Department of Biochemistry, University of California, Riverside, California, USA.
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA.
J Biol Chem. 2022 Jun;298(6):101927. doi: 10.1016/j.jbc.2022.101927. Epub 2022 Apr 13.
The EtfAB components of two bifurcating flavoprotein systems, the crotonyl-CoA-dependent NADH:ferredoxin oxidoreductase from the bacterium Megasphaera elsdenii and the menaquinone-dependent NADH:ferredoxin oxidoreductase from the archaeon Pyrobaculum aerophilum, have been investigated. With both proteins, we find that removal of the electron-transferring flavin adenine dinucleotide (FAD) moiety from both proteins results in an uncrossing of the reduction potentials of the remaining bifurcating FAD; this significantly stabilizes the otherwise very unstable semiquinone state, which accumulates over the course of reductive titrations with sodium dithionite. Furthermore, reduction of both EtfABs depleted of their electron-transferring FAD by NADH was monophasic with a hyperbolic dependence of reaction rate on the concentration of NADH. On the other hand, NADH reduction of the replete proteins containing the electron-transferring FAD was multiphasic, consisting of a fast phase comparable to that seen with the depleted proteins followed by an intermediate phase that involves significant accumulation of FAD⋅, again reflecting uncrossing of the half-potentials of the bifurcating FAD. This is then followed by a slow phase that represents the slow reduction of the electron-transferring FAD to FADH, with reduction of the now fully reoxidized bifurcating FAD by a second equivalent of NADH. We suggest that the crossing and uncrossing of the reduction half-potentials of the bifurcating FAD is due to specific conformational changes that have been structurally characterized.
两种分叉黄素蛋白系统的 EtfAB 组分，即细菌 Megasphaera elsdenii 中的丙二酰辅酶 A 依赖型 NADH：黄素氧化还原酶和古菌 Pyrobaculum aerophilum 中的依赖甲萘醌的 NADH：黄素氧化还原酶，已经过研究。对于这两种蛋白质，我们发现从两种蛋白质中去除电子转移黄素腺嘌呤二核苷酸（FAD）部分会导致剩余分叉 FAD 的还原电势不交叉；这显著稳定了 otherwise very unstable semiquinone state，该状态在与连二亚硫酸钠的还原滴定过程中会积累。此外，通过 NADH 还原耗尽电子转移 FAD 的 EtfAB 是单相的，反应速率对 NADH 浓度呈双曲线依赖性。另一方面，含有电子转移 FAD 的饱满蛋白质的 NADH 还原是多相的，由一个快速相组成，与耗尽蛋白质的快速相相似，然后是一个中间相，涉及分叉 FAD 的 significant accumulation，再次反映了分叉 FAD 的半电势不交叉。然后是一个缓慢的阶段，代表电子转移 FAD 缓慢还原为 FADH，现在完全再氧化的分叉 FAD 被第二个当量的 NADH 还原。我们认为分叉 FAD 的还原半电势的交叉和不交叉是由于已经结构表征的特定构象变化引起的。
