Cryo-EM structures define ubiquinone-10 binding to mitochondrial complex I and conformational transitions accompanying Q-site occupancy.

Abstract

Mitochondrial complex I is a central metabolic enzyme that uses the reducing potential of NADH to reduce ubiquinone-10 (Q₁₀) and drive four protons across the inner mitochondrial membrane, powering oxidative phosphorylation. Although many complex I structures are now available, the mechanisms of Q₁₀ reduction and energy transduction remain controversial. Here, we reconstitute mammalian complex I into phospholipid nanodiscs with exogenous Q₁₀. Using cryo-EM, we reveal a Q₁₀ molecule occupying the full length of the Q-binding site in the 'active' (ready-to-go) resting state together with a matching substrate-free structure, and apply molecular dynamics simulations to propose how the charge states of key residues influence the Q₁₀ binding pose. By comparing ligand-bound and ligand-free forms of the 'deactive' resting state (that require reactivating to catalyse), we begin to define how substrate binding restructures the deactive Q-binding site, providing insights into its physiological and mechanistic relevance.