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

Abstract

We previously demonstrated that amilorides bind to the quinone binding pocket of bovine mitochondrial complex I, not to the hitherto suspected Na⁺/H⁺ antiporter-like subunits (ND2, ND4, and ND5) [Murai, M., et al. (2015) Biochemistry 54, 2739-2746]. To characterize the binding position of amilorides within the pocket in more detail, we conducted specific chemical labeling [alkynylation (-C≡CH)] of complex I via ligand-directed tosyl (LDT) chemistry using a newly synthesized amide-type amiloride AAT as a LDT chemistry reagent. The inhibitory potency of AAT, in terms of its IC50 value, was markedly higher (∼1000-fold) than that of prototypical guanidine-type amilorides such as commercially available EIPA and benzamil. Detailed proteomic analyses in combination with click chemistry revealed that the chemical labeling occurred at Asp160 of the 49 kDa subunit (49 kDa Asp160). This labeling was significantly suppressed in the presence of an excess amount of other amilorides or ordinary inhibitors such as quinazoline and acetogenin. Taking into consideration the fact that 49 kDa Asp160 was also specifically labeled by LDT chemistry reagents derived from acetogenin [Masuya, T., et al. (2014) Biochemistry 53, 2307-2317, 7816-7823], we found this aspartic acid to elicit very strong nucleophilicity in the local protein environment. The structural features of the quinone binding pocket in bovine complex I are discussed on the basis of this finding.

A Self‐Assembled Respiratory Chain that Catalyzes NADH Oxidation by Ubiquinone‐10 Cycling between Complex I and the Alternative Oxidase

“…The mechanism of Q 10 reduction, an integral part of the unknown coupling mechanism, presents a much greater challenge owing to the extreme hydrophobicity of Q 10 . Previous studies have addressed the effects of inhibitors2 and mutations in and around the substrate binding site,3, 4 and made use of spectroscopy to search for ubiqsemiquinone intermediates 5. However, they have relied on either Q 10 in native membranes5, 6 (which contain many different enzymes, thus complicating spectroscopic and kinetic analyses) or on non‐physiological hydrophilic Q 10 analogues such as ubiquinone‐1, ubiquinone‐2 and decylubiquinone (DQ)4, 7 (which must be added in excessive concentrations to maintain steady‐state catalysis and that react adventitiously at the flavin to generate damaging reactive oxygen and semiquinone species8).…”

Exploring the quinone/inhibitor-binding pocket in mitochondrial respiratory complex I by chemical biology approaches

“…Based on the results of structure-activity relationship studies on amilorides (31,32), we newly synthesized four photoreactive 125 I-labeled amilorides ( Fig. 2 and Schemes S4 and S5).…”

“…The synthetic procedures for these amilorides are described in the supporting information. We named the derivatives possessing a photolabile azido group in the toxophoric pyrazinoyl ring ( I]PRA6 are amide-type amiloride derivatives that elicit more potent inhibitory activities than guanidine-type derivatives with bovine complex I (32). The inhibitory potencies determined in terms of IC 50 values using their cold derivatives are listed in the parentheses in Fig.…”

“…Membrane potential formation coupled with NADHquinone oxidoreduction was measured by following changes in the absorbance of oxonol VI (601-630 nm) with a Shimadzu UV-3000 instrument in the dual-wavelength mode in reaction medium (2.5 ml) containing 0.25 M sucrose, 1.0 mM MgCl 2 , 0.8 M antimycin A, 4.0 mM KCN, 2.5 mM oligomycin, 0.10 M nigericin, 1.0 M oxonol VI, and 50 mM phosphate buffer (pH 7.4) at 30°C (16,32). The final mitochondrial protein concentration was set to 90 g of proteins/ml.…”

A Self‐Assembled Respiratory Chain that Catalyzes NADH Oxidation by Ubiquinone‐10 Cycling between Complex I and the Alternative Oxidase

“…Mitochondrial complex I(NADH:ubiquinone oxidoreductase) is ac rucial energy-transducing respiratory enzyme.I t catalyzes NADH oxidation in the matrix and ubiquinone-10 (Q 10 )r eduction in the inner membrane by ap rocess coupled to energy-conserving proton transfer across the membrane. [1] Themechanism of NADH oxidation by the flavin-containing active site has been characterized in detail by using soluble electron acceptors.T he mechanism of Q 10 reduction, an integral part of the unknown coupling mechanism, presents am uch greater challenge owing to the extreme hydrophobicity of Q 10 .P revious studies have addressed the effects of inhibitors [2] and mutations in and around the substrate binding site, [3,4] and made use of spectroscopy to search for ubiqsemiquinone intermediates. [5] However, they have relied on either Q 10 in native membranes [5,6] (which contain many different enzymes,t hus complicating spectroscopic and kinetic analyses) or on non-physiological hydrophilic Q 10 analogues such as ubiquinone-1, ubiquinone-2 and decylubiquinone (DQ) [4,7] (which must be added in excessive concentrations to maintain steady-state catalysis and that react adventitiously at the flavin to generate damaging reactive oxygen and semiquinone species [8] ).…”