Abstract
The purified electron-transferring flavoprotein (ETF) from the anaerobic bacterium Megasphaera elsdenii has one tightly bound FAD molecule as the cofactor and binds another FAD molecule added exogenously. Thus holo-ETF contains two FAD molecules. Here the FAD contained in purified ETF is named FAD-1 and the additionally bound FAD is named FAD-2. The function of ETF is receiving electrons from NADH and donating them to other flavoenyzmes, enoyl-CoA reductase and D-lactate dehydrogenase. When purified ETF is mixed with NADH, FAD-1 is reduced. When holo-ETF is mixed with NADH, both FAD-1 and FAD2 are reduced. These findings clarifies that FAD-1 directly receives electrons from NADH. However, it is unclear whether FAD-2 receives electrons from NADH or FAD-1. The reduction of FAD-1 and FAD-2 by NADH was too fast to investigate kinetically even with stopped flow apparatus. In this study, we used 8-CN-FAD, which has more positive standard redox potential than FAD. The holo-ETF whose FAD-1 was exchanged for 8-CN-FAD was prepared. If FAD-2 receives electrons from FAD-1, the reduction of FAD-2 should be perturbed by the exchange. When this holo-ETF was mixed with NADH, fully reduced 8-CN-FAD and one electron-reduced FAD-2 were formed in the dead time of the measurement, followed by slow additional one-electron reduction of FAD-2. This result indicates that FAD-2 does not receive electrons from NADH but from FAD-1, because the electron transfer from NADH must be simultaneous two-electron transfer. [J Physiol Sci. 2007;57 Suppl:S122]
