2-Azido-4-nitrophenol is a light-sensitive antibacterial protonophore

The azide-containing analogue 2-azido-4-nitrophenol (NPA) of the classical mitochondrial uncoupler 2,4-dinitrophenol (DNP) was actively used in 1970s as a tool for searching the binding sites of OxPhos uncouplers on mitochondrial proteins. Here, we report data on the protonophoric, uncoupling and cytotoxic activity of this compound. Artificial bilayer lipid membrane (BLM) experiments revealed much higher potency of NPA compared to DNP in the induction of proton-selective transmembrane electric current. Accordingly, NPA was more effective than DNP in both decreasing membrane potential and stimulating respiration of isolated rat liver mitochondria. NPA also exhibited higher depolarizing and cytotoxic activity towards mammalian cell culture, and was more potent than DNP in suppressing growth of both gram-negative (Escherichia coli) and gram-positive (Bacillus subtilis) bacteria. In all these systems, the activity of NPA but not that of DNP was abolished upon UV illumination. As shown by capillary electrophoresis and LC-MS analysis, UV light induced degradation of NPA to products lacking protonophoric activity, unlike DNP, which appeared to be light-insensitive. Therefore, NPA can be considered as a light-sensitive protonophore that is able to inhibit bacterial growth and promote mitophagy in mammalian cells.