Kinetic mechanisms of electron bifurcation with electron transfer flavoprotein, NADH, butyryl-CoA dehydrogenase, and ferredoxin reveal a semiquinone cycle

Electron transfer flavoprotein (EtfAB, with α-FAD and β-FAD) and tetrameric butyryl-CoA dehydrogenase (Bcd, with δ-FAD in each subunit) from Acidaminococcus fermentans catalyze electron bifurcation which reduces low potential ferredoxin (Fd) and high potential crotonyl-CoA using NADH as an electron donor. Our previous rapid kinetic studies have demonstrated "pseudo-electron bifurcation" where NADH and two EtfAB molecules generate EtfA_SQB (A_SQ contains α-FAD^•-) and the charge-transfer complex of EtfA_SQB_HQ:NAD⁺ (B_HQ contains β-FADH^-). Since the radical in EtfA_SQB inhibits the further reduction of β-FAD with NADH, the question arises as to how the five components of the complete system interact to mediate the whole flavin-based electron bifurcation. This study shows that Bcd releases the inhibition effect of α-FAD^•-, allowing fast β-FAD reduction for turnover. In the presence of both Bcd and Fd, the total β-FADH^- of EtfAB bifurcates to afford α-FAD^•- and Fd^-; a second bifurcation yields α-FADH^- in the Bcd-EtfA_HQB complex and additional Fd^-. In the presence of crotonyl-CoA, two simultaneous one-electron transfers from both EtfA_HQB yield reduced Bcd and two EtfA_SQB, confirmed by electron paramagnetic resonance spectroscopy. This step is proposed to require a slow conformational change of the Bcd-EtfAB complex for electron transfer with a limiting rate constant of 0.0098 s^-1 at 4 °C, but increases about 14-fold to 0.14 s^-1 at 30 °C, the optimal growth temperature of A. fermentans. The final reduction of crotonyl-CoA to butyryl-CoA completes the cycle, which we call the semiquinone cycle of electron bifurcation, because it starts and ends with a semiquinone.