temporal control of the regeneration process, creation of controlled environments with an enhanced catalytic efficiency due to confinement and concentration effects or from the bottom‐up perspective in synthetic biology, understanding of the effects of compartmentalization in catalytic systems might help us to unveil the constraints and challenges possibly faced by the first cells regarding the development of compartmentalized metabolic networks . Most NAD + regeneration strategies considered lipid vesicles as compartments . The NAD + regeneration was driven by light by chemical energy or electrochemically and regeneration of both oxidized and reduced forms of NAD + was demonstrated.…”

“…Regeneration of NAD + was chemically driven in recent studies by Wang et al and Bonfia et al In the first example, NADH was encapsulated inside of lipid vesicles. Alike other mentioned examples where soluble redox mediators were used, in this contribution, the redox mediator was confined inside of the lipid membrane.…”

“…The authors demonstrated a straightforward strategy for the oxidation of entrapped NADH by ferricyanides as an external electron acceptor. A hydrophobic redox shuttle (tetracyanoquinodimethane (TCNQ)), embedded in the liposome bilayer, mediated electron transfer across the membrane, and thus allowed to shortcut and emulate part of the electron transfer chain functionality without the involvement of membrane proteins . In the recent work by Bonfia et al, NAD + was regenerated by NADH oxidation in the presence of iron‐sulfur peptide catalysts and hydrogen peroxide as the final electron acceptor .…”

Towards Design of Self‐Organizing Biomimetic Systems

“…One advantage of bottom‐up synthetic biology is the development of completely synthetic biomimetic modules that do not occur in nature and have a simpler structure with the same functionality as their natural counterparts. In this context, a mathematical model for a synthetic electron transport chain (ETC), as the coupling module between metabolism and energy supply modules, was developed where the functionalities of the ETC membrane proteins are mimicked by membrane‐situated tetracyanochinodimethan (TCNQ) . TCNQ oxidizes the reduction equivalent NADH and transfers electrons through a vesicle membrane which are needed to pump protons in mitochondria .…”