Visualization of an Endogenous Mitochondrial Azoreductase Activity under Normoxic Conditions Using a Naphthalimide Azo-Based Fluorogenic Probe

• Published in: Analytical chemistry (Washington) 2024-01, Vol.96 (4), p.1774-1780
• Main Authors: Michel, Laurane, Auvray, Marie, Askenatzis, Laurie, Badet-Denisot, Marie-Ange, Bignon, Jérôme, Durand, Philippe, Mahuteau-Betzer, Florence, Chevalier, Arnaud
• Format: Article
• Language: English
• Subjects: Azo compounds; Azoreductase; Biochemistry; Biochemistry, Molecular Biology; Cell lines; Chemical bonds; Chemical compounds; Fluorophores; Life Sciences; Mitochondria; Oxygenation; Reducing agents; Reductases
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.3c05030

In this paper, we demonstrate the existence of an endogenous mitochondrial azoreductase (AzoR) activity that can induce the cleavage of NN double bonds of azobenzene compounds under normoxic conditions. To this end, 100% OFF–ON azo-based fluorogenic probes derived from 4-amino-1,8-naphthalimide fluorophores were synthesized and evaluated. The in vitro study conducted with other endogenous reducing agents of the cell, including reductases, demonstrated both the efficacy and the selectivity of the probe for AzoR. Confocal experiments with the probe revealed an AzoR activity in the mitochondria of living cells under normal oxygenation conditions, and we were able to demonstrate that this endogenous AzoR activity appears to be expressed at different levels across different cell lines. This discovery provides crucial information for our understanding of the biochemical processes occurring within the mitochondria. It thus contributes to a better understanding of its function, which is implicated in numerous pathologies.