The final step of aerobic respiration, oxidative phosphorylation, combines the activities of the electron transport chain and of ATP synthase. The electron transport chain is composed of membrane-bound energy transducers, which are organized in supramolecular assemblies known as respiratory supercomplexes. 

In this work we determined the cryo-EM structure of the obligate III₂IV₂ supercomplex from the Gram-positive bacterium Corynebacterium glutamicum. The structure shows that the individual complexes are intertwined and that the electron transfer between them occurs via a di-heme cc subunit instead of via soluble cytochrome c. The structure reveals additional features that distinguish the supercomplex from its canonical counterpart. These are a cytoplasmic QcrB loop that occludes the proton-entry point of the complex IV D-pathway, and an FeS cluster in a fixed position. These characteristics are conserved among actinobacteria. 

With the goal to elucidate the structure-function relationship for complexes III and IV in actinobacteria, we also investigated electron and proton transfer kinetics of an obligate respiratory supercomplex from Mycobacterium smegmatis, which is a model organism for Mycobacterium tuberculosis. The results show that the sequence of reactions involved in electron transfer in complex IV is similar to that observed in other A1-type oxidases, but the F to O transition of the catalytic cycle is slower than that reported for canonical complex IV. We also observed that reaction steps previously shown to display pH dependence in canonical complex IV were pH independent in Mycobacterium smegmatis. In addition, proton uptake kinetics through the D-pathway of complex IV were altered with no proton uptake during the F to O step. These findings can be attributed to the presence of the QcrB loop and point towards a possible unique regulatory mechanism for mycobacterial supercomplexes.

As the mycobacterial supercomplex is a promising drug target for tuberculosis treatment, we studied its interaction with the drug candidate Telacebec and the metabolite of an already approved drug, lansoprazole sulfide. We determined the cryo-EM structures of the III₂IV₂ supercomplex with Telacebec and with lansoprazole sulfide bound in the Q_P site of the QcrB subunit of complex III. In both structures the inhibitor replaces the natural substrate menaquinol in the inner position of the Q_P binding pocket and makes multiple interactions with the QcrA and QcrB subunits of complex III. Multiple turnover assays showed that this binding mode inhibits the supercomplex of Mycobacterium smegmatis. Results from our in silico studies show that lansoprazole sulfide is likely to bind to the supercomplex of Mycobacterium tuberculosis in a similar way as was observed for Mycobacterium smegmatis.