Mechanisms driving the hypoxic rescue of ferredoxin and lipoate deficiency

Abstract

The introduction of environmental oxygen on earth opened new avenues of biochemistry that enabled rapid evolution of greater complexity across the tree of life. However, oxygen levels must be maintained carefully to prevent toxic reactions that can result from toxic radical accumulation and oxidation reactions of critical cofactors. Oxygen is the most utilized substrate in the human body, and the largest consumer of oxygen is the electron transport chain in the mitochondria. Thus, mitochondrial function and oxygen tensions are inextricably linked together, and much of the work done in studying cellular adaptations to varying oxygen tensions has uncovered critical mitochondrial adaptations that alter functional capacity to maintain critical energetic functions under varying oxygen tensions. Recent work from the lab has suggested that various defects in mitochondrial function may benefit from exposure to low ambient oxygen. These defects not only included lesions in the electron transport chain, but also in other mitochondrial pathways such as the synthesis of iron sulfur clusters. Whether these diverse models of mitochondrial dysfunction are rescued by shared or distinct mechanisms under hypoxia remains unknown. I initially began my thesis studies examining the mitochondrial ferredoxin FDX2 and its role in iron sulfur cluster (ISC) synthesis. Ferredoxins are iron sulfur cluster proteins that serve as single electron donors. FDX2 is known to contribute electrons to the mitochondrial iron sulfur cluster assembly complex. A screen done in C. elegans in the lab had identified FDX2 mutations as suppressing phenotypes caused by the loss of FXN, an allosteric regulator of the ISC machinery. FXN had previously been found to be dispensable under low oxygen tensions in human cells and C. elegans. We investigated these FDX2 mutants in human cells and surprisingly found that overexpression of FDX2 resulted in a suppression of ISC synthesis in cells with normal FXN expression in normoxic, but not hypoxic oxygen tensions. Further work from collaborators confirmed that FDX2 and FXN required a fixed stoichiometry for optimum ISC machinery activity. Studies from other labs’ revealed the structure of the ISC machinery and showed that FDX2 and FXN competed for the same binding site on the structure. These pieces of evidence helped clarify our findings, suggesting that FDX2 overexpression inhibited FXN binding and subsequent activity. Our findings may provide clues into the potential mechanisms of FXN dispensability in low oxygen tensions. I was next intrigued by the fact that human mitochondria possessed a second ferredoxin – FDX1 – which was also localized to the mitochondrial matrix and had 50% identity with FDX2. Both ferredoxins were reported to receive electrons from the same reductase, FDXR. However, despite the similarities, the two ferredoxins were always reported to deliver electrons to distinct pathways, with FDX1 previously found to function in sterol synthesis and FDX2 in iron sulfur cluster synthesis. Additionally, FDX1 scored as dispensable in our labs’ previously published low/high oxygen CRISPR screen, while FDX2 did not. I was curious about investigating the discrepancies between these two proteins. Using a combination of CRISPR knockout studies in low and high oxygen tensions and proteomics, I confirmed that FDX1, but not FDX2, was dispensable for human cell proliferation under low oxygen tensions. Using TMT Proteomics, I found that FDX1 and FDX2 knockouts had different proteomic profiles. Finally, using protein modeling by Alpha Fold, I found that FDX1, but not FDX2 was required for the synthesis of the mitochondrial cofactor lipoate. Intriguingly, lipoate levels were not restored in these knockouts under low oxygen, and we confirmed that the lipoate synthesis enzyme LIAS was also dispensable under low O2, leading us to conclude that the cofactor might in fact be dispensable for human cell proliferation under low oxygen, identifying yet another model of mitochondrial dysfunction that could be dispensable under low oxygen tensions. Finally, I sought to understand the mechanism behind hypoxic rescue of lipoate deficiency, and how this mechanism was similar or different to that driving rescue of ETC and OXPHOS disruptions. I found that the inhibition of multiple ETC complexes, OXPHOS (CV) and loss of LIAS all lead to fitness defects at 21% O2 that are alleviated at 1% O2 in HepG2 cells. HIF activation was sufficient for rescue of LIAS and Complex II inhibition, and partially sufficient for rescue of other ETC or OXPHOS defects. Hypoxia broadly remodeled our HepG2 model cell line, and LIAS KO cells were rescued through a combination of increased glycolytic flux, reductive carboxylation through pyruvate carboxylase (PC), and activation of carbonic anhydrase 9 (CA9) under hypoxia. However, neither PC nor CA9 are needed for rescue of ETC or OXPHOS under hypoxia. Thus, we were able to identify a specific mechanism for hypoxic rescue of a defect in mitochondrial metabolism and conclude that the broad remodeling under hypoxia allowed for a myriad of distinct mechanisms to concurrently exist, allowing rescue of diverse mitochondrial insults.Biological and Biomedical Science