Department of Chemistry, Texas A&M University, College Station, Texas 77843-3255, United States.
Department of Biochemistry and Biophysics, Texas A&M University, College Station Texas 77843, United States.
J Am Chem Soc. 2023 Jun 28;145(25):13556-13569. doi: 10.1021/jacs.2c13439. Epub 2023 Jun 20.
Iron-sulfur cluster (ISC) assembly occurs in both mitochondria and cytosol. Mitochondria are thought to export a low-molecular-mass (LMM) iron and/or sulfur species which is used as a substrate for cytosolic ISC assembly. This species, called X-S or (Fe-S), has not been directly detected. Here, an assay was developed in which mitochondria were isolated from Fe-enriched cells and incubated in various buffers. Thereafter, mitochondria were separated from the supernatant, and both fractions were investigated by ICP-MS-detected size exclusion liquid chromatography. Aqueous Fe in the buffer declined upon exposure to intact Fe-enriched mitochondria. Some Fe was probably surface-absorbed but some was incorporated into mitochondrial iron-containing proteins when mitochondria were activated for ISC biosynthesis. When activated, mitochondria exported/released two LMM nonproteinaceous iron complexes. One species, which comigrated with an Fe-ATP complex, developed faster than the other Fe species, which also comigrated with phosphorus. Both were enriched in Fe and Fe, suggesting that the added Fe entered a pre-existing pool of Fe, which was also the source of the exported species. When Fe-loaded Fe-enriched mitochondria were mixed with isolated cytosol and activated, multiple cytosolic proteins became enriched with Fe. No incorporation was observed when Fe was added directly to the cytosol in the absence of mitochondria. This suggests that a different Fe source in mitochondria, the one enriched mainly with Fe, was used to export a species that was ultimately incorporated into cytosolic proteins. Iron from buffer was imported into mitochondria fastest, followed by mitochondrial ISC assembly, LMM iron export, and cytosolic ISC assembly.
铁硫簇（ISC）的组装发生在线粒体和细胞质中。人们认为线粒体将一种低分子量（LMM）的铁和/或硫物种输出，作为细胞质 ISC 组装的底物。这种物质称为 X-S 或（Fe-S），尚未被直接检测到。在这里，开发了一种测定方法，其中从富含铁的细胞中分离出线粒体，并在各种缓冲液中孵育。此后，将线粒体与上清液分离，并通过 ICP-MS 检测的尺寸排阻液相色谱法研究这两个部分。缓冲液中的水相铁在暴露于完整的富含铁的线粒体时下降。当线粒体被激活以进行 ISC 生物合成时，一些铁可能被表面吸附，但一些铁被整合到线粒体含铁蛋白中。当被激活时，线粒体输出/释放了两种 LMM 非蛋白铁复合物。一种与 Fe-ATP 复合物共迁移的物质比另一种也与磷共迁移的 Fe 物质更快地发展。这两种物质都富含 Fe 和 Fe，表明添加的 Fe 进入了一个预先存在的 Fe 池，这也是输出物质的来源。当负载 Fe 的富含 Fe 的线粒体与分离的细胞质混合并被激活时，多种细胞质蛋白富含 Fe。当 Fe 直接添加到没有线粒体的细胞质中时，没有观察到掺入。这表明线粒体中的一种不同的 Fe 源，主要富含 Fe，被用来输出一种最终被整合到细胞质蛋白中的物质。铁从缓冲液最快地被导入线粒体，其次是线粒体 ISC 组装、LMM 铁输出和细胞质 ISC 组装。
