Mycobacterium tuberculosis
(
Mtb
), the causative agent of human tuberculosis, harbors a branched electron transport chain preventing the bactericidal action of cytochrome
bc
1
inhibitors (e.g. TB47). Here, we investigated, using luminescent mycobacterial strains, the
in vitro
combination activity of cytochrome
bc
1
inhibitors and nitric oxide (NO) donors including pretomanid (PMD) and explored the mechanisms of combination activity. The TB47 and PMD combination quickly abolished the light emission of luminescent bacilli, as was the case for the combination of TB47 and aurachin D, a putative cytochrome
bd
inhibitor. The TB47 and PMD combination inhibited
Mtb
oxygen consumption, decreased ATP levels, and had a delayed bactericidal effect. The NO scavenger carboxy-PTIO prevented the bactericidal activity of the drug combination, suggesting the requirement for NO. In addition, cytochrome
bc
1
inhibitors were largely bactericidal when administered with DETA NONOate, another NO donor. Proteomic analysis revealed that the cotreated bacilli had a compromised expression of the dormancy regulon proteins, PE/PPE proteins and proteins required for the biosynthesis of several cofactors, including mycofactocin. Some of these proteomic changes, e.g. the impaired dormancy regulon induction, were attributed to PMD. In conclusion, combination of cytochrome
bc
1
inhibitors with PMD inhibited
Mtb
respiration and killed the bacilli. The activity of cytochrome
bc
1
inhibitors can be greatly enhanced by NO donors. Monitoring of luminescence may be further exploited to screen cytochrome
bd
inhibitors.