Related Experiment Video
Updated: Jun 12, 2025
08:57
Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases
Published on: February 24, 2018
10.1K
Engineering Oxygen-Independent NADH Oxidase Integrated with Electrocatalytic FAD Cofactor Regeneration
Mengjie Hou1,2, Jing Yuan2, Xinyu Dong2
1School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen 518055, China.
JACS Au
|September 27, 2024
Summary
This study presents a novel electrochemically mediated enzyme process for nicotinamide adenine dinucleotide (NADH) oxidation, replacing oxygen dependence with electrochemical cofactor regeneration for enhanced enzyme stability and improved NADH biosensing.
Area of Science:
- Biocatalysis
- Electrochemistry
- Biosensors
Background:
- Traditional NADH oxidase enzymes rely on oxygen, limiting stability in biosynthetic applications.
- Oxygen dependence necessitates continuous air or oxygen supply, complicating processes and reducing enzyme longevity.
Purpose of the Study:
- To develop an oxygen-independent electrochemically mediated enzyme process for NADH oxidation.
- To engineer NADH oxidase for enhanced stability and efficient electrochemical cofactor regeneration.
- To create responsive electrochemical biosensors for NADH detection.
Main Methods:
- Enzyme immobilization on electrodes coupled with electrochemical oxidation of FADH2 via ferrocene carboxylic acid (FcCA) mediator.
- Site-directed mutagenesis of Leuconostoc mesenteroides (LmNOx) at Leu40 and Cys42 to block oxygen entry and eliminate native FAD regeneration.
- Development of electrochemical biosensors utilizing engineered enzymes.
Main Results:
- Engineered LmNOx enzymes, inactive in solution, exhibit electrocatalytic activity for NADH to NAD+ conversion.
- Successful electrochemical regeneration of the FAD cofactor within the enzyme's active site.
- Developed two highly responsive electrochemical biosensors for NADH detection with a 1-3 μM detection limit.
Conclusions:
- The developed bioelectrocatalytic system offers an oxygen-independent alternative for NADH oxidation and biosensing.
- Mutated enzymes demonstrate efficient electrocatalytic activity, highlighting the success of active site engineering.
- The novel biosensors provide a substrate-specific and sensitive platform for NADH detection.
Related Concept Videos
01:29Role of Reduced Coenzymes NADH and FADH₂
11.2K
The energy released from the breakdown of the chemical bonds within nutrients can be stored either through the reduction of electron carriers or in the bonds of adenosine triphosphate (ATP). In living systems, a small class of compounds functions as mobile electron carriers, molecules that bind to and shuttle high-energy electrons between compounds in pathways. The principal electron carriers that will be considered originate from the B vitamin group and are derivatives of nucleotides; they are...
11.2K
01:43Electron Transport Chain: Complex III and IV
7.2K
During the electron transport chain, electrons from NADH and FADH2 are first transferred to complexes I and II, respectively. These two complexes then transfer the electrons to ubiquinol, which carries them further to complex III. Complex III passes the electrons across the intermembrane space to Cyt c, which carries them further to complex IV. Complex IV donates electrons to oxygen and reduces it to water. As electrons pass through complexes I, III, and IV, the energy released aids the pumping...
7.2K
01:19Oxidation and Reduction of Organic Molecules
6.2K
Energy production within a cell involves many coordinated chemical pathways. Most of these pathways are combinations of oxidation and reduction reactions, which occur at the same time. An oxidation reaction strips an electron from an atom in a compound, and the addition of this electron to another compound is a reduction reaction. Because oxidation and reduction usually occur together, these pairs of reactions are called redox reactions.
The removal of an electron from a molecule, results in a...
The removal of an electron from a molecule, results in a...
6.2K
01:30The Electron Transport Chain
16.3K
The electron transport chain or oxidative phosphorylation is an exothermic process in which free energy released during electron transfer reactions is coupled to ATP synthesis. This process is a significant source of energy in aerobic cells, and therefore inhibitors of the electron transport chain can be detrimental to the cell's metabolic processes.
Inhibitors of the electron transport chain
Rotenone, a widely used pesticide, prevents electron transfer from Fe-S cluster to ubiquinone or Q...
Inhibitors of the electron transport chain
Rotenone, a widely used pesticide, prevents electron transfer from Fe-S cluster to ubiquinone or Q...
16.3K
01:46Electron Transport Chain: Complex I and II
12.2K
The mitochondrial electron transport chain (ETC) is the main energy generation system in the eukaryotic cells. However, mitochondria also produce cytotoxic reactive oxygen species (ROS) due to the large electron flow during oxidative phosphorylation. While Complex I is one of the primary sources of superoxide radicals, ROS production by Complex II is uncommon and may only be observed in cancer cells with mutated complexes.
ROS generation is regulated and maintained at moderate levels necessary...
ROS generation is regulated and maintained at moderate levels necessary...
12.2K
01:24Electron Carriers
84.2K
Electron carriers can be thought of as electron shuttles. These compounds can easily accept electrons (i.e., be reduced) or lose them (i.e., be oxidized). They play an essential role in energy production because cellular respiration is contingent on the flow of electrons.
Over the many stages of cellular respiration, glucose breaks down into carbon dioxide and water. Electron carriers pick up electrons lost by glucose in these reactions, temporarily storing and releasing them into the electron...
Over the many stages of cellular respiration, glucose breaks down into carbon dioxide and water. Electron carriers pick up electrons lost by glucose in these reactions, temporarily storing and releasing them into the electron...
84.2K