Structural basis of a regulatory switch in mammalian complex I

Respiratory complex I powers oxidative phosphorylation in mammalian mitochondria by using the reducing potential of NADH to reduce ubiquinone-10 and drive protons across the inner mitochondrial membrane. High-resolution cryoEM structures have provided a molecular framework for complex I catalysis, but controversies about how to assign functional properties to the states identified in single-particle analyses are preventing progress on its energy-converting mechanism. Here, we combine precise biochemical definition with high-resolution cryoEM structures in the phospholipid bilayer of coupled vesicles and show that the closed and open states observed in mammalian complex I preparations are components of the deactive transition that occurs during ischaemia. Populations of the cryoEM open state and biochemical deactive state match exactly. Deactivation switches the enzyme off, converting the closed state that is capable of rapid, reversible catalysis into an open, dormant state that is unable to start up in reverse. The deactive state is switched back on by slow priming reactions with NADH and ubiquinone-10. Thus, by developing a versatile membrane system to unite structure and function, we define the role of large-scale conformational transitions in complex I and establish a new gold standard for structure-based investigations of catalysis by energy-coupled proteins.