Structure of the Deactive State of Mammalian Respiratory Complex I

Abstract

SummaryComplex I (NADH:ubiquinone oxidoreductase) is central to energy metabolism in mammalian mitochondria. It couples NADH oxidation by ubiquinone to proton transport across the energy-conserving inner membrane, catalyzing respiration and driving ATP synthesis. In the absence of substrates, active complex I gradually enters a pronounced resting or deactive state. The active-deactive transition occurs during ischemia and is crucial for controlling how respiration recovers upon reperfusion. Here, we set a highly active preparation of Bos taurus complex I into the biochemically defined deactive state, and used single-particle electron cryomicroscopy to determine its structure to 4.1 Å resolution. We show that the deactive state arises when critical structural elements that form the ubiquinone-binding site become disordered, and we propose reactivation is induced when substrate binding to the NADH-reduced enzyme templates their reordering. Our structure both rationalizes biochemical data on the deactive state and offers new insights into its physiological and cellular roles.

Exploring the quinone/inhibitor-binding pocket in mitochondrial respiratory complex I by chemical biology approaches

“…7C). The tryptic digests of this protein contained the sequence corre-sponding to the 39-kDa supernumerary subunit (Table S1), which is positioned adjacent to the 49-kDa, PSST, and ND1 core subunits (7)(8)(9)(10)(11).…”

“…The findings of chemical biology studies previously conducted in our laboratory (15)(16)(17)(18) via different techniques using bovine heart SMPs are difficult to be reconciled with the quinone/inhibitor-access channel models (5)(6)(7)(8)(9)(10)(11), as summarized under the "Discussion." Therefore, our studies raise the question of whether the channel models fully reflect physiologically relevant states present throughout the catalytic cycle.…”

“…The X-ray crystallographic structures of complex I from Thermus thermophilus (5) and Yarrowia lipolytica (6) were modeled at resolutions of 3.3 and 3.6 Å, respectively. The entire structures of mammalian complex I, including all 45 subunits (31 of which are the supernumerary subunits), from bovine (Bos taurus) (7,8), ovine (Ovis aries) (9), porcine (Sus scrofa domesticus) (10), and mouse (Mus musculus) (11) hearts and human (Homo sapiens) HEK293F cells (12) were modeled by single-particle cryoelectron microscopy (cryo-EM). One of the striking findings obtained with T. thermophilus complex I (5) is the identification of a long and narrow channel, which extends from the membrane interior to the Fe-S cluster N2 (ϳ30 Å long) and is a completely enclosed tunnel with only a narrow entry point (ϳ3 ϫ 5 Å diameter) for quinone/inhibitors; however, this has not yet been confirmed experimentally.…”

“…From this, the yeast and ovine enzymes were supposed to be in the deactive state. Hirst and co-workers (8,11) recently reported that the structural changes accompanying deactivation may be common to the bovine and mouse enzymes. Considering the unusually long substrate-binding channel, definitions of how UQs of varying isoprenyl chain length (UQ 1 -UQ 10 ) enter and transit the channel to be reduced, thereby eliciting the same proton-pumping stoichiometry, remain elusive (13,14).…”

“…According to the current channel models (5)(6)(7)(8)(9), complex I only catalyzes the reduction of UQs that enter and transit the narrow channel to the reaction site near the Fe-S cluster N2 located at the "top" of the channel. Given this convincing experiment to verify the physiological relevance of the models may prove that the enzyme is indeed unable to catalyze the reduction of UQs, which are incapable of reaching the reaction site for some physical reason.…”