Isolation and Characterization of a Hybrid Respiratory Supercomplex Consisting of Mycobacterium tuberculosis Cytochrome bcc and Mycobacterium smegmatis Cytochrome aa ₃*

Abstract

Background: Mycobacteria have no soluble cytochrome c; the electron transfer chain involves a Complex III-IV “supercomplex.”

Results: Expression of the M. tuberculosis Complex III in M. smegmatis lacking native complex yields a functional hybrid supercomplex.

Conclusion: This supercomplex is a dimer of protomers containing two each of hemes A, B, and C.

Significance: This is the first purification of respiratory Complex III or IV from Mycobacterium.

Abstract

Recently, energy production pathways have been shown to be viable antitubercular drug targets to combat multidrug-resistant tuberculosis and eliminate pathogen in the dormant state. One family of drugs currently under development, the imidazo[1,2- a]pyridine derivatives, is believed to target the pathogen's homolog of the mitochondrial bc ₁ complex. This complex, denoted cytochrome bcc, is highly divergent from mitochondrial Complex III both in subunit structure and inhibitor sensitivity, making it a good target for drug development. There is no soluble cytochrome c in mycobacteria to transport electrons from the bcc complex to cytochrome oxidase. Instead, the bcc complex exists in a “supercomplex” with a cytochrome aa ₃-type cytochrome oxidase, presumably allowing direct electron transfer. We describe here purification and initial characterization of the mycobacterial cytochrome bcc-aa ₃ supercomplex using a strain of M. smegmatis that has been engineered to express the M. tuberculosis cytochrome bcc. The resulting hybrid supercomplex is stable during extraction and purification in the presence of dodecyl maltoside detergent. It is hoped that this purification procedure will potentiate functional studies of the complex as well as crystallographic studies of drug binding and provide structural insight into a third class of the bc complex superfamily.