We previously succeeded in site-specific chemical modifications of the inner part of the quinone binding pocket of bovine mitochondrial complex I through ligand-directed tosylate (LDT) chemistry using specific inhibitors as high-affinity ligands for the enzyme [Masuya, T., et al. (2014) Biochemistry 53, 2304-2317, 7816-7823]. To investigate whether a short-chain ubiquinone, in place of these specific inhibitors, serves as a ligand for LDT chemistry, we herein synthesized a LDT reagent QT possessing ubiquinone scaffold and performed LDT chemistry with bovine heart submitochondrial particles (SMP). Detailed proteomic analyses revealed that QT properly guides the tosylate group into the quinone binding pocket and transfers a terminal alkyne to nucleophilic amino acids His150 and Asp160 in the 49 kDa subunit. This result clearly indicates that QT occupies the inner part of the quinone binding pocket. Nevertheless, we noted that QT is a unique electron acceptor from complex I distinct from typical short-chain ubiquinones such as ubiquinone-1 (Q1) for several reasons; for example, QT reduction in NADH-QT oxidoreduction was almost completely insensitive to quinone-site inhibitors (such as bullatacin and piericidin A), and this reaction did not produce a membrane potential. On the basis of detailed comparisons of the electron transfer features between QT and typical short-chain quinones, we conclude that QT may accept electrons from an N2 cluster at a position different from that of typical short-chain quinones because of its unique side-chain structure; accordingly, QT reduction is unable to induce putative structural changes inside the quinone binding pocket, which are critical for driving proton translocation. Thus, QT is the first ubiquinone analogue, to the best of our knowledge, the catalytic reduction of which is decoupled from proton translocation through the membrane domain. Implications for mechanistic studies on QT are also discussed.