Work

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

Year: 2025

Type: article

Abstract: 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 SQ B (A SQ = α-FAD ●− ) and the charge-transfer complex of EtfA SQ B HQ :NAD + (B HQ = β-FADH − ). Since the radical in EtfA SQ B 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 HQ B complex and additional Fd − . In the presence of crotonyl-CoA, two simultaneous one-electron transfers from both EtfA HQ B yield reduced Bcd and two EtfA SQ B, confirmed by EPR 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.

Source: Journal of Biological Chemistry

DOI: 10.1016/j.jbc.2025.110727