Fenazaquin Acaricide Specific Binding Sites in NADH: Ubiquinone Oxidoreductase and Apparently the ATP Synthase Stalk

Abstract

Fenazaquin [4-(4-tert-butylphenethoxy)quinazoline] is one of several acaricides and insecticides, including rotenone and pyridaben, that are reported to act by inhibiting NADH:ubiquinone oxidoreductase (Complex I) (EC 1.6.99.3) in the range 1–10 nM. [3H]Fenazaquin is used here at 0.8 nMas a radioligand with electron transport particles (ETP) of bovine heart mitochondria to develop a new and rapid filtration assay with 42% specific binding (displaced by 2 μMrotenone). The potency of rotenoids, quinazolines, pyridaben, acetogenins, and several other disrupters of electron transport as inhibitors of NADH:ubiquinone oxidoreductase activity generally correlates with their effectiveness in displacing [3H]fenazaquin from its binding site in ETP (r2= 0.96,n= 18). This correlation extends to the fenazaquin analog with (trifluoromethyl)-diazirinyl replacing thetert-butyl substituent assayed at 6 nM,indicating that it also acts at a high-affinity site in Complex I. This diazirinyl analog of fenazaquin with tritium at theO-methylene position (4.7 Ci/mmol), on incubation at 435 nMwith ETP then irradiation at 350 nm, results in photoactivatable, irreversible specific binding which is fully protectable with rotenone. The site of labeling was examined by sequential isolation of the only band of specifically labeled protein(s) (which appeared at 22–24 kDa on SDS–PAGE),in situtryptic digestion of the protein(s) in the band, fractionation of the generated peptides by microbore HPLC, and amino acid sequence analysis. The single labeled HPLC peak of peptides in the tryptic digest was derived from two proteins, both components of ATP synthase, identified as subunit d and the oligomycin sensitivity conferral protein. An unusually low recovery of tryptophan at residue 12 of subunit d of specifically labeled ETP versus control ETP leads to the tentative proposal that labeling occurs at that site. Fenazaquin (and presumably rotenone as well) therefore has at least two specific binding sites in ETP, the anticipated high–affinity site in NADH:ubiquinone oxidoreductase and apparently a low-affinity site of unknown function, photoaffinity labeled in the stalk region of ATP synthase.