Department of Structural Biology, Van Andel Institute, Grand Rapids, MI 49503.
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602.
Proc Natl Acad Sci U S A. 2021 Jan 12;118(2). doi: 10.1073/pnas.2016978118.
The electron-transferring flavoprotein-menaquinone oxidoreductase ABCX (EtfABCX), also known as FixABCX for its role in nitrogen-fixing organisms, is a member of a family of electron-transferring flavoproteins that catalyze electron bifurcation. EtfABCX enables endergonic reduction of ferredoxin (°' ∼-450 mV) using NADH (°' -320 mV) as the electron donor by coupling this reaction to the exergonic reduction of menaquinone (°' -80 mV). Here we report the 2.9 Å structure of EtfABCX, a membrane-associated flavin-based electron bifurcation (FBEB) complex, from a thermophilic bacterium. EtfABCX forms a superdimer with two membrane-associated EtfCs at the dimer interface that contain two bound menaquinones. The structure reveals that, in contrast to previous predictions, the low-potential electrons bifurcated from EtfAB are most likely directly transferred to ferredoxin, while high-potential electrons reduce the quinone via two [4Fe-4S] clusters in EtfX. Surprisingly, EtfX shares remarkable structural similarity with mammalian [4Fe-4S] cluster-containing ETF ubiquinone oxidoreductase (ETF-QO), suggesting an unexpected evolutionary link between bifurcating and nonbifurcating systems. Based on this structure and spectroscopic studies of a closely related EtfABCX, we propose a detailed mechanism of the catalytic cycle and the accompanying structural changes in this membrane-associated FBEB system.
电子传递黄素蛋白-menaquinone 氧化还原酶 ABCX（也称为 FixABCX，因其在固氮生物中的作用）是电子传递黄素蛋白家族的成员，该家族的蛋白能够催化电子分叉。EtfABCX 能够利用 NADH（°' -320 mV）作为电子供体，将铁氧还蛋白（°' ∼-450 mV）的内吸还原与menaquinone（°' -80 mV）的外吸还原偶联，从而实现内吸还原。在这里，我们报道了来自嗜热细菌的膜相关黄素基电子分叉（FBEB）复合物 EtfABCX 的 2.9 Å 结构。EtfABCX 在二聚体界面处形成一个由两个膜相关 EtfC 组成的超二聚体，每个 EtfC 都含有两个结合的menaquinone。该结构表明，与之前的预测相反，来自 EtfAB 的低电位电子很可能直接转移到铁氧还蛋白，而高电位电子则通过 EtfX 中的两个 [4Fe-4S] 簇还原醌。令人惊讶的是，EtfX 与哺乳动物含 [4Fe-4S] 簇的 ETF 泛醌氧化还原酶（ETF-QO）具有显著的结构相似性，这表明分叉和非分叉系统之间存在出人意料的进化联系。基于该结构和对密切相关的 EtfABCX 的光谱研究，我们提出了该膜相关 FBEB 系统中催化循环和伴随结构变化的详细机制。
