Moxifloxacin-mediated killing of M. tuberculosis involves accumulation of NADH-dependent ROS, which is further enhanced by NAC. (A) Moxifloxacin enters M. tuberculosis and traps gyrase on DNA as reversible, bacteriostatic drug-enzyme-DNA complexes in which the DNA is broken. The bacterium responds by downregulating the expression of genes involved in respiration. The transcriptional changes result in reduced rate of respiration. NADH levels and the ratio of NADH to NAD+ increase. NADH increases the free Fe2+ pool by releasing Fe from ferritin-bound forms and keeps it in a reduced state. ROS damage macromolecules in a self-amplifying process, as indicated by exogenous catalase blocking killing when added after removal of moxifloxacin. (B) Addition of N-acetyl cysteine to cells stimulates respiration and provides more ROS from moxifloxacin-mediated lesions. NAC alone does not induce ROS or trigger death. The additional ROS increases killing by moxifloxacin. Repair of moxifloxacin-mediated lesions, NADH dissipation, Fe sequestration, and ROS detoxification mechanisms contribute to survival. The image was created using BioRender.com.