Wang Kevin, Hirschenson Jonathan, Moore Amanda, Mailloux Ryan J
The School of Human Nutrition, Faculty of Agricultural and Environmental Sciences, McGill University, Sainte-Anne-de-Bellevue, Montreal, QC H9X 3V9, Canada.
Antioxidants (Basel). 2022 Oct 17;11(10):2043. doi: 10.3390/antiox11102043.
Mitochondrial complex I can produce large quantities of reactive oxygen species (ROS) by reverse electron transfer (RET) from the ubiquinone (UQ) pool. Glutathionylation of complex I does induce increased mitochondrial superoxide/hydrogen peroxide (O/HO) production, but the source of this ROS has not been identified. Here, we interrogated the glutathionylation of complex I subunit NDUFS1 and examined if its modification can result in increased ROS production during RET from the UQ pool. We also assessed glycerol-3-phosphate dehydrogenase (GPD) and proline dehydrogenase (PRODH) glutathionylation since both flavoproteins have measurable rates for ROS production as well. Induction of glutathionylation with disulfiram induced a significant increase in O/HO production during glycerol-3-phosphate (G3P) and proline (Pro) oxidation. Treatment of mitochondria with inhibitors for complex I (rotenone and S1QEL), complex III (myxothiazol and S3QEL), glycerol-3-phosphate dehydrogenase (iGP), and proline dehydrogenase (TFA) confirmed that the sites for this increase were complexes I and III, respectively. Treatment of liver mitochondria with disulfiram (50-1000 nM) did not induce GPD or PRODH glutathionylation, nor did it affect their activities, even though disulfiram dose-dependently increased the total number of protein glutathione mixed disulfides (PSSG). Immunocapture of complex I showed disulfiram incubations resulted in the modification of NDUFS1 subunit in complex I. Glutathionylation could be reversed by reducing agents, restoring the deglutathionylated state of NDUFS1 and the activity of the complex. Reduction of glutathionyl moieties in complex I also significantly decreased ROS production by RET from GPD and PRODH. Overall, these findings demonstrate that the modification of NDUFS1 can result in increased ROS production during RET from the UQ pool, which has implications for understanding the relationship between mitochondrial glutathionylation reactions and induction of oxidative distress in several pathologies.
线粒体复合物I可通过泛醌（UQ）池的逆向电子传递（RET）产生大量活性氧（ROS）。复合物I的谷胱甘肽化确实会导致线粒体超氧化物/过氧化氢（O₂⁻/H₂O₂）生成增加，但这种ROS的来源尚未确定。在这里，我们研究了复合物I亚基NDUFS1的谷胱甘肽化，并检查其修饰是否会导致UQ池RET过程中ROS生成增加。我们还评估了甘油-3-磷酸脱氢酶（GPD）和脯氨酸脱氢酶（PRODH）的谷胱甘肽化，因为这两种黄素蛋白也具有可测量的ROS生成速率。用双硫仑诱导谷胱甘肽化会导致甘油-3-磷酸（G3P）和脯氨酸（Pro）氧化过程中O₂⁻/H₂O₂生成显著增加。用复合物I抑制剂（鱼藤酮和S1QEL）、复合物III抑制剂（粘噻唑和S3QEL）、甘油-3-磷酸脱氢酶抑制剂（iGP）和脯氨酸脱氢酶抑制剂（TFA）处理线粒体证实，这种增加的位点分别是复合物I和复合物III。用双硫仑（50 - 1000 nM）处理肝线粒体不会诱导GPD或PRODH的谷胱甘肽化，也不会影响它们的活性，尽管双硫仑剂量依赖性地增加了蛋白质-谷胱甘肽混合二硫化物（PSSG）的总数。复合物I的免疫捕获显示，双硫仑孵育导致复合物I中NDUFS1亚基的修饰。谷胱甘肽化可以被还原剂逆转，恢复NDUFS1的去谷胱甘肽化状态和复合物的活性。复合物I中谷胱甘肽部分的还原也显著降低了GPD和PRODH通过RET产生的ROS。总体而言，这些发现表明，NDUFS1的修饰可导致UQ池RET过程中ROS生成增加，这对于理解线粒体谷胱甘肽化反应与几种病理状态下氧化应激诱导之间的关系具有重要意义。
