Lipoylation is dependent on the ferredoxin FDX1 and dispensable under hypoxia in human cells

Iron sulfur clusters (ISC) are essential cofactors that participate in electron transfer, environment sensing, and catalysis. Amongst the most ancient ISC containing proteins are the ferredoxin family of electron carriers. Humans have two ferredoxins, FDX1 and FDX2, localized to the mitochondria and important for ISC synthesis itself. We previously showed that hypoxia can bypass the requirement for some, but not all, components of the ISC biosynthetic pathway, but ferredoxins were not tested at that time. Here we report that FDX1 and its reductase FDXR, but not FDX2, are dispensable under ambient 1% O2 in cultured cells. We find that FDX1 is essential for production of the lipoic acid cofactor, which is synthesized by the ISC containing enzyme lipoyl synthase (LIAS). While hypoxia can rescue the growth phenotype of either FDX1 or LIAS knockout cells, lipoylation is not rescued, arguing against an alternative biosynthetic route or salvage pathway for lipoate in hypoxia. Our work identifies a role for FDX1/LIAS in lipoate synthesis and surprisingly reveals dispensability of lipoic acid altogether under low oxygen tensions in cell culture.

铁硫簇（Iron sulfur clusters, ISC）是一类不可或缺的辅因子，参与电子传递、环境感应及催化反应。在含ISC的蛋白中，最为古老的一类是作为电子载体的铁氧还蛋白家族。人体中存在两种铁氧还蛋白：铁氧还蛋白1（FDX1）与铁氧还蛋白2（FDX2），二者定位于线粒体，且对ISC自身的合成具有关键作用。我们此前的研究证实，低氧环境可绕过ISC生物合成通路中部分（而非全部）组分的功能需求，但当时并未针对铁氧还蛋白开展相关实验验证。本研究发现，在体外培养细胞的1%常氧环境下，铁氧还蛋白1（FDX1）及其还原酶FDXR并非必需，而铁氧还蛋白2（FDX2）则无此特性。我们进一步揭示，铁氧还蛋白1（FDX1）是脂酰辅因子生成的必需因子，而该辅因子由含ISC的酶——脂酰合酶（lipoyl synthase, LIAS）催化合成。尽管低氧环境能够挽救铁氧还蛋白1（FDX1）或脂酰合酶（LIAS）敲除细胞的生长表型，但无法恢复其脂酰化修饰水平，这一结论排除了低氧环境下存在脂酸替代生物合成通路或补救途径的可能。本研究明确了铁氧还蛋白1（FDX1）/脂酰合酶（LIAS）在脂酸合成中的功能角色，并意外发现，在低氧张力的细胞培养体系中，脂酸完全不具有必需性。