Sucharitakul Jeerus, Mangkalee Montisa, Intasian Pattarawan, Pornsuwan Soraya, Ermler Ulrich, Buckel Wolfgang, Chaiyen Pimchai
Department of Biochemistry, Bangkok, Thailand; Center of Excellence in Integrative Immuno-Microbial Biochemistry and Bioresponsive Nanomaterials, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand.
Center of Excellence in Integrative Immuno-Microbial Biochemistry and Bioresponsive Nanomaterials, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand; Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.
J Biol Chem. 2025 Oct;301(10):110727. doi: 10.1016/j.jbc.2025.110727. Epub 2025 Sep 16.
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 EtfAB (A contains α-FAD) and the charge-transfer complex of EtfAB:NAD (B contains β-FADH). Since the radical in EtfAB 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-EtfAB complex and additional Fd. In the presence of crotonyl-CoA, two simultaneous one-electron transfers from both EtfAB yield reduced Bcd and two EtfAB, 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 at 4 °C, but increases about 14-fold to 0.14 s 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.
来自发酵氨基酸球菌的电子传递黄素蛋白（EtfAB，含α-FAD和β-FAD）和四聚体丁酰辅酶A脱氢酶（Bcd，每个亚基含δ-FAD）催化电子分支反应，该反应以NADH作为电子供体，将低电位铁氧化还原蛋白（Fd）还原，并使高电位巴豆酰辅酶A还原。我们之前的快速动力学研究表明存在“假电子分支”现象，即NADH和两个EtfAB分子生成EtfAB（A含α-FAD）以及EtfAB:NAD的电荷转移复合物（B含β-FADH）。由于EtfAB中的自由基会抑制β-FAD被NADH进一步还原，因此就出现了整个系统的五个组分如何相互作用以介导整个基于黄素的电子分支反应这一问题。本研究表明，Bcd可消除α-FAD的抑制作用，使β-FAD能快速被还原以进行周转。在同时存在Bcd和Fd的情况下，EtfAB的总β-FADH发生分支反应，生成α-FAD和Fd；第二次分支反应在Bcd-EtfAB复合物中产生α-FADH和额外的Fd。在存在巴豆酰辅酶A的情况下，电子顺磁共振光谱证实，来自两个EtfAB的两个同步单电子转移生成了还原型Bcd和两个EtfAB。这一步骤被认为需要Bcd-EtfAB复合物发生缓慢的构象变化以进行电子转移，在4℃时极限速率常数为0.0098 s⁻¹，但在30℃（发酵氨基酸球菌的最适生长温度）时增加约14倍，达到0.14 s⁻¹。巴豆酰辅酶A最终还原为丁酰辅酶A完成了这个循环，我们将其称为电子分支反应的半醌循环，因为它以半醌开始和结束。
