Oversized ubiquinones as molecular probes for structural dynamics of the ubiquinone reaction site in mitochondrial respiratory complex I

Abstract

NADH-quinone oxidoreductase (complex I) couples electron transfer from NADH to quinone with proton translocation across the membrane. Quinone reduction is a key step for energy transmission from the site of quinone reduction to the remotely located proton-pumping machinery of the enzyme. Although structural biology studies have proposed the existence of a long and narrow quinone-access channel, the physiological relevance of this channel remains debatable. We investigated here whether complex I in bovine heart submitochondrial particles (SMPs) can catalytically reduce a series of oversized ubiquinones (OS-UQs), which are highly unlikely to transit the narrow channel because their side chain includes a bulky “block” that is ∼13 Å across. We found that some OS-UQs function as efficient electron acceptors from complex I, accepting electrons with an efficiency comparable with ubiquinone-2. The catalytic reduction and proton translocation coupled with this reduction were completely inhibited by different quinone-site inhibitors, indicating that the reduction of OS-UQs takes place at the physiological reaction site for ubiquinone. Notably, the proton-translocating efficiencies of OS-UQs significantly varied depending on their side-chain structures, suggesting that the reaction characteristics of OS-UQs affect the predicted structural changes of the quinone reaction site required for triggering proton translocation. These results are difficult to reconcile with the current channel model; rather, the access path for ubiquinone may be open to allow OS-UQs to access the reaction site. Nevertheless, contrary to the observations in SMPs, OS-UQs were not catalytically reduced by isolated complex I reconstituted into liposomes. We discuss possible reasons for these contradictory results.

IACS-010759, a potent inhibitor of glycolysis-deficient hypoxic tumor cells, inhibits mitochondrial respiratory complex I through a unique mechanism

“…Taken together, the action mechanisms of complex I inhibitors with widely different chemical frameworks are far more diverse than hitherto considered based on the channel model (13,40,47,48). We recently demonstrated that complex I in bovine SMPs can catalytically reduce oversized ubiquinones, which are highly unlikely to transit the narrow channel because their side chain includes a significantly bulky block (30,49). This fact may question whether the current channel model fully reflects the physiologically relevant states.…”

Cryo-electron microscopy reveals how acetogenins inhibit mitochondrial respiratory complex I

“…Ubiquinone binding to complex I has also been explored using synthesized ubiquinone derivatives of varying lengths, with bulky groups (1-methoxy-2,6-di(3-methoxy-3-methyl-1-butynyl)benzene) added to the end of their tails—known as ‘oversized ubiquinones’ (OS-UQs). The bulky group is too large to pass through the entrance to the ubiquinone-binding channel from the membrane, and if the linking tail is not long enough, the ubiquinone headgroup is unable to reach its catalytic site at the end of the channel ( 51 , 52 ). Bovine complex I reconstituted into proteoliposomes was only able to catalyze the inhibitor-sensitive reduction of OS-UQ8, which has a tail length equivalent to eight isoprenoid units, predicted by the structure to be just long enough.…”

Ubiquinone Binding and Reduction by Complex I—Open Questions and Mechanistic Implications

“…Taken together, the evidence for a single narrow Q access pathway in complex I seems to be compelling. However, it should be noted that Uno et al (2020) showed inhibitor-sensitive reduction of Q analogs which are too bulky to enter the Q tunnel. It is currently unclear how these results can be reconciled with the structural data and more work is needed to resolve this issue.…”