Cyazofamid-dessulfonamide (CCIM) is an anionic uncoupler of oxidative phosphorylation in mitochondria and a protonophore on lipid membranes

Cyazofamid is a commonly used fungicide degrading in plants and soil into various compounds, one of which is CCIM (4-chloro-5-p-tolylimidazole-2-carbonitrile) that lacks a sulfonamide moiety. Using NMR spectroscopy, we directly showed the conversion of cyazofamid into CCIM in DMSO/water solution, which allowed us to attribute the previously reported mitochondrial uncoupling action of cyazofamid to the formation of CCIM. In this study, CCIM increased the respiration rate and decreased the membrane potential of isolated rat liver mitochondria at submicromolar concentrations. It also depolarized mitochondrial membranes in HepG2 cells at micromolar concentrations. The ability of CCIM to electrogenically transport hydrogen ions across membranes was demonstrated here by the generation of transmembrane electric current. CCIM exhibited pH-dependent fluorescence with a pKa value of 5.7 in aqueous solution. Therefore, we concluded that CCIM uncouples oxidative phosphorylation of mitochondria via the protonophoric mechanism. The present data provide insight into the mechanism of CCIM's toxic action and extend the list of known protonophores that affect the mitochondrial function. © 2025 Elsevier B.V., All rights reserved.