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Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases
Published on: February 24, 2018
Interaction between NADH and electron-transferring flavoprotein from Megasphaera elsdenii
Kyosuke Sato1, Yasuzo Nishina, Kiyoshi Shiga
1Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto, Kumamoto 860-8556, Japan. satok@kumamoto-u.ac.jp
Journal of Biochemistry
|April 2, 2013
Summary
Electron-transferring flavoprotein (ETF) from Megasphaera elsdenii contains two FAD molecules. FAD-2 receives electrons from FAD-1, not directly from NADH, revealing its role in electron transfer.
Area of Science:
- Biochemistry
- Microbiology
- Enzyme kinetics
Background:
- Electron-transferring flavoprotein (ETF) is a crucial enzyme in anaerobic energy metabolism.
- Megasphaera elsdenii ETF is a heterodimer with two FAD cofactors, but one is often lost during purification.
- Understanding the distinct roles of the two FAD molecules is essential for elucidating ETF function.
Purpose of the Study:
- To characterize the two FAD molecules in holoETF from Megasphaera elsdenii.
- To determine the electron transfer pathway between NADH, FAD-1, and FAD-2.
- To compare the bacterial ETF system with its human counterpart.
Main Methods:
- Spectrophotometric titration with NADH to monitor FAD reduction.
- Enzyme assays with modified FAD analogs (8-cyano-FAD).
- Midpoint potential measurements at pH 7.
Main Results:
- FAD-1 is directly reduced by NADH via a two-electron process.
- FAD-2 is reduced after FAD-1, first to a semiquinone, then fully reduced.
- The reduction of FAD-2 is dependent on FAD-1, indicating sequential electron transfer.
- Midpoint potentials for FAD-2 reduction were +81 mV and -136 mV; for FAD-1, -279 mV.
Conclusions:
- FAD-2 acts as an electron acceptor from FAD-1, not directly from NADH.
- The electron transfer pathway involves sequential reduction: NADH → FAD-1 → FAD-2.
- FAD-2 in M. elsdenii ETF likely corresponds to the FAD/AMP component in human ETF.
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