Terminal Respiratory Oxidases: A Targetables Vulnerability of Mycobacterial Bioenergetics?

Abstract

Recently, ATP synthase inhibitor Bedaquiline was approved for the treatment of multi-drug resistant tuberculosis emphasizing the importance of oxidative phosphorylation for the survival of mycobacteria. ATP synthesis is primarily dependent on the generation of proton motive force through the electron transport chain in mycobacteria. The mycobacterial electron transport chain utilizes two terminal oxidases for the reduction of oxygen, namely the bc₁-aa₃ supercomplex and the cytochrome bd oxidase. The bc₁-aa₃ supercomplex is an energy-efficient terminal oxidase that pumps out four vectoral protons, besides consuming four scalar protons during the transfer of electrons from menaquinone to molecular oxygen. In the past few years, several inhibitors of bc₁-aa₃ supercomplex have been developed, out of which, Q203 belonging to the class of imidazopyridine, has moved to clinical trials. Recently, the crystal structure of the mycobacterial cytochrome bc₁-aa₃ supercomplex was solved, providing details of the route of transfer of electrons from menaquinone to molecular oxygen. Besides providing insights into the molecular functioning, crystal structure is aiding in the targeted drug development. On the other hand, the second respiratory terminal oxidase of the mycobacterial respiratory chain, cytochrome bd oxidase, does not pump out the vectoral protons and is energetically less efficient. However, it can detoxify the reactive oxygen species and facilitate mycobacterial survival during a multitude of stresses. Quinolone derivatives (CK-2-63) and quinone derivative (Aurachin D) inhibit cytochrome bd oxidase. Notably, ablation of both the two terminal oxidases simultaneously through genetic methods or pharmacological inhibition leads to the rapid death of the mycobacterial cells. Thus, terminal oxidases have emerged as important drug targets. In this review, we have described the current understanding of the functioning of these two oxidases, their physiological relevance to mycobacteria, and their inhibitors. Besides these, we also describe the alternative terminal complexes that are used by mycobacteria to maintain energized membrane during hypoxia and anaerobic conditions.

Keywords: Aurachin D; Mycobacterium; Q203; bc1-aa3 supercomplex; cytochrome bd oxidase; electron transport chain; oxidative phoshorylation; respiratory inhibitors.

Figure 1

Schematic representation of the electron transport chain of mycobacteria. NADH dehydrogenase (Complex I) oxidizes NADH and transfers the electrons to cytochrome bc₁-aa₃ oxidoreductase (complex III-IV) by reducing menaquinone. Alternatively, succinate dehydrogenase (complex II) uses succinate as substrate and transfers electrons to complex III via menaquinone. Electrons are then transferred to cytochrome bc₁-aa₃ and cytochrome bd-type menaquinol oxidase (bd oxidase), which finally passes the electrons to the terminal electron acceptor, oxygen. During this process, a proton gradient is generated, which helps in the synthesis of ATP by ATP synthase. Inhibitors of the respiratory complexes are shown in red color.

Figure 2

The cytochrome bc₁-aa₃oxidoreductase supercomplex. (A) The passage of electron through various components of the cytochrome bc₁-aa₃oxidoreductase. The crystal structure of cytochrome bc₁-aa₃ complex reveals its homo dimeric form. Each of the monomers is represented by a different color (orange and blue). The complex III is composed of a [2Fe-2S] cluster present on the Riske protein (QcrA), a cytochrome b (QcrB) containing two b-type heme groups (low and high potential), and a di-heme c-type cytochrome c₁(QcrC). Electrons are initially accepted by the QcrB subunit, which is transferred to the Fe-S complex in QcrA, releasing proton in the periplasmic space. Electrons are then passed to cytochrome c of the QcrC subunit via cytochrome c₁. CuA (located on CtaC, subunit II) harvests electrons from the cytochrome c and transfers them to heme a (located on CtaS, subunit I), which then passes them to the dinuclear site composed of heme a3 and CuB (located on CtaD). The dinuclear site activates oxygen for reduction and rapid transfer of four electrons converting it to water. (B) Schematic representation of cytochrome bc₁-aa₃subunits and their inhibitors. Cytochrome bc₁-aa₃forms complex III and IV of the ETC of Mycobacterium species and has proved to be novel drug target against the bacteria.

Figure 3

Genetic regulation of cytochrome bd complex. The expression of cydB component of terminal oxidase cytochrome bd depends upon SenX₃ – RegX₃ two-component system specific to Mycobacterium. During the aerobic condition, phosphorylated RegX₃ binds to the promoter region of cydB and inhibits transcription. However, during hypoxia, RegX₃ gets dephosphorylated and does not bind to the promoter of cydB, thus facilitating its expression.

Figure 4

Mycobacterial cytochrome bd oxidase. The components and inhibitors of terminal oxidase cytochrome bd and the passage of electron through its various subcomponents. The electron is finally accepted by oxygen, which accepts proton from the periplasm to form water.

Figure 5

Schematic representation of the effect of inhibition of both the terminal oxidases of Mycobacterium species. Inhibition of both the terminal oxidases leads to cell death. A combination of various drugs could directly target both of these terminal oxidases, which are specific to Mycobacterium and could effectively kill the bacteria, thus providing a novel drug target against Mycobacterium species.

Figure 6

Nitrate reductase pathway in M. tuberculosis. Figure shows dependence of nitrate reductase pathway upon DosS-DosR two component system of Mtb. In stress conditions such as hypoxia, DosR upregulates expression of NarK2, which in turn, transports nitrate into the cytoplasm. This nitrate is reduced to nitrite with the help of NarGHI. In this process, electron from menaquinol are used for reduction of nitrate. Nitrate produced in the process is transported out, however the nitrate transporter remains to be identified.

Figure 7

Role of fumarate reductase/succinate dehydrogenase as an alternate respiratory pathway. Under hypoxic conditions, fumarate can act as an alternate electron acceptor. For this either fumarate reductase is utilized or succinate dehydrogenase perform the reverse reaction. Succinate is secreted out of the cells.