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.

Tunnel dynamics of quinone derivatives and its coupling to protein conformational rearrangements in respiratory complex I

“…2). This position of Q has previously been observed in bacterial and mammalian complex I structures (Chung et al, 2022;Gu et al, 2022;Gutiérrez-Fernández et al, 2020), but not in Y. lipolytica complex I structural data. Here, our MD simulations show that oxidized Q (Qox) can indeed bind closer to the N2 FeS cluster also in Yarrowia complex I, which may enhance efficiency of electron transfer from N2 to Q (Moser, Farid, Chobot, & Dutton, 2006).…”

“…At these sites Q is expected to be reduced by electron transfer(s) from N2. Both sites have been confirmed by structural data (Chung et al, 2022;Gu et al, 2022;Gutiérrez-Fernández et al, 2020;Kampjut & Sazanov, 2020;Parey et al, 2021) as well as MD simulations (Haapanen et al, 2019;Warnau et al, 2018).…”

“…Computational studies have indicated the role of electrostatics and conformational dynamics, protein hydration and Q binding in the long-range electron-proton coupling in complex I (Galemou Yoga, Schiller, & Zickermann, 2021; Haapanen, Reidelbach, & Sharma, 2020; Haapanen & Sharma, 2021). Recent high resolution structural data from cryo electron microscopy (Chung et al, 2022; Grba & Hirst, 2020; Gu, Liu, Guo, Zhang, & Yang, 2022; Kampjut & Sazanov, 2020; Parey et al, 2018; Parey et al, 2019; Parey et al, 2021; Yoga, Parey, et al, 2020) have provided new insights into the role of Q binding, loop dynamics and water molecules in proton pumping by complex I.…”

Structural Basis of Mammalian Respiratory Complex I Inhibition by Medicinal Biguanides

“…S2), but which exhibited such a high degree of heterogeneity that it was not possible to model parts of the map. Subsequent global classification yielded three major classes resembling the active and deactive states mentioned above and a state called slack (see Supplementary Text), which is of unknown functional relevance but has been described previously in cryo-EM studies of bovine complex I ( 22, 24, 25 ). Inhibitor-free reference maps of bovine complex I from separate inhibitor-free preparations in detergent (Fig.…”

“…4 & S23). The pocket is occupied by the N-terminal 7 residues of NDUFC2 in the inhibitor-free enzyme, both here in n-Dodecyl-B-D-maltoside (DDM) and in all states of the bovine enzyme in nanodiscs ( 22 ). The NDUFC2 N-terminus is displaced by the biguanide, with inhibitor densities observed in most classes here (Fig S23).…”