TMAO converts cytochrome c into a pro-apoptotic peroxidase by destabilizing the heme-Met80 ligation.

Trimethylamine N-oxide (TMAO), a gut microbiota-derived metabolite, has been linked to cardiovascular, renal, and hepatic disorders, but its direct impact on mitochondrial apoptotic machinery remains unclear. Here, we show that TMAO binds cytochrome c (Cyt c), disrupting its structural integrity and converting it into an apoptotically competent species. Spectroscopic analyses revealed that TMAO destabilizes the heme-Met80 axial ligation, shifting Cyt c from its native hexacoordinate to a pentacoordinate state. This conformational change enhances peroxidase activity, exposes hydrophobic clusters, and perturbs the Trp microenvironment, marking Cyt c's transition from electron carrier to pro-apoptotic catalyst. Absorption spectra further showed splitting of the native 530 nm band into peaks at 520 and 550 nm, consistent with heme reduction. These alterations facilitate Cyt c release from the mitochondrial membrane and engagement in intrinsic apoptosis. Given that TMAO accumulates at higher concentrations in tissues enriched with oxygen transporters, such as kidney and liver, our findings provide mechanistic insight into its role in organ-specific toxicity, including chronic kidney disease (CKD) and non-alcoholic fatty liver disease (NAFLD). This study establishes a direct molecular link between TMAO and mitochondrial apoptosis via Cyt c destabilization, suggesting that stabilizing Cyt c could represent a therapeutic strategy against TMAO-associated pathologies.