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.

Charge translocation by mitochondrial NADH:ubiquinone oxidoreductase (complex I) from Yarrowia lipolytica measured on solid-supported membranes

“…These data suggest a binding of amilorides to the Q-module rather than to one of the membrane integral subunits [44]. In line with these investigations, we observed that EIPA inhibited the NADH:ubiquinone oxidoreductase activity of solubilized lipid-activated Yarrowia complex I with an I 50 of 23 µM (results not shown) with some residual activity present at 100 µM.…”

“…Indeed, uncoupling activity of EIPA and other amilorides has been observed before [42,43]. Recently, photoreactive amilorides were synthesized to locate their binding site(s) within bovine heart complex I [44]. None of the Na + /H + -antiporter-like subunits (ND2, ND4 and ND5) was labelled, but a specific interaction with the accessory subunit B14.5a and the central 49 kDa subunit was observed.…”

Identification of the Binding Position of Amilorides in the Quinone Binding Pocket of Mitochondrial Complex I

“…It should be, however, realized that the photoaffinity labeling studies previously showed that amiloride and acetogenin occupy somewhat different positions inside the quinone binding pocket of bovine complex I (12,(32)(33)(34). These findings lead to the question of why 49 kDa Asp160 elicit such a strong nucleophilicity against the tosylate group of different types of LDT reagents.…”

“…Since the previous findings strongly suggested that the binding position of amilorides within the quinone binding pocket is considerably different from that of ordinary inhibitors (12), there is the possibility that our synthetic amilorides may inhibit the quinone redox reaction and proton translocation to different extents (the so-called decoupling effect). The decoupling effect was claimed by Batista et al for the action of EIPA against complex I in Rhodothermus marinus membrane vesicles (26).…”

“…Since the subunits ND2, ND4, and ND5 (in bovine complex I) in the membrane domain are homologous to subunits of the multidrug resistance protein (Mrp) family Na + /H + antiporters (11), amilorides are considered to block complex I activity by binding to any or all of the antiporter-like subunits. However, we recently revealed through photoaffinity labeling experiments that amilorides inhibit complex I by occupying the quinone binding pocket rather than directly blocking proton translocation through the antiporter-like subunits (ND2, ND4, and ND5) (12). We also showed that a photoreactive amiloride (PRA1) possessing a photolabile group in the alkyl side chain moiety labeled to the accessory subunit B14.5a, indicating that the binding position of amilorides within the pocket is somewhat different form that of traditional inhibitors.…”