*2.4. Terminal Oxidases*

MKH2 is reoxidised with the transfer of electrons to branched routes of terminal respiratory oxidases or reductases. In *M. tb*, there are two terminal oxidases present which catalyse the four-electron reduction of oxygen to water, namely the proton-pumping Cyt-bcc-aa3 supercomplex and an alternative cytochrome bd oxidase (Cyt-bd) [39].

Cytochromes bcc and aa3 (also known as complexes III and IV) are encoded by qcrCAB and ctaB, ctaC, ctaD, ctaE, respectively, and form a supercomplex of Cyt-bcc-aa3 in mycobacteria [55–57]. As this complex is proton-pumping, it is energetically more favourable for optimum growth and seems to act as the primary respiratory route in *M. tb* under aerobic conditions [15]. Through inducible repression and genetic deletion, elegant studies revealed that, while the Cyt-bcc-aa3 complex is required for optimal *M. tb* growth rates in vitro and in mice, it is not strictly essential for growth nor persistence as long as the alternate Cyt-bd oxidase is expressed [35].

Following the first purification and initial characterisation of a hybrid respiratory complex consisting of *M. tb* cytochrome bcc and *Mycobacterium smegmatis* (*M. smeg*) cytochrome aa3 [56], the crystal structure of the Cyt-bcc-aa3 supercomplex of *M. smeg* has been recently solved by cryo-EM by two independent groups to a resolution of 3.3–3.5 Å [57,58]. This has enabled the full visualization of the supercomplex with its associated subunits, including cytochrome bcc subunits QcrC, QcrA (the Rieske iron-sulphur protein) and QcrB in a dimeric form with their respective prosthetic groups [57,58]. These studies shed light on the direct internal transfer of electrons from MKH2 to oxygen without the need for a separate cytochrome c electron shuttle as in other respiratory systems, and identifies novel subunits that contribute to the stability of the supercomplex, including an enzymatically active superoxide dismutase (SOD1). The presence of SOD1 in the bcc-aa3 supercomplex suggests the need for efficient ROS detoxification at a site dealing with a high amount of oxygen. Altogether, the structural information obtained from these studies is possibly relevant for further rationale-based TB drug discovery studies for inhibitors of this complex due to the high degree of sequence similarity of the quinol oxidation (Qp) site between *M. tb* and *M. smeg*, the site of inhibition of the TB drug candidate Q203 and other known QcrB inhibitors discovered thus far.

The bacterial-specific Cyt-bd is less characterised in *M. tb* [59]. The pathogen harbours genes encoding the two main subunits, CydA and CydB, and a putative ABC transporter CydDC, which has been proposed to be important for the assembly of the cytochrome in *E. coli* [60–63]. Genetic inactivation of the Cyt-bd-encoding genes in *M. tb* presented no loss of bacterial fitness and did not significantly impact ATP homeostasis under standard aerobic growth conditions, nor did it affect growth and persistence of *M. tb* in mice [35,64,65]. To date, two structures of the Cyt-bd have been reported in *Geobacillus thermodenitrificans* and *E. coli* [66,67], and these studies have highlighted the structural diversity within this family of enzymes. Given the low sequence homology of mycobacterial Cyt-bd to that of *G. thermodenitrificans* and *E. coli* [68], further structural studies of these enzymes in mycobacteria would be necessary to aid in drug development against the *M. tb* Cyt-bd oxidase. While structural and biochemical information are currently lacking for the *M. tb* Cyt-bd, similarities can be drawn with the *E. coli* homologue, which exhibits a very high affinity to oxygen and is non-proton-pumping, making it less efficient energetically compared to proton-pumping oxidases [69–71]. These characteristics would indicate a role of this terminal oxidase in bacterial survival in environments of low oxygen tension and protection against oxidative stress, even though it appears to function efficiently under normoxic conditions as well [65]. The upregulation of the cydAB operon has been reported for *M. tb* under hypoxia and in the presence of NO, as well as during the chronic phase of infection in mice [72–74], and when the function of the Cyt-bcc-aa3 is compromised [32,59–61,75–77].
