Single‐Molecule Imaging of Active Mitochondrial Nitroreductases Using a Photo‐Crosslinking Fluorescent Sensor

Abstract Many biomacromolecules are known to cluster in microdomains with specific subcellular localization. In the case of enzymes, this clustering greatly defines their biological functions. Nitroreductases are enzymes capable of reducing nitro groups to amines, and play a role in detoxification and pro‐drug activation. Although nitroreductase activity has been detected in mammalian cells, the subcellular localization of this activity remains incompletely characterized. Here, we report a fluorescent probe that enables super‐resolved imaging of pools of nitroreductase activity within mitochondria. This probe is activated sequentially by nitroreductases and light to give a photo‐crosslinked adduct of active enzymes. In combination with a general photoactivatable marker of mitochondria, we performed two‐color, three‐dimensional, single‐molecule localization microscopy. These experiments allowed us to image the sub‐mitochondrial organization of microdomains of nitroreductase activity.