Respiratory complex III₂ assembles complex I via toxic intermediate in mitochondrial disease

Mutations in mitochondrial complex I can cause severe metabolic disease. Although no treatments are available for complex I deficiencies, chronic hypoxia improves lifespan and function in a mouse model of the severe mitochondrial disease Leigh syndrome caused by mutation of complex I subunit NDUFS4. To understand the molecular mechanism of NDUFS4 mutant pathophysiology and hypoxia rescue, we investigated the structure of complex I in respiratory supercomplexes isolated from NDUFS4 mutant mice. We identified complex I assembly intermediates bound to complex III₂, proving the cooperative assembly model. Further, an accumulated complex I intermediate is structurally consistent with pathological oxygen-dependent reverse electron transfer, revealing unanticipated pathophysiology and hypoxia rescue mechanisms. Thus, the build-up of toxic intermediates and not simply decreases in complex I levels underlie mitochondrial disease.