title: Amilorides bind to the quinone binding pocket of bovine mitochondrial complex I.
abstract: Amilorides, well-known inhibitors of Na(+)/H(+) antiporters, were previously shown to inhibit bacterial and mitochondrial NADH-quinone oxidoreductase (complex I) but were markedly less active for complex I. Because membrane subunits ND2, ND4, and ND5 of bovine complex I are homologous to Na(+)/H(+) antiporters, amilorides have been thought to bind to any or all of the antiporter-like subunits; however, there is currently no direct experimental evidence that supports this notion. To identify the binding site of amilorides in bovine complex I, we synthesized two photoreactive amilorides (PRA1 and PRA2), which have a photoreactive azido (-N3) group and terminal alkyne (-C≡CH) group at the opposite ends of the molecules, respectively, and conducted photoaffinity labeling with bovine heart submitochondrial particles. The terminal alkyne group allows various molecular tags to covalently attach to it via Cu(+)-catalyzed click chemistry, thereby allowing purification and/or detection of the labeled peptides. Proteomic analyses revealed that PRA1 and PRA2 label none of the antiporter-like subunits; they specifically label the accessory subunit B14.5a and core subunit 49 kDa (N-terminal region of Thr25-Glu115), respectively. Suppressive effects of ordinary inhibitors (bullatacin, fenpyroximate, and quinazoline), which bind to the putative quinone binding pocket, on labeling were fairly different between the B14.5a and 49 kDa subunits probably because the binding positions of the three inhibitors differ within the pocket. The results of this study clearly demonstrate that amilorides inhibit complex I activity by occupying the quinone binding pocket rather than directly blocking translocation of protons through the antiporter-like subunits (ND2, ND4, and ND5). The accessory subunit B14.5a may be located adjacent to the N-terminal region of the 49 kDa subunits. The structural features of the quinone binding pocket in bovine complex I were discussed on the basis of these results.
authors: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
authors: Masatoshi
authors: M
authors: Murai
authors: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
authors: Sonomi
authors: S
authors: Murakami
authors: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
authors: Takeshi
authors: T
authors: Ito
authors: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
authors: Hideto
authors: H
authors: Miyoshi
mesh_terms: D062646:Acid Sensing Ion Channel Blockers / Q000378:metabolism*; D000584:Amiloride / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D016227:Benzoquinones / Q000378:metabolism*; D001665:Binding Sites; D002417:Cattle; D042967:Electron Transport Complex I / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D019920:Photoaffinity Labels; D013367:Submitochondrial Particles / Q000378:metabolism
chemical_list: D062646:Acid Sensing Ion Channel Blockers; D016227:Benzoquinones; D019920:Photoaffinity Labels; C004532:quinone; D000584:Amiloride; D042967:Electron Transport Complex I