Uncovering the link between ATP synthase and the TCA cycle by crosslinking mass spectrometry

Mitochondrial oxidative phosphorylation (OXPHOS) comprises a series of multi-subunit protein complexes that operate in coordination with the tricarboxylic acid (TCA) cycle to generate ATP. Although these systems are metabolically interconnected, complex II is generally regarded as the only direct structural link between the OXPHOS and TCA cycle. Here, we combine in-solution crosslinking mass-spectrometry (XL-MS), quantitative proteomics, and blue native PAGE (BN-PAGE) to explore how ATP synthase (complex V) integrates within the mitochondrial metabolic network under physiological and pathological conditions. We demonstrate that in murine wild-type hearts, the F catalytic head of ATP synthase forms extensive contacts with TCA cycle enzymes, establishing a previously unanticipated link between the OXPHOS and central carbon metabolism. We also found that under mitochondrial dysfunction, in this case LRPPRC-deficient hearts, where defective mitochondrial DNA gene expression destabilizes ATP synthase, these interactions become strengthened. Moreover, ATP synthase dysfunction promotes binding of the ATPase inhibitory factor 1 (ATIF1) to the F head via its N-terminal inhibitory region, shifting the ATP synthase toward an energy-preserving state. Together, our findings show that ATP synthase deficiency drives remodeling of the F interactome, revealing how mitochondrial structure and regulation adapt to preserve energy homeostasis under stress.