F₄₂₀H₂ dependent reductases (FDRs) are found in just Actinobacteria and Archaea including mycobacterial pathogens such as Mycobacterium tuberculosis. These proteins utilise the rare cofactor F₄₂₀ and can be divided into two broad families, FDRAs and FDRBs, which can include as many as 30 different homologues in any individual species. FDRAs reduce oxidative stress against mycobacteria by reducing endogenous quinones and naturally occurring contaminants like aflatoxins though some of them also activate bicyclic 4’-nitroimidazole pro-drugs used against M. tuberculosis. A subset of FDRAs called FDRAAs have no demonstrated activity with any of these substrates and the reason for this maybe a missing loop in the active site of the FDRAAs. To investigate this, the structure of the FDRA msmeg_2027 from Mycobacterium smegmatis has been solved and the FDRAA msmeg_1077 is currently being crystallised for structural comparison. Both FDRAs and FDRBs have split β-barrel structures that are closely related to the highly conserved FMN-dependent proteins called pyridoxamine 5’-phosphate oxidases (PnPOxs) and several FDRB-like proteins appear to be present in organisms that do not synthesise F₄₂₀. As there is a possibility that not all mycobacterial FDRB homologues are F₄₂₀H₂ dependent, several FDRBs from Mycobacterium smegmatis are currently being tested for cofactor preference to identify the amino acid motifs responsible for F₄₂₀H_­2­ dependence.