Remission spectroscopy resolves the mechanism of action of bedaquiline within living mycobacteria

Bedaquiline, an ATP synthase inhibitor, is the spearhead of transformative therapies against drug-resistant Mycobacterium tuberculosis. Here, we use remission spectroscopy to measure the energy-transducing cytochromes within unperturbed, respiring suspensions of mycobacterial and human cells, allowing spectroscopic measurements of electron transport chains as they power living cells and respond to bedaquiline. No evidence is found for protonophoric or ionophoric uncoupling. Rather, by directly inhibiting ATP synthase, bedaquiline slows the respiratory supercomplex (Qcr:Cta; bcc:aa 3 ) by increasing the proton-motive force, causing sub-second redirection of electron flux through the cytochrome bd oxidase (CydAB) to O 2 . Electron flux redirection explains the idiosyncratic bedaquiline-induced increase in O 2 consumption rates previously observed. Redirection occurs as CydAB is present even in cells grown in plentiful O 2 . Applying the same approach to human cells did not detect bedaquiline-induced inhibition of mitochondrial function despite such inhibition being seen in isolated systems. Overall, we clarify how bedaquiline works, why different models for its action developed, and the mechanisms underlying the synergy of bedaquiline in combination regimes.