Pathogenesis of mtDNA point mutation m.10191T>C affecting complex I function is a multifactorial process leading to metabolic remodeling of mitochondria

Inherited mitochondrial disorders are of multiple genetic origins and may lead to a broad range of frequently severe disease phenotypes. Yet, how molecular causes ultimately present as a clinical phenotype is poorly understood. To address this conundrum starting from the molecular defect, we thoroughly investigated the consequences of the well-known pathogenic mitochondrial DNA mutation m.10191T>C. The mutation changes serine-45 in subunit ND3 of respiratory chain complex I to proline and causes Leigh syndrome, which is one of the most devastating mitochondrial diseases. Human mitochondria carrying the mutation ND3^S45P retained 30-40 % of complex I activity and oxidative phosphorylation capacity. In stark contrast, intact mutant cells exhibited only minimal oxygen consumption and a massively increased NADH/NAD⁺ ratio. Since the energy barrier for the Active/Deactive transition of complex I was reduced by ∼20 kJ∙mol^-1 in mutant cells, we concluded that complex I was shut-off by malfunctioning of an as yet unknown regulatory pathway. Comprehensive analysis of the mitochondrial complexome of cybrids, patient fibroblasts and muscle biopsies rendered other causes for the accumulation of NADH unlikely. The complexome datasets provide a rich resource for further studies to discover possible additional factors involved in regulating complex I. We propose that the derailed regulation of complex I is the main culprit leading to NADH accumulation and eventually the severity of the disease phenotype caused by mutation ND3^S45P.