Department of Biochemistry, Chulalongkorn University, Patumwan, Bangkok, Thailand; Skeletal Disorders Research Unit, Faculty of Dentistry, Chulalongkorn University, Patumwan, Bangkok, Thailand.
Laboratorium für Mikrobiologie, Fachbereich Biologie and Synmikro, Philipps-Universität, Marburg, Germany; Max-Plank-Institut für terrestrische Mikrobiologie, Marburg, Germany.
J Biol Chem. 2021 Jan-Jun;296:100124. doi: 10.1074/jbc.RA120.016017. Epub 2020 Dec 2.
Electron bifurcation uses free energy from exergonic redox reactions to power endergonic reactions. β-FAD of the electron transfer flavoprotein (EtfAB) from the anaerobic bacterium Acidaminococcus fermentans bifurcates the electrons of NADH, sending one to the low-potential ferredoxin and the other to the high-potential α-FAD semiquinone (α-FAD). The resultant α-FAD hydroquinone (α-FADH) transfers one electron further to butyryl-CoA dehydrogenase (Bcd); two such transfers enable Bcd to reduce crotonyl-CoA to butyryl-CoA. To get insight into the mechanism of these intricate reactions, we constructed an artificial reaction only with EtfAB containing α-FAD or α-FAD to monitor formation of α-FAD or α-FADH, respectively, using stopped flow kinetic measurements. In the presence of α-FAD, we observed that NADH transferred a hydride to β-FAD at a rate of 920 s, yielding the charge-transfer complex NAD:β-FADH with an absorbance maximum at 650 nm. β-FADH bifurcated one electron to α-FAD and the other electron to α-FAD of a second EtfAB molecule, forming two stable α-FAD. With α-FAD, the reduction of β-FAD with NADH was 1500 times slower. Reduction of β-FAD in the presence of α-FAD displayed a normal kinetic isotope effect (KIE) of 2.1, whereas the KIE was inverted in the presence of α-FAD. These data indicate that a nearby radical (14 Å apart) slows the rate of a hydride transfer and inverts the KIE. This unanticipated flavin chemistry is not restricted to Etf-Bcd but certainly occurs in other bifurcating Etfs found in anaerobic bacteria and archaea.
电子分支利用放能氧化还原反应中的自由能来驱动吸能反应。来自厌氧细菌产氨棒杆菌的电子传递黄素蛋白（EtfAB）中的β-FAD 使 NADH 的电子发生分支，一部分传递给低电势铁氧还蛋白，另一部分传递给高电势α-FAD 半醌（α-FAD）。生成的α-FAD 氢醌（α-FADH）进一步将一个电子传递给丁酰基辅酶 A 脱氢酶（Bcd）；两个这样的电子传递使 Bcd 将丁烯酰辅酶 A 还原为丁酰辅酶 A。为了深入了解这些复杂反应的机制，我们构建了一个仅包含 EtfAB 的人工反应，该反应分别含有α-FAD 或α-FAD，以使用停流动力学测量来监测α-FAD 或α-FADH 的形成。在存在α-FAD 的情况下，我们观察到 NADH 以 920 s 的速率将一个氢化物转移到β-FAD 上，生成具有 650nm 最大吸收的电荷转移复合物 NAD：β-FADH。β-FADH 将一个电子分支到α-FAD，另一个电子分支到第二个 EtfAB 分子的α-FAD，形成两个稳定的α-FAD。有α-FAD 时，β-FAD 与 NADH 的还原速度慢 1500 倍。在存在α-FAD 的情况下，β-FAD 的还原显示出正常的动力学同位素效应（KIE）为 2.1，而在存在α-FAD 的情况下，KIE 则被反转。这些数据表明，附近的自由基（相距 14Å）会降低氢化物转移的速率并反转 KIE。这种出乎意料的黄素化学不仅限于 Etf-Bcd，而且肯定会发生在厌氧细菌和古细菌中发现的其他分支 Etfs 中。
