Loss of mitochondrial fatty acid β‐oxidation protein short‐chain Enoyl‐CoA hydratase disrupts oxidative phosphorylation protein complex stability and function

Short‐chain enoyl‐CoA hydratase 1 (ECHS1) is involved in the second step of mitochondrial fatty acid β‐oxidation (FAO), catalysing the hydration of short‐chain enoyl‐CoA esters to short‐chain 3‐hyroxyl‐CoA esters. Genetic deficiency in ECHS1 (ECHS1D) is associated with a specific subset of Leigh Syndrome, a disease typically caused by defects in oxidative phosphorylation (OXPHOS). Here, we examined the molecular pathogenesis of ECHS1D using a CRISPR/Cas9 edited human cell ‘knockout’ model and fibroblasts from ECHS1D patients. Transcriptome analysis of ECHS1 ‘knockout’ cells showed reductions in key mitochondrial pathways, including the tricarboxylic acid cycle, receptor‐mediated mitophagy and nucleotide biosynthesis. Subsequent proteomic analyses confirmed these reductions and revealed additional defects in mitochondrial oxidoreductase activity and fatty acid β‐oxidation. Functional analysis of ECHS1 ‘knockout’ cells showed reduced mitochondrial oxygen consumption rates when metabolising glucose or OXPHOS complex I‐linked substrates, as well as decreased complex I and complex IV enzyme activities. ECHS1 ‘knockout’ cells also exhibited decreased OXPHOS protein complex steady‐state levels (complex I, complex III(2), complex IV, complex V and supercomplexes CIII(2)/CIV and CI/CIII(2)/CIV), which were associated with a defect in complex I assembly. Patient fibroblasts exhibit varied reduction of mature OXPHOS complex steady‐state levels, with defects detected in CIII(2), CIV, CV and the CI/CIII(2)/CIV supercomplex. Overall, these findings highlight the contribution of defective OXPHOS function, in particular complex I deficiency, to the molecular pathogenesis of ECHS1D.