Modulations of the reduction potentials of flavin-based electron bifurcation complexes and semiquinone stabilities...

The flavin-based electron bifurcation (FBEB) system from Acidaminococcus fermentans is composed of the electron transfer flavoprotein (EtfAB) and butyryl-CoA dehydrogenase (Bcd). α-FAD binds to domain II of the A-subunit of EtfAB, β-FAD to the B-subunit of EtfAB and δ-FAD to Bcd. NADH reduces β-FAD to β-FADH^- , which bifurcates one electron to the high potential α-FAD^•- semiquinone followed by the other to the low potential ferredoxin (Fd). As deduced from crystal structures, upon interaction of EtfAB with Bcd, the formed α-FADH^- approaches δ-FAD by rotation of domain II, yielding δ-FAD^•- . Repetition of this process leads to a second reduced ferredoxin (Fd^- ) and δ-FADH^- , which reduces crotonyl-CoA to butyryl-CoA. In this study, we measured the redox properties of the components EtfAB, EtfaB (Etf without α-FAD), Bcd, and Fd, as well as of the complexes EtfaB:Bcd, EtfAB:Bcd, EtfaB:Fd, and EftAB:Fd. In agreement with the structural studies, we have shown for the first time that the interaction of EtfAB with Bcd drastically decreases the midpoint reduction potential of α-FAD to be within the same range of that of β-FAD and to destabilize the semiquinone of α-FAD. This finding clearly explains that these interactions facilitate the passing of electrons from β-FADH^- via α-FAD^•- to the final electron acceptor δ-FAD^•- on Bcd. The interactions modulate the semiquinone stability of δ-FAD in an opposite way by having a greater semiquinone stability than in free Bcd.