title: Interaction between NADH and electron-transferring flavoprotein from Megasphaera elsdenii.
abstract: Electron-transferring flavoprotein (ETF) from the anaerobic bacterium Megasphaera elsdenii is a heterodimer containing two FAD cofactors. Isolated ETF contains only one FAD molecule, FAD-1, because the other, FAD-2, is lost during purification. FAD-2 is recovered by adding FAD to the isolated ETF. The two FAD molecules in holoETF were characterized using NADH. Spectrophotometric titration of isolated ETF with NADH showed a two-electron reduction of FAD-1 according to a monophasic profile indicating that FAD-1 receives electrons from NADH without involvement of FAD-2. When holoETF was titrated with NADH, FAD-2 was reduced to an anionic semiquinone and then was fully reduced before the reduction of FAD-1. The midpoint potential values at pH 7 were +81, -136 and -279 mV for the reduction of oxidized FAD-2 to semiquinone, semiquinone to the fully reduced FAD-2 and the two-electron reduction of FAD-1, respectively. Both FAD-1 and FAD-2 in holoETF were reduced by excess NADH very rapidly. The reduction of FAD-2 was slowed by replacement of FAD-1 with 8-cyano-FAD indicating that FAD-2 receives electrons from FAD-1 but not from NADH directly. The present results suggest that FAD-2 is the counterpart of the FAD in human ETF, which contains one FAD and one AMP.
authors.affiliation: Department 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
authors.forename: Kyosuke
authors.initials: K
authors.lastname: Sato
authors.forename: Yasuzo
authors.initials: Y
authors.lastname: Nishina
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Shiga
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D019281:Dimerization; D004579:Electron Transport; D044927:Electron-Transferring Flavoproteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D045851:Megasphaera / Q000378:metabolism*; D009243:NAD / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction
chemical_list: D001426:Bacterial Proteins; D044927:Electron-Transferring Flavoproteins; D009243:NAD
keywords: 8-CN-FAD; Megasphaera elsdenii; NADH; electron-transferring flavoprotein; midpoint potential