Interaction between NADH and electron-transferring flavoprotein from Megasphaera elsdenii
Interaction between NADH and electron-transferring flavoprotein from Megasphaera elsdenii
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DOI：
10.1093/jb/mvt026
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发表时间：
2013-06-01
影响因子：
2.7
通讯作者：
Shiga, Kiyoshi
中科院分区：
文献类型：
--
作者：
Sato, Kyosuke;Nishina, Yasuzo;Shiga, Kiyoshi
来自厌氧细菌艾氏巨球菌的电子转移黄素蛋白 (ETF) 是一种含有两个 FAD 辅因子的异二聚体。分离的 ETF 仅包含一种 FAD 分子 FAD-1，因为另一种 FAD-2 在纯化过程中丢失。通过将 FAD 添加到隔离 ETF 来恢复 FAD-2。 HoloETF 中的两个 FAD 分子使用 NADH 进行表征。用 NADH 对分离的 ETF 进行分光光度滴定显示，根据单相曲线，FAD-1 发生双电子还原，表明 FAD-1 从 NADH 接收电子，而没有 FAD-2 的参与。当holoETF用NADH滴定时，FAD-2被还原为阴离子半醌，然后在FAD-1被还原之前被完全还原。氧化的FAD-2还原为半醌、半醌还原为完全还原的FAD-2和FAD-1的双电子还原的中点电位值在pH 7时分别为+81、-136和-279 mV。 HoloETF 中的 FAD-1 和 FAD-2 都被过量的 NADH 迅速还原。通过用 8-氰基-FAD 替代 FAD-1，FAD-2 的还原速度减慢，表明 FAD-2 接收来自 FAD-1 的电子，但不直接接收来自 NADH 的电子。目前的结果表明，FAD-2 是人类 ETF 中 FAD 的对应物，其中包含一个 FAD 和一个 AMP。
Electron-transferring flavoprotein (ETF) from the anaerobic bacterium Megasphaera elsdenii is a heterodimer containing two FAD cofactors. Isolated ETF contains only one FAD molecule, FAD-1, because the other, FAD-2, is lost during purification. FAD-2 is recovered by adding FAD to the isolated ETF. The two FAD molecules in holoETF were characterized using NADH. Spectrophotometric titration of isolated ETF with NADH showed a two-electron reduction of FAD-1 according to a monophasic profile indicating that FAD-1 receives electrons from NADH without involvement of FAD-2. When holoETF was titrated with NADH, FAD-2 was reduced to an anionic semiquinone and then was fully reduced before the reduction of FAD-1. The midpoint potential values at pH 7 were +81, -136 and -279 mV for the reduction of oxidized FAD-2 to semiquinone, semiquinone to the fully reduced FAD-2 and the two-electron reduction of FAD-1, respectively. Both FAD-1 and FAD-2 in holoETF were reduced by excess NADH very rapidly. The reduction of FAD-2 was slowed by replacement of FAD-1 with 8-cyano-FAD indicating that FAD-2 receives electrons from FAD-1 but not from NADH directly. The present results suggest that FAD-2 is the counterpart of the FAD in human ETF, which contains one FAD and one AMP.