两个分叉黄素蛋白系统的EtfAB成分,巴豆酰辅酶A依赖的NADH:来自埃氏巨细菌的铁氧还蛋白氧化还原酶和甲萘醌依赖的NADH:来自古细菌嗜热杆菌的铁氧还蛋白氧化还原酶,已被调查。有了这两种蛋白质,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)部分会导致剩余的分叉FAD的还原电位不相交;这显着稳定了原本非常不稳定的半醌状态,在连二亚硫酸钠的还原滴定过程中积累。此外,NADH对两个消耗其电子转移FAD的EtfAB的还原是单相的,反应速率对NADH浓度的双曲线依赖性。另一方面,含有电子转移FAD的大量蛋白质的NADH还原是多相的,由与耗尽的蛋白质相当的快速阶段组成,随后是涉及FAD·-大量积累的中间阶段-,再次反映了分叉FAD的半电位的不交叉。然后是缓慢阶段,表示电子转移FAD到FADH的缓慢还原-,现在完全再氧化的分叉FAD减少了第二个当量的NADH。我们建议,分叉FAD的还原半电位的交叉和不交叉是由于已在结构上表征的特定构象变化。
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.