Investigating the relationship between ATP synthase and the TCA cycle by crosslinking mass spectrometry

Abstract Mitochondrial oxidative phosphorylation (OXPHOS) comprises 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 OXPHOS and TCA cycle. Here, we combine in-solution crosslinking mass-spectrometry (XL-MS), quantitative proteomics, complexome profiling and blue native PAGE (BN-PAGE) to explore how ATP synthase (complex V) is positioned within the mitochondrial metabolic network under physiological and pathological conditions. We demonstrate that in murine wild-type hearts, the F₁ catalytic head of ATP synthase forms extensive contacts with TCA cycle enzymes, establishing a previously unanticipated spatial link between OXPHOS and central carbon metabolism. We further report that loss of the mitochondrial RNA-stabilizing protein LRPPRC, which disrupts mtDNA gene expression in the mouse heart, results in ATP synthase destabilization and enhanced F 1 -TCA cycle interactions. Moreover, ATP synthase dysfunction promotes binding of the ATPase inhibitory factor 1 (ATIF1) to the F₁ head via its N-terminal inhibitory region, shifting the ATP synthase toward an energy-preserving state. Together, our findings show that impaired mitochondrial gene expression leads to secondary ATP synthase remodeling and reshaping of its interaction landscape, revealing how mitochondria may adapt to bioenergetic stress.