Corynebacterium glutamicum is a preferentially aerobic gram-positive bacterium belonging to the phylum Actinobacteria, which also includes the pathogen Mycobacterium tuberculosis. In these bacteria, respiratory complexes III and IV form a CIII₂CIV₂ supercomplex that catalyzes oxidation of menaquinol and reduction of dioxygen to water. We isolated the C. glutamicum supercomplex and used cryo-EM to determine its structure at 2.9 Å resolution. The structure shows a central CIII₂ dimer flanked by a CIV on two sides. A menaquinone is bound in each of the Q_N and Q_P sites in each CIII and an additional menaquinone is positioned ∼14 Å from heme b_L. A di-heme cyt. cc subunit electronically connects each CIII with an adjacent CIV, with the Rieske iron-sulfur protein positioned with the iron near heme b_L. Multiple subunits interact to form a convoluted sub-structure at the cytoplasmic side of the supercomplex, which defines a path for proton transfer into CIV.

The M. smegmatis respiratory III₂IV₂ supercomplex consists of a complex III (CIII) dimer flanked on each side by a complex IV (CIV) monomer, electronically connected by a di-heme cyt. cc subunit of CIII. The supercomplex displays a quinol oxidation‑oxygen reduction activity of ~90 e⁻/s. In the current work we have investigated the kinetics of electron and proton transfer upon reaction of the reduced supercomplex with molecular oxygen. The data show that, as with canonical CIV, oxidation of reduced CIV at pH 7 occurs in three resolved components with time constants ~30 μs, 100 μs and 4 ms, associated with the formation of the so-called peroxy (P), ferryl (F) and oxidized (O) intermediates, respectively. Electron transfer from cyt. cc to the primary electron acceptor of CIV, Cu_A, displays a time constant of ≤100 μs, while re-reduction of cyt. cc by heme b occurs with a time constant of ~4 ms. In contrast to canonical CIV, neither the P → F nor the F → O reactions are pH dependent, but the P → F reaction displays a H/D kinetic isotope effect of ~3. Proton uptake through the D pathway in CIV displays a single time constant of ~4 ms, i.e. a factor of ~40 slower than with canonical CIV. The slowed proton uptake kinetics and absence of pH dependence are attributed to binding of a loop from the QcrB subunit of CIII at the D proton pathway of CIV. Hence, the data suggest that function of CIV is modulated by way of supramolecular interactions with CIII.

The imidazopyridine telacebec, also known as Q203, is one of only a few new classes of compounds in more than 50 years with demonstrated antituberculosis activity in humans. Telacebec inhibits the mycobacterial respiratory supercomplex composed of complexes III and IV (CIII₂CIV₂). In mycobacterial electron transport chains, CIII₂CIV₂ replaces canonical CIII and CIV, transferring electrons from the intermediate carrier menaquinol to the final acceptor, molecular oxygen, while simultaneously transferring protons across the inner membrane to power ATP synthesis. We show that telacebec inhibits the menaquinol:oxygen oxidoreductase activity of purified Mycobacterium smegmatis CIII₂CIV₂ at concentrations similar to those needed to inhibit electron transfer in mycobacterial membranes and Mycobacterium tuberculosis growth in culture. We then used electron cryomicroscopy (cryoEM) to determine structures of CIII₂CIV₂ both in the presence and absence of telacebec. The structures suggest that telacebec prevents menaquinol oxidation by blocking two different menaquinol binding modes to prevent CIII₂CIV₂ activity.

Tuberculosis is one of the most common causes of death worldwide, with a rapid emergence of multi-drug-resistant strains underscoring the need for new antituberculosis drugs. Recent studies indicate that lansoprazole—a known gastric proton pump inhibitor and its intracellular metabolite, lansoprazole sulfide (LPZS)—are potential antituberculosis compounds. Yet, their inhibitory mechanism and site of action still remain unknown. Here, we combine biochemical, computational, and structural approaches to probe the interaction of LPZS with the respiratory chain supercomplex III₂IV₂ of Mycobacterium smegmatis, a close homolog of Mycobacterium tuberculosis supercomplex. We show that LPZS binds to the Q_o cavity of the mycobacterial supercomplex, inhibiting the quinol substrate oxidation process and the activity of the enzyme. We solve high-resolution (2.6 Å) cryo-electron microscopy (cryo-EM) structures of the supercomplex with bound LPZS that together with microsecond molecular dynamics simulations, directed mutagenesis, and functional assays reveal key interactions that stabilize the inhibitor, but also how mutations can lead to the emergence of drug resistance. Our combined findings reveal an inhibitory mechanism of LPZS and provide a structural basis for drug development against tuberculosis.