Department of Biological Sciences, Louisiana State University, Baton Rouge, LA, 70803, USA.
Department of Biological Sciences, Louisiana State University, Baton Rouge, LA, 70803, USA.
Free Radic Biol Med. 2020 Aug 20;156:11-19. doi: 10.1016/j.freeradbiomed.2020.05.004. Epub 2020 May 20.
MitoNEET is a mitochondrial outer membrane protein that hosts a redox active [2Fe-2S] cluster in the C-terminal cytosolic domain. Increasing evidence has shown that mitoNEET has an essential role in regulating energy metabolism in human cells. Previously, we reported that the [2Fe-2S] clusters in mitoNEET can be reduced by the reduced flavin mononucleotide (FMNH) and oxidized by oxygen or ubiquinone-2, suggesting that mitoNEET may act as a novel redox enzyme catalyzing electron transfer from FMNH to oxygen or ubiquinone. Here, we explore the FMN binding site in mitoNEET by using FMN analogs and find that lumiflavin, like FMN, at nanomolar concentrations can mediate the redox transition of the mitoNEET [2Fe-2S] clusters in the presence of flavin reductase and NADH (100 μM) under aerobic conditions. The electron paramagnetic resonance (EPR) measurements show that both FMN and lumiflavin can dramatically change the EPR spectrum of the reduced mitoNEET [2Fe-2S] clusters and form a covalently bound complex with mitoNEET under blue light exposure, suggesting that FMN/lumiflavin has specific interactions with the [2Fe-2S] clusters in mitoNEET. In contrast, lumichrome, another FMN analog, fails to mediate the redox transition of the mitoNEET [2Fe-2S] clusters and has no effect on the EPR spectrum of the reduced mitoNEET [2Fe-2S] clusters under blue light exposure. Instead, lumichrome can effectively inhibit the FMNH-mediated reduction of the mitoNEET [2Fe-2S] clusters, indicating that lumichrome may act as a potential inhibitor to block the electron transfer activity of mitoNEET.
MitoNEET 是一种线粒体外膜蛋白，在其 C 端胞质域含有一个具有氧化还原活性的 [2Fe-2S] 簇。越来越多的证据表明，MitoNEET 在调节人类细胞的能量代谢中起着至关重要的作用。之前，我们报道过 MitoNEET 中的 [2Fe-2S] 簇可以被还原型黄素单核苷酸 (FMNH) 还原，被氧或泛醌-2 氧化，这表明 MitoNEET 可能作为一种新型的氧化还原酶，催化 FMNH 向氧或泛醌的电子转移。在这里，我们通过使用 FMN 类似物来探索 MitoNEET 中的 FMN 结合位点，发现类似于 FMN 的光黄素在纳米摩尔浓度下，可以在黄素还原酶和 NADH（100 μM）存在下，在有氧条件下介导 MitoNEET [2Fe-2S] 簇的氧化还原转换。电子顺磁共振（EPR）测量表明，FMN 和光黄素都可以显著改变还原态 MitoNEET [2Fe-2S] 簇的 EPR 谱，并在蓝光照射下与 MitoNEET 形成共价结合复合物，表明 FMN/光黄素与 MitoNEET 中的 [2Fe-2S] 簇具有特异性相互作用。相比之下，另一种 FMN 类似物光色烯不能介导 MitoNEET [2Fe-2S] 簇的氧化还原转换，并且在蓝光照射下对还原态 MitoNEET [2Fe-2S] 簇的 EPR 谱没有影响。相反，光色烯可以有效地抑制 FMNH 介导的 MitoNEET [2Fe-2S] 簇的还原，表明光色烯可能作为一种潜在的抑制剂来阻断 MitoNEET 的电子转移活性。
