Isolation and Characterization of a Hybrid Respiratory Supercomplex Consisting of Mycobacterium tuberculosis Cytochrome bcc and Mycobacterium smegmatis Cytochrome aa3

Mi-Sun Kim,Jichan Jang,Nurlilah Binte Ab Rahman,Kevin Pethe,Edward A Berry,Li-Shar Huang

doi:10.1074/jbc.m114.624312

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 bc1 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 aa3-type cytochrome oxidase, presumably allowing direct electron transfer. We describe here purification and initial characterization of the mycobacterial cytochrome bcc-aa3 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.

Highlights

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
The molecular weight is too high for any known subunits of the supercomplex, but as discussed below, an incompletely dissociated complex containing cytochrome cc is often seen near this molecular weight
The strong band at 50 kDa due to the supercomplex becomes visible after the DEAE-Sepharose chromatography and is slightly enriched by Q-Sepharose and Sepharose 6B chromatography

Summary

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. The leading drug candidate, Q203, combines this low toxicity with high activity against multidrug-resistant and extensively drug-resistant clinical isolates and high potency at low dose in a mouse model of tuberculosis (8, 9, 12) Both mitochondrial cytochrome bc[1] (Complex III) and mycobacterial cytochrome bcc belong to an evolutionarily related superfamily of “cytochrome bc” complexes catalyzing electron transfer from a hydrophobic quinol to a proteinaceous acceptor (13). We report here the purification and initial characterization of a stable supercomplex consisting of the cytochrome bcc of M. tuberculosis and cytochrome aa[3] of M. smegmatis, using untagged proteins and methods suitable for purification on a scale large enough to support crystallographic studies We believe that this is the first reported isolation an untagged Complex III or IV from actinobacteria and the first tagless isolation of a stable Complex III-IV supercomplex from any organism

Experimental Procedures

Results and Discussion

Conclusions