Cytochrome bd from Mycobacterium tuberculosis (Mtbd) is a menaquinol oxidase that has gained considerable interest as an antibiotic target due to its importance in survival under infectious conditions. Mtbd contains a characteristic disulfide bond that has been hypothesized to confer a redox regulatory role during infection by constraining the movement of the menaquinone-binding Q-loop. Interference of reductants used in the standard activity assay of quinol oxidases has prevented testing of this hypothesis. Here, the role of the disulfide bond and quinone specificity of Mtbd has been determined by the reconstitution of a minimal respiratory chain consisting of a NADH dehydrogenase and Mtbd, both in detergent and native-like lipid environments. Comparison to cytochrome bd from Escherichia coli (Ecbd) confirms that Mtbd is under tight redox regulation and is selective for menaquinol, unable to oxidize either ubiquinol or demethylmenaquinol. Reduction of the Mtbd disulfide bond resulted in a decrease in oxidase activity up to 90%, depending on menaquinol concentrations. In addition, the catalytic rates of Ecbd and Mtbd are over 10 times lower with the natural lipophilic quinones in comparison to their often-used hydrophilic analogs. Additionally, unlike Ecbd, the activity of Mtbd is substrate inhibited at physiologically relevant menaquinol concentrations. We signify Mtbd as the first redox sensory terminal oxidase and propose that this enables Mtbd to adapt its activity in defence against reactive oxygen species encountered during infection by M. tuberculosis.

Menaquinone-specific oxidation by M. tuberculosis cytochrome bd is redox regulated by the Q-loop disulfide bond