Barth syndrome (BTHS) is an X-linked disorder characterized by cardio- and skeletal myopathy, neutropenia, and mitochondrial dysfunction, which limit the quality of life for BTHS patients. Despite improvements in diagnosis, premature death is still commonplace due to the lack of effective therapies. Recently, clinical trials for two potential BTHS drug treatments, bezafibrate and elamipretide, were conducted in BTHS patients. Neither of these drugs showed a beneficial effect in their respective double-blind, placebo-controlled trials. These results underscore the central problem - the leading pathogenic mechanism of BTHS is unknown. The gene mutated in BTHS is TAFAZZIN, which codes for the transacylase tafazzin (Taz), which remodels the signature mitochondrial lipid, cardiolipin (CL), a unique lipid containing four acyl chains and localized almost exclusively in the matrix- facing leaflet of the inner mitochondrial membrane. Taz deficiency leads to decreased CL and accumulation of the remodeling intermediate, monolyso-cardiolipin (MLCL), However, the mechanism linking defective CL remodeling to the pathology in BTHS is not known. We established that both CL and MLCL can form a complex with the hemoprotein, cytochrome c (cyt c), localized in the intermembrane space (IMS) of mitochondria, and this complex acts as a peroxidase towards polyunsaturated (PUFA)-CL . Normally, low steady-state levels of MLCL and compartmentalization of CL and cyt c preclude the formation of this peroxidase complex. In Taz deficiency, robust accumulation of MLCL in the IMS-facing leaflet of the IMM, the site of the Taz-catalyzed reaction, makes it available to interact with cyt c. We further discovered that polyunsaturated MLCL, CL and other major phospholipids undergo robust peroxidation by the peroxidase complex. Given the well-documented membrane toxicity of lipid peroxidation products, we hypothesize that MLCL/cyt c complexes represent the primary pathogenic mechanism underlying BTHS, and suppression of this peroxidase activity and lipid peroxidation will protect mitochondria from BTHS-associated injury. We propose three mechanistic approaches and small molecule inhibitors to quench the peroxidase activity of MLCL/cyt c complexes as a new therapeutic strategy that will be tested in three Specific Aims: 1: Inhibiting MLCL/cyt c peroxidase catalytic activity. 2: Blocking complex formation by competitively inhibiting MLCL-cyt c binding. 3: Eliminating sources of H2O2 required for peroxidase activity. Our multi-PI team of experts in molecular genetics (M. Greenberg), redox biology (V. Kagan) and LC-MS based redox lipidomics and MS-imaging (H. Bayir) will provide the interdisciplinary scientific expertise required to implement the proposed experiments. Discovery of the MLCL/cyt c peroxidase complex as the primary mitochondrial mechanism of BTHS pathogenesis will lead to new medicines - clinically relevant inhibitors of pro-oxidant peroxidase activity.