Abstract
NADH-quinone oxidoreductase (respiratory complex I) couples NADH-to-quinone electron transfer to the translocation of protons across the membrane. Even though the architecture of the quinone-access channel in the enzyme has been modeled by X-ray crystallography and cryo-EM, conflicting findings raise the question whether the models fully reflect physiologically relevant states present throughout the catalytic cycle. To gain further insights into the structural features of the binding pocket for quinone/inhibitor, we performed chemical biology experiments using bovine heart sub-mitochondrial particles. We synthesized ubiquinones that are oversized (SF-UQs) or lipid-like (PC-UQs) and are highly unlikely to enter and transit the predicted narrow channel. We found that SF-UQs and PC-UQs can be catalytically reduced by complex I, albeit only at moderate or low rates. Moreover, quinone-site inhibitors completely blocked the catalytic reduction and the membrane potential formation coupled to this reduction. Photoaffinity-labeling experiments revealed that amiloride-type inhibitors bind to the interfacial domain of multiple core subunits (49 kDa, ND1, and PSST) and the 39-kDa supernumerary subunit, although the latter does not make up the channel cavity in the current models. The binding of amilorides to the multiple target subunits was remarkably suppressed by other quinone-site inhibitors and SF-UQs. Taken together, the present results are difficult to reconcile with the current channel models. On the basis of comprehensive interpretations of the present results and of previous findings, we discuss the physiological relevance of these models.
Highlights
NADH– quinone oxidoreductase couples NADH-to-quinone electron transfer to the translocation of protons across the membrane
Because the substituted phenol moiety of SF–UQ1–SF–UQ3 and SF–UQ6 is weakly acidic due to the presence of strong electron-withdrawing substituents at the para position (i.e. ϪC'N and ϪCOOR) [21], ϳ25% of SF–UQs exists as anionic form in the reaction buffer, a negative charge is delocalized over the long-electron– conjugated system
Proteins equivalent to ϳ50 g of SMPs were loaded onto each well
Summary
NADH– quinone oxidoreductase (respiratory complex I) couples NADH-to-quinone electron transfer to the translocation of protons across the membrane. 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.” our studies raise the question of whether the channel models fully reflect physiologically relevant states present throughout the catalytic cycle In this context, it is important to note that the channel in the static state was postulated to undergo structural rearrangement to allow UQs to move into and out of the channel because the planar quinone head-ring is wider (ϳ6 Å across) than the diameter of the entry point [5, 11]. On the basis of the comprehensive interpretations of the present results and previous findings [15,16,17,18], we discussed the physiological relevance of the channel models
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