Tuberculosis (TB), a leading cause of global mortality from infectious diseases, poses a significant public health challenge. The emergence of drug-resistant strains necessitates the development of novel, fast-acting drugs. Bedaquiline, approved in 2012, was the first new drug in 40 years to target a novel mechanism in TB, opening avenues for exploring the mycobacterial respiratory chain. Simultaneously targeting multiple respiratory enzyme complexes in *Mycobacterium tuberculosis* is considered an effective strategy to shorten treatment and reduce drug resistance. The cytochrome *bd* oxidase, a quinol-oxidizing terminal oxygen reductase crucial for mycobacterial survival under low oxygen conditions, has emerged as a promising target. Deletion mutants show increased susceptibility to inhibitors, highlighting its clinical relevance. Previous structural insights into cytochrome *bd* oxidases were limited to structures from *Geobacillus thermodenitrificans* and *Escherichia coli*, which, while sharing core architecture, differ in structural details. This study aims to determine the high-resolution structure of the *M. tuberculosis* cytochrome *bd* oxidase to provide a framework for structure-guided drug design.

Previous structural studies of cytochrome *bd* oxidase have focused on the enzymes from *Geobacillus thermodenitrificans* (G. th) and *Escherichia coli* (E. coli). The G. th structure, at 3.8 Å resolution, and the E. coli structures, at 2.7-3.3 Å resolution, revealed a similar core architecture. However, significant differences exist in side-chain details, prosthetic group arrangement, and accessory subunits. These variations emphasize the need for a detailed understanding of the *M. tuberculosis* enzyme for effective drug design. Studies have also demonstrated the importance of cytochrome *bd* oxidase in mycobacterial pathophysiology, with deletion mutants showing increased susceptibility to inhibitors. This synergistic lethal interaction between respiratory branches underscores the importance of this enzyme as a drug target for combating drug-resistant TB.

The cytochrome *bd* oxidase from *M. tuberculosis* (cyt. *bd_Mtb*) was recombinantly produced in a *Mycobacterium smegmatis* strain lacking its native cytochrome *bd* oxidase. Cofactor assembly and activity were confirmed spectroscopically and by oxygen consumption measurements. The 3D structure was determined using single-particle electron cryo-microscopy (cryo-EM), reaching a resolution of 2.5 Å. Atomistic molecular dynamics (MD) simulations were performed in a POPC lipid environment to assess solvent accessibility and dynamics. Cryo-EM structures were also determined in the presence of Aurachin D and AD3-11 inhibitors to investigate inhibitor binding. Multiple sequence alignments and phylogenetic analyses were conducted using 561 CydA sequences to assess the conservation of identified structural features. The purification process involved several steps: cell disruption using a high-pressure homogenizer, centrifugation to separate membranes, solubilization with dodecyl-β-D-maltoside (DDM), FLAG tag affinity chromatography, and size exclusion chromatography (SEC). For cryo-EM, the purified *bd* oxidase was reconstituted into lipid nanodiscs. Grids were prepared using a Vitrobot IV device and imaged using a Titan Krios G3i microscope. Image processing involved motion correction, CTF determination, particle picking using crYOLO, and 3D classification and refinement using RELION. Model building and refinement were performed using Coot and Phenix. Analysis of tunnels and cavities used MOLE 2.5. Structural alignments employed the DALI server. MD simulations used CHARMM-GUI, CHARMM36m force field, and GROMACS 2019.6. Menaquinone-9 identification was done via high-resolution mass spectrometry. UV-Vis absorption spectroscopy was used to analyze heme cofactors. Oxygen consumption measurements employed an Oroboros O2k respirometer.

The cryo-EM structure reveals a pseudo-symmetrical heterodimer of CydA and CydB subunits, each with nine transmembrane helices. The CydA subunit harbors the three heme groups (b₅₅₈, b₅₉₅, and d) and the quinol binding and oxidation domain (Q-loop). Unlike proteobacterial *bd* oxidases, the mycobacterial enzyme lacks a CydX subunit homolog, yet retains full cofactor assembly and function. MD simulations identified a major water-filled channel for proton delivery to the oxygen reduction site. The Q-loop architecture is unique, exhibiting a disulfide bond between Cys^266.A and Cys^285.A, which rigidifies the Q-loop and prevents substrate quinol binding at the canonical site. A novel MK-9 binding site near heme b₅₉₅ was identified. MD simulations suggest that MK-9, in both oxidized and reduced states, interacts with Trp^9.A, and that oxidized MK-9 can form H-bonds with Arg^8.A and Trp^9.A. Phylogenetic analysis shows that the disulfide bond and the MK-9 binding site are conserved among Actinobacteria. The MK-9 binding site may serve as an alternative quinol oxidation site, bypassing the inactive Q-loop.

The unique structural features of the *M. tuberculosis* cytochrome *bd* oxidase, specifically the disulfide bond in the Q-loop and the MK-9 binding site, offer new avenues for drug development. The disulfide bond-mediated rigidity of the Q-loop likely inhibits the canonical quinol oxidation pathway. The MK-9 binding site near heme b₅₉₅ suggests a possible alternative electron transfer pathway. Inhibitors could potentially target the Qc-PL8 interaction, the oxygen-conducting channel, or the MK-9 binding site. Further research is needed to elucidate the detailed mechanism of MK-9's role in electron transfer and its potential as a drug target. The conservation of these features among Actinobacteria suggests that these findings might have broader implications for targeting related pathogens.

This study provides the first high-resolution structure of the *M. tuberculosis* cytochrome *bd* oxidase, revealing unique structural features like a Q-loop disulfide bond and a novel MK-9 binding site. These findings offer promising targets for developing highly specific anti-tuberculosis drugs. Future research should focus on characterizing the mechanistic role of the MK-9 binding site and exploring the potential of disrupting the Qc-PL8 interaction to inhibit enzyme function.

While the study provides detailed structural insights, it does not fully elucidate the precise mechanistic role of the MK-9 binding site in electron transfer. Further functional studies are needed to confirm the hypothesis of an alternative electron transfer pathway bypassing the Q-loop. The study focuses on the *M. tuberculosis* enzyme, and the generalizability of these findings to other mycobacterial species needs further investigation.