Edited by Ruma Banerjee 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. Figure 2. Structures of photoreactive amilorides synthesized in this study. PRA1 and PRA2 used in our previous work (17) are also shown. A photolabile azido group attached to each compound is indicated by a gray circle. The concentration in parentheses is the average IC 50 value, which is the molar concentration (nanomolar) needed to reduce the control NADH oxidase activity in bovine heart SMPs (30 g of proteins/ml) by 50%. As a reference, the IC 50 value of bullatacin was 1.1 (Ϯ 0.09) nM under the same experimental conditions. Quinone/inhibitor-binding pocket in respiratory complex I

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.

Remodeling of the acyl chain compositions of cardiolipin (CL) species by the transacylase tafazzin is an important process for maintaining optimal mitochondrial functions. The results of mechanistic studies on the tafazzin-mediated transacylation from phosphatidylcholine (PC) to monolyso-CL (MLCL) in artificial lipid membranes are controversial. The present study investigated the role of the acyl chain composition of PC in the Saccharomyces cerevisiae tafazzin-mediated remodeling of CL by examining the structural factors responsible for the superior acyl donor ability of dipalmitoleoyl (16:1) PC over dipalmitoyl (16:0) PC. To this end, we synthesized systematic derivatives of dipalmitoleoyl PC; for example, the location of the cis double bond was migrated from the Δ9-position toward either end of the acyl chains (the Δ5- or Δ13-position), the cis double bond in the sn-1 or sn-2 position or both, was changed to a trans form, and palmitoleoyl and palmitoyl groups were exchanged in the sn-1 and sn-2 positions, maintaining similar PC fluidities. Analyses of the tafazzin-mediated transacylation from these PCs to sn-2'-MLCL(18:1-18:1/18:1-OH) in the liposomal membrane revealed that tafazzin strictly discriminates the molecular configuration of the acyl chains of PCs, including their glycerol positions (sn-1 or sn-2); however, the effects of PC fluidity on the reaction may not be neglected. On the basis of the findings described herein, we discuss the relevance of the so-called thermodynamic remodeling hypothesis that presumes no acyl selectivity of tafazzin.

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The ubiquinone reduction step in NADH-ubiquinone oxidoreductase (complex I) is the key to triggering proton translocation in its membrane part. Although the existence of a long and narrow quinoneaccess channel has been identified, it remains debatable whether the channel model can account for binding of various ligands (ubiquinones and inhibitors) to the enzyme. We previously proposed that the matrix-side interfacial region of the 49 kDa, ND1, PSST, and 39 kDa subunits, which is covered by a loop connecting transmembrane helices (TMHs) 1 and 2 of ND3, may be the area for entry of some bulky ligands into the quinone reaction cavity. However, this proposition lacks direct evidence that the cavity is accessible from the putative matrix-side region, which allows ligands to pass. To address this, we examined whether Cys 39 of ND3 and Asp 160 of 49 kDa can be specifically cross-linked by bifunctional cross-linkers (tetrazine-maleimide hybrid, named TMBC). On the basis of the structural models of complex I, such dual cross-linking is unexpected because ND3 Cys 39 and 49 kDa Asp 160 are located on the TMH1−2 loop and deep inside the channel, respectively, and hence, they are physically separated by peptide chains forming the channel wall. However, three TMBCs with different spacer lengths did cross-link the two residues, resulting in the formation of new cross-linked ND3/49 kDa subunits. Chemical modification of either ND3 Cys 39 or 49 kDa Asp 160 blocked the dual cross-linking, ensuring the specificity of the cross-linking. Altogether, this study provides direct evidence that the quinone reaction cavity is indeed accessible from the proposed matrix-side region covered by the ND3 TMH1−2 loop.