Department of Biochemistry, The University of California, Riverside, California, USA.
Department of Biochemistry, The University of California, Riverside, California, USA.
J Biol Chem. 2024 Oct;300(10):107745. doi: 10.1016/j.jbc.2024.107745. Epub 2024 Sep 3.
We have investigated the kinetic behavior of the electron-bifurcating crotonyl-CoA-dependent NADH: ferredoxin oxidoreductase EtfAB:bcd from Megasphaera elsdenii. The overall behavior of the complex in both the reductive and the oxidative half-reactions is consistent with that previously determined for the individual EtfAB and bcd components. This includes an uncrossing of the half-potentials of the bifurcating flavin of the EtfAB component in the course of ferredoxin-reducing catalysis, ionization of the bcd flavin semiquinone and the appearance of a charge transfer complex upon binding of the high potential acceptor crotonyl-CoA. The observed rapid-reaction rates of ferredoxin reduction are independent of [NADH], [crotonyl-CoA], and [ferredoxin], with an observed rate of ∼0.2 s, consistent with the observed steady-state kinetics. In enzyme-monitored turnover experiments, an approach to steady-state where the complex's flavins become reduced but no ferredoxin is generated is followed by a steady-state phase characterized by extensive ferredoxin reduction but little change in overall levels of flavin reduction. The approach to the steady-state phase can be eliminated by prior reduction of the complex, in which case there is no lag in the onset of ferredoxin reduction; this is consistent with the et FAD needing to be reduced to the level of the (anionic) semiquinone for bifurcation and concomitant ferredoxin reduction to occur. Single-turnover experiments support this conclusion, with the accumulation of the anionic semiquinone of the et FAD apparently required to prime the system for subsequent bifurcation and ferredoxin reduction.
我们研究了源自巨球形菌的电子分叉丁烯酰基辅酶 A 依赖的 NADH：铁氧还蛋白氧化还原酶 EtfAB：bcd 的动力学行为。该复合物在还原和氧化半反应中的整体行为与之前确定的 EtfAB 和 bcd 单个组件的行为一致。这包括 EtfAB 组件中分叉黄素的半电位在铁氧还蛋白还原催化过程中交叉，bcd 黄素半醌的离解以及高电势受体丁烯酰辅酶 A 结合时出现电荷转移复合物。观察到的铁氧还蛋白还原的快速反应速率与 [NADH]、[丁烯酰辅酶 A] 和 [铁氧还蛋白] 无关，观察到的速率约为 0.2 s，与观察到的稳态动力学一致。在酶监测的周转实验中，复合物的黄素被还原但没有产生铁氧还蛋白的接近稳态的方法，随后是特征为铁氧还蛋白广泛还原但黄素还原的总水平变化不大的稳态阶段。通过预先还原复合物，可以消除接近稳态阶段，在这种情况下，铁氧还蛋白还原没有延迟；这与 EtFAD 需要还原到（阴离子）半醌的水平以发生分叉和伴随的铁氧还蛋白还原一致。单轮实验支持这一结论，显然需要积累 EtFAD 的阴离子半醌来为随后的分叉和铁氧还蛋白还原做好准备。
